Denise M.

Harmening

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FIFTH EDITION
 HEMATOLOGIC VALUES*

Reference Range

Determination Conventional SI

Hematology
“Complete” blood count
(CBC): Automated
Methodology Coulter
Model STKS
WBC 4.8-10.8 X 103/uL 4.8-10.8 X 10°/L
RBC Male: 4.7-6.1 * 10°/uL 4.7-6.1 * 10?/L
Female: 4.2-5.4 < 10°/uL 4.2-5.4 * 10?/L
Hemoglobin (Hgb) Male: 14-18 g/dL 140-180 g/L Direct measurement:
Female: 12-16 g/dL 120-160 g/L Cyanide pigment
Infant: 14-22 g/dL 140-220 g/L
Hematocrit (Hct) Male: 42%-52% 0.42-0.52 L/L Calculation:
Female: 37%-47% 0.37-0.47 L/L RBC X MCV/10
Mean corpuscular 80-100 fL 80-100 fL Derived from RBC
volume (MCV) histogram: Measured
volume of 1 RBC
Calculation: Hct/RBC x 10
Mean corpuscular 27-31 pg 27-31 pg Calculation:
hemoglobin (MCH) Hgb/RBC X 10
Mean corpuscular 32%-36% 32.0-36.0 g/dL Calculation:
hemoglobin concentration Hegb/Hct 100
(MCHC)
Red cell distribution 11.5%-14.5% 11.5-14.5 g/L Derived from RBC
width (RDW) histogram:
CV of RBC distribution =
(SD X 100)
Mean
Platelets (PLT) 150,000-400,000/uL 150-400 * 10°/L Direct count: Cells 2-20 fL
Mean platelet volume (MPV) 7.4-10.4 fL 7.4-10.4 fL Derived from PLT histogram

WBC Differential
Lymphocytes 20%-44%
Monocytes 2%-9%
Neutrophils 50%-10%
Bands 2%-6%
Eosinophils O%4%
Basophils 0%—2%

Absolute Counts
Lymphocytes 1.2-3.4 X 10%/uL 1.2-3.4 X 10°/L Lymphs %/100 X WBC
Monocytes 0.11-0.59 X 103/uL 0.11-0.59 X 10°/L Mono %/100 * WBC
Neutrophils 1.4-6.5 X 10% 1.4-6.5 X 10°/L Neutrophils %/100 * WBC
Bands 0-0.7 X 103/uL 0-0.7 X 10°/L Bands %/100 X WBC
Eosinophils 0-0.5 X 103/uL 0-0.5 X 10°/L Eos %/100 * WBC
Basophils 0-0.2 X 10°/uL 0-0.2 10°/L Baso %/100 X WBC
Manual Methodology: CBC
Hematocrit Newborn: 53%-65% 0.53-0.65 L/L Centrifugal microhemato-
Infant/child: 30%43% 0.30-0.43 L/L crit method
Adult male: 42%-—52% 0.42-0.52 L/L
Adult female: 37%47% 0.37-0.47 L/L
Hemoglobin Newborn: 17-23 g/dL 170-230 g/L Cyanmethemeglobin
3 mo: 9-14 g/dL 90-140 g/L method
10 yr: 12-14.5 g/dL 120-145 g/L
Adult male: 14-17 g/dL 140-170 g/L
Adult female: 12.5-15 g/dL 125-150 g/L
*Please note: Normal values vary by institution, patient population, and testing methodology.

Continued on next page

 HEMATOLOGIC VALUES (Continued)

Reference Range

Determination Conventional Notes

Leukocyte count Newborn: 9000—30,000/uL 9.0-30.0 * 10°/L Hemacytometer method

1 wk: 5000-21 ,000/uL 5.0-21.0 * 10°/L
1 mo: 5000-19,500/uL 5.0-19.5 X 10°/L
6-12 mo: 6000-17,500/uL 6.0-17.5 * 10°/L
2 yr: 6200-17,000/uL 6.2-17.0 X 10°/L
Erythrocyte count Newborn: 4.4—5.8 million/uL 4.4-5.8 X 10°%/L Hemacytometer method
Infant/child: 3.8-5.5 million/uL 3.8-5.5 X 10”/L
Adult male: 4.7-6.1 million/uL 4.7-6.1 X 10°/L
Adult female: 4.2—5.4 million/uL 4.2-5.4 * 10°/L
Eosinophil count 50—400/uL 0.05-0.4 * 10°/L Hemacytometer method
Reticulocyte count Newborn: 2.5%-—6.0% 2.5-6.0 g/L New methylene blue
Adult: 0.5%-2.0% 0.5-2.0 g/L
Absolute reticulocyte 24,000-84,000/uL 24-84 X 10°/L Calculation: %Retic X RBC
Erythrocyte sedimentation Male: 0-15 mm/hr Westergren method
rate (ESR) Female: 0—20 mm/hr EDTA (anticoagulant)
Osmotic fragility of Initial hemolysis: 0.45% Heparin (anticoagulant)
erythrocytes Complete hemolysis:
0.30%-0.35%
Acidified serum test No hemolysis
(Ham’s test) for PNH
Sugar water test for PNH <5% hemolysis
Autohemolysis test Lysis at 48 hr: without added
dextrose (0.2%-—2.0%)
Lysis at 48 hr: with added
dextrose (O—1.0%)
Lysis at 48 hr: with added
ATP (0-0.8%)

RBC Enzymes
G6PD 3.4-8.0 U/g Hgb 0.22-0.52 MU/mol Hgb Bishop, modified method
Pyruvate kinase 13-17 U/g Hgb 0.84-1.1 MU/mol Hgb ICSH method

Iron Studies
Serum iron Adult male: 65-170 ug/dL 11.63-30.43 umol/L Colorimetric
Adult female: 50-170 pg/dL 8.95-30.43 pmol/L
TIBC 250-450 g/dL 44.75-80.55 umol/L Colorimetric
Transferrin saturation Male: 20%-50%
Female: 15%—50%
Serum TfR (transferrin 1.5-2.75 mg/L
receptor)
Ferritin (serum) 12-30 ng/mL 12 ug/L-30 ug/L
Iron deficiency 0-12 ng/mL 12 ug/L
Borderline 13-20 ng/mL 13-20 ug/L
Iron excess >400 ng/L >400 pg/L
Zinc protoporphyrin 16-65 pg/dL 0.28 pmol/L-1.17 mol/L
(free erythrocyte
protoporphyrin)

Folic Acid
Normal = s.onp/18 >7.3 nmol/L RIA
Borderline 2.5—3.2 ng/mL 5.75-7.39 nmol/L
Red cell folate 150-450 mg/mL 340-1020 nmol/L Collect in EDTA

Haptoglobin 40-336 mg/dL 0.4-3.6 g/L

SI = System of International Units; CV = coefficient of variation; SD = standard deviation; PNH = paroxysmal
nocturnal hemoglobinuria; ATP = adenosine triphosphate; G6PD = glucose-6-phosphate dehydrogenase
deficiency; TIBC = total iron-binding capacity; RIA = radioimmunoassay; RID = radioimmunodiffusion;
ICSH = International Committee for Standardization of Hematology; EDTA = ethylenediamine tetraacetic acid.
 Digitized by the Internet Archive
in 2022 with funding from
Kahle/Austin Foundation

httos://archive.org/details/clinicalhematolo0000unse_d5f7
Clinical
Hematology and
Fundamentals of
Hemostasis
FIFTH EDITION
~
 FIFTH EDITION

Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)

Director of Instruction and Customer Learning, Heritage Division
American Red Cross
and :
Professor |
Department of Medical and Research Technology
University of Maryland School of Medicine
Baltimore, Maryland

CONSORTIUM LIBRARY, ANCHORAGE, AK

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Library of Congress Cataloging-in-Publication Data

Clinical hematology’ and fundamentals of hemostasis / [edited by] Denise M. Harmening. —Sth ed.
p.;cm.
Includes bibliographical references and index.
ISBN-13: 978-0-8036-1732-2
ISBN-10: 0-8036-1732-1
1. Hematology. 2. Blood—Diseases. 3. Homeostasis. I. Harmening, Denise.
[DNLM: |. Hematologic Diseases. 2. Blood Cells—physiology. 3. Hemostasis. WH 100 C6413 2009]
RB145.C536 2009
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To all students—full-time, part-time, past, present,
and future—who have touched and will continue to
touch the lives of so many educators... .

It is to you this book is dedicated in the hope of

inspiring an unquenchable thirst for knowledge and
love of mankind.
 Preface

The fifth edition of this text continues to set the standard for guide with student exercises; and all the color illustrations
teaching and learning in clinical hematology and fundamen- and figures by chapter to use in PowerPoint lectures. A stu-
tals of hemostasis. Thorough, yet concise, a total of 36 chap- dent website is also available for this edition which includes
ters serve as a combination of text, laboratory procedure Picture Match, Flash Cards, and Case Histories.
manual, and atlas of cell morphology. This textbook is unique This new edition, like the first, second, third, and fourth,
in its five-part format, featuring an introduction to clinical is a culmination of the dedicated efforts of many prominent
hematology and sections on anemias, white blood cell disor- laboratory professionals. More than fifty-five contributors
ders, hemostasis and thrombosis, and laboratory methods. committed themselves to this project by donating their time
Outlines, educational objectives, case histories, summary and expertise out of concern for a common goal: improving
charts, and study guide questions support each chapter. patient care by providing a high-quality, practical, and usable
The laboratory methods section includes two new chap- textbook. To all of these contributors, thank you and congrat-
ters, “Quality Management, Quality Assurance, and Quality ulations on meeting this goal. A very special thank you and
Control” and “Body Fluid Examination,” in addition to chap- acknowledgment must also be given to my husband, Jesse
ters on hematology methods, automated differential analysis, Javens, who always supports my writing and provides en-
coagulation methods, flow cytometry, molecular diagnostic couragement which always motivates me. I would also like to
techniques in hematopathology, and cytochemistry. Numer- thank the following students who helped with this edition:
ous illustrations, including 375 color photographs, 600 line Ernest Frank, Laura Little, Nathan Marchiano, Abiola
drawings, and 355 tables throughout the book, aid in the Oderinlo, and especially Laura Simpson, who spent many
process of learning. The text has retained the popular listing hours assisting me.
of normal hematologic values on the inside covers for a quick Finally, my sincere appreciation is also extended to
reference. In addition, an invaluable glossary provides easy the educators, clinicians, and industry representatives
access to definitions of medical terms unique to hematology who gave thorough and thoughtful reviews of selected
and hemostasis. Summary charts have been added at the end manuscripts.
of each chapter to identify for students the most important In summary, this book has been designed to inspire an
information to know for clinical rotation. ACD-Rom is avail- unquenchable thirst for knowledge in every medical technol-
able to educators who adopt the text. This CD includes an ogist, hematologist, clinician, and practitioner whose knowl-
interactive test-generating question bank containing over edge, skills, and ongoing education provide the public with
2000 questions with assigned taxonomy levels; an instructor’s excellent health care.
D.M. Harmening, PhD, MT(ASCP), CLS(NCA)

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 Contributors

Ashraf Z. Badros, MD Thérésa L. Coetzer, PHD

Associate Professor Assistant Professor
University of Maryland School of Medicine Department of Molecular Medicine and Hematology
Oncology Program University of Witwatersrand Medical School
Baltimore, Maryland Parktown, Johannesburg, South Africa

Ann Bell, Ms, SH(ASCP), CLSpH(NCA) Frank Cruz IT, mp

Emeritus Professor, Clinical Laboratory Sciences Hematopathology Fellow
Emeritus Assistant Professor, Department of Medicine Department of Pathology
University of Tennessee, Memphis Oregon Health and Science University
Memphis, Tennessee Portland, Oregon

Melissa Bethel, MT(ASCP) Naomi B. Culp, DA, MT(ASCP)SH

Laboratory Manager Manager, Scientific Marketing
Esoterix Coagulation Clinical Cellular Analysis Business Center
Aurora, Colorado Beckman Coulter, Inc.
Brea, California
Angelica Black, USAF, BSC, MS, MT(ASCP)
Laboratory and Pathology Flight Commander Krista M. Curcio, MT(ASCP)
Malcolm Grow Medical Center Manager, Scientific Affairs
Andrews Air Force Base, Maryland Sysmex America, Inc.
Mundelein, Illinois
Rita Braziel, MD
Professor, Department of Pathology Giovanni D’ Angelo, FCMLS
Oregon Health and Science University Associate Director, Quebec Leukemia Cell Bank
Portland, Oregon Department of Hematology
Hopital Maisonneuve-Rosemont
Carlos E. Bueso-Ramos, MD, PHD Montreal, Quebec, Canada
Associate Professor
Department of Hematopathology Aamir Ehsan, MD
Division of Pathology and Laboratory Medicine Assistant Professor
The University of Texas Department of Pathology
M.D. Anderson Cancer Center The University of Texas Health Science Center
Houston, Texas at San Antonio
San Antonio, Texas
Lambert Busque, MD, FRCP(C)
Associate Professor of Medicine Claudia E. Escobar, MT(ASCP)SH
University of Montreal Technical Service Specialist
Director, Molecular Hematology and Staff Hematologist Diagnostica Stago
H6pital Maisonneuve-Rosemont Parsippany, New Jersey
Montreal, Quebec, Canada
Kathleen Finnegan, MS, MT(ASCP)SH
Kathrina Chua, MD Chair and Clinical Associate Professor
Hematology/Oncology Clinical Laboratory Sciences Program
Bayhealth Medical Center State University of New York, Stony Brook
Milford, Delaware Stony Brook, New York
Ye Contributors

Donna M. Gandour, PHD Josée Hébert, MD, (FRCP)C

Manager, Customer Education Services Associate Professor of Medicine
BD Biosciences—Immunocytometry Systems University of Montreal
San Jose, California Hematologist, H6pital Maisonneuve-Rosemont
Director, Cytogenetic Laboratory
Ken Gatter, MD, JD Hopital Maisonneuve-Rosemont
Associate Professor Director, Quebec Leukemia Cell Bank
Department of Pathology Research Center, H6pital Maisonneuve-Rosemont
Oregon Health and Science University Montreal, Quebec, Canada
Portland, Oregon
Adjunct Professor of Law Jennifer L. Herrick, MD
Willamette University School of Law Hematopathology Fellow
Salem, Oregon The University of Texas Health Science Center
at San Antonio
Ian Giles, MD San Antonio, Texas
Director of Scientific Affairs
Sysmex America Inc. Meyer R. Heyman, MD
Mundelein, Illinois Associate Professor
University of Maryland School of Medicine
Armand B. Glassman, MD Oncology Program
Olla S. Striblin Distinguished Chair for Cancer Research Baltimore, Maryland
Kiawah Island, South Carolina
Russell Aaron Higgins, MD
Rick Gooch, MBA, MT(ASCP) Assistant Professor of Pathology
Manager, Global Scientific Leadership The University of Texas Health Science Center
Abbott Hematology at San Antonio
Santa Clara, California San Antonio, Texas

Ralph Green, BAPPSCI(MLS), FAIMLS Laurel D. Holmer, MED, MT(ASCP)SH

Associate Professor Formerly from
Division and Program Leader Department of Clinical Laboratory Science
Division of Laboratory Medicine University of Nevada School of Medicine
School of Medical Sciences Reno, Nevada
Royal Melbourne Institute of Technology University
Bundoora, Victoria, Australia Virginia C. Hughes, Ms, MT(ASCP)SBB, CLS(NCA)I
Assistant Professor
Margaret L. Gulley, mp Division of Clinical Laboratory Sciences
Department of Pathology Auburn University at Montgomery
University of North Carolina Montgomery, Alabama
Chapel Hill, North Carolina
Dan M. Hyder, Mp
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA) Director, Department of Pathology and Laboratory Medicine
Director of Instruction and Customer Learning Southwest Washington Medical Center
Heritage Division Vancouver, Washington
American Red Cross
Professor, Department of Medical and Research Technology Kathy W. Jones, MS, MT(ASCP), CLS(NCA)
University of Maryland School of Medicine Division of Clinical Laboratory Science
Baltimore, Maryland Auburn University at Montgomery
Montgomery, Alabama
Chantal Ricaud Harrison, MD
Medical Director/Blood Bank Carmen J. Julius, mp
and Professor of Pathology AmeriPath, Inc.
The University of Texas Health Science Center Vice Chair, Department of Pathology
at San Antonio St. Elizabeth Health Center
San Antonio, Texas Youngstown, Ohio
Assistant Professor of Pathology
Northeastern Ohio Universities College of Medicine
Rootstown, Ohio
 Contributors Xl

Richard Kendall, FIBMs, PHD Mary Loring Perkins, Ms, MT(ASCP)SH

Director of Global Scientific Affairs System Manager, Hematology
Abbott Hematology Manager, Cytogenetics, Molecular Diagnostics
Santa Clara, California and Flow Cytometry
Legacy Laboratory Services
Charles L. Knupp, mp Legacy Health System
Professor of Medicine Portland, Oregon
Division of Hematology/Oncology
East Carolina University School of Medicine Sherrie L. Perkins, MD, PHD
Greenville, North Carolina Medical Director of Hematopathology
Division of Anatomic Pathology
Jolanta Kunicka, PHD The University of Utah Health Sciences Center
Scientific Affairs Salt Lake City, Utah
Siemens Healthcare Diagnostics
Tarrytown, New York Kim A. Przekop, BS, MT(ASCP)
Hematology Supervisor
Louann W. Lawrence, DRPH, MT(ASCP)SH, CLSP(NCP) Saint Michael’s Medical Center
Associate Professor and Department Head Newark, New Jersey
Department of Clinical Laboratory Sciences
School of Allied Health Professions Carl R. Schaub, MD
Louisiana State University Health Science Center AmeriPath, Inc.
New Orleans, Louisiana Chair, Department of Pathology
Health Center, Youngstown, Ohio
John Lazarchick, MD Youngstown, Ohio
Professor/Director Associate Professor of Pathology
Hematopathology/Hemostasis Northeastern Ohio Universities College of Medicine
Medical University of South Carolina Rootstown, Ohio
Pathology and Laboratory Medicine
Charleston, South Carolina Sharon L. Schwartz, MS, MT(ASCP)SH, CLS(NCA)
Technical Specialist
Darla K. Liles, mp Special Coagulation, Special Hematology Section
Assistant Professor of Medicine North Shore-Long Island Jewish Health Systems Laboratories
Division of Hematology/Oncology Lake Success, New York
East Carolina University School of Medicine Adjunct Associate Professor
Greenville, North Carolina Department of Biomedical Sciences
School of Health Professions and Nursing
Joe Marty, MS, MT(ASCP) Clinical Laboratory Science Program
Formerly from Long Island University
Department of Pathology C.W. Post Campus
The University of Utah Health Sciences Center Brookville, New York
Salt Lake City, Utah
Catherine M. Spier, MD
David L. McGlasson, MS, CLS(NCA) Formerly from
Clinical Research Scientist Department of Pathology
59th Clinical Research Squadron/MSRL University of Arizona
Lackland Air Force Base, Texas Health Science Center
Tucson, Arizona
Luigina Mollica, MD, FRCP(C), PHD
Department of Hematology Ronald G. Strauss, MD
H6pital Maisonneuve-Rosemont Formerly from
Montreal, Quebec, Canada University of lowa Hospitals and Clinics
Iowa City, lowa
Melanie Oswald, MHS, MT(ASCP)SH
Coagulation Technical Coordinator Mitra Taghizadeh, Ms, MT(ASCP), CLS(NCA)
Fast Flow Laboratory Clinical Assistant Professor
Medical University of South Carolina Department of Medical and Research Technology
Charleston, South Carolina University of Maryland School of Medicine
Baltimore, Maryland
Xil Contributors

David Yang, MD Hallye Zeringer, MT(ASCP)SH

Formerly Formerly from
Hematopathology Fellow Southern Baptist Hospital
University of Utah Health Sciences New Orleans, Louisiana
Salt Lake City, Utah
Frank Xianfeng Zhao, MD, PHD
Stan Zail, MBBCH, MD, FCRPATH(London) Assistant Professor of Pathology/Attending Hematopathologist
Department of Molecular Medicine and Haematology Director of Hematopathology Fellowship
South African Institute for Medical Research Department of Pathology
School of Pathology University of Maryland School of Medicine
University of Witwatersrand Medical School Baltimore, Maryland
Johannesburg, South Africa
 Keviewers

Hassan Aziz, PHD Theresa D. Foster, MPH, MT(ASCP)SH

Department Head, Associate Professor Program Director
Medical Technology Medical Technology
Armstrong Atlantic State University Saint Francis Hospital
Savannah, Georgia Tulsa, Oklahoma

Marvin D. Bearden, MA, CLS(NCA), MT(ASCP) Jan Fox, BSC, ART MLT
Assistant Professor, Education Coordinator Coordinator
Clinical Laboratory Sciences Medical Laboratory Sciences
The University of Texas Health Science Center St. Lawrence College
at San Antonio Kingston, Ontario, Canada
San Antonio, Texas
Mildred K. Fuller, PHD, MT(ASCP)
Bernadette Bekken, CLS(NCA), MT(ASCP)BB Chair and Professor
Program Director * Allied Health
Clinical Laboratory Sciences Norfolk State University
Augusta Medical Center Norfolk, Virginia
Fishersville, Virginia
Andrea G. Gordon, MED, MT(ASCP)SH
Rose V. Brown, MED, CLS, MT(ASCP) Professor
Program Director Health and Human Services
Clinical Laboratory Sciences New Hampshire Community Technical College
Penrose-St. Francis Hospital Claremont, New Hampshire
Colorado Springs, Colorado
Karen G. Gordon, BS, CLS, MLT(ASCP)SLS
Donna D. Castellone, MS, MT(ASCP)SH Program Manager, Assistant Professor
Clinical Project Manager Medical Laboratory Technology
Hemostasis/Hematology Northern Virginia Community College
Siemens Healthcare Diagnostics Springfield, Virginia
Tarrytown, New York
Michele G. Harms, MS, MT(ASCP)
Faye E. Coleman, MS, CLS, MT(ASCP) Program Director
Program Director, Associate Professor Medical Technology
Medical Laboratory and Radiation Sciences Women’s Christian Association Hospital
Old Dominion University Jamestown, New York
Norfolk, Virginia
Sheryl B. Herring, BS, MSA, MT(ASCP)
Linda Collins, MS, MT(ASCP) Program Coordinator
Instructor Clinical Laboratory Technology
Medical Laboratory Technology Louisiana State University
Delaware Technical & Community College Alexandria, Virginia
Dover, Delaware
Virginia C. Hughes, Ms, MT(ASCP)SBB, CLS(NCA)I
Jane B. Finley, BS, MT(ASCP) Assistant Professor
Clinical Assistant Professor Division of Clinical Laboratory Sciences
Clinical Laboratory Sciences Auburn University at Montgomery
The University of Texas Medical Branch Montgomery, Alabama
Galveston, Texas Xiil
X1V Reviewers

Stephen M. Johnson, Ms, MT(ASCP) Jan C. Rogers, MS, MT(ASCP)

Program Director Program Coordinator
Medical Technology Medical Laboratory Technology
Saint Vincent Health Center Morrisville State College
Erie, Pennsylvania Morrisville, New York

Gail I. Jones, PHD, MT(ASCP)SC Stacey Rohrbaugh, MED, CLS(NCA), MT(ASCP)

Instructor Associate Professor
Biology Department Medical Laboratory Technology
Texas Christian University Allegany College of Maryland
Fort Worth, Texas Cumberland, Maryland

Jennifer B. Jones, BS, MT(ASCP)SC John K. Scariano, PHD

Clinical Instructor Assistant Professor
Clinical Laboratory Sciences Pathology and Internal Medicine
The University of Kansas Medical Center University of New Mexico
Kansas City, Kansas Albuquerque, New Mexico

Art Keehnle, Ms, CLS(NCA), MT(ASCP) Karen Senechal, BSC, MLT

Program Director Medical Laboratory Sciences
Medical Laboratory Technology St. Clair College
Beaufort County Community College Windsor, Ontario, Canada
Washington, North Carolina
Lauren I. Strader, MED, MT(ASCP)
Pam Kieffer, Ms, CLS(NCA), MT(ASCP) Program Director, Chair
Program Director Allied Health and Medical Laboratory Technology
Clinical Laboratory Sciences North Georgia Technical College
Rapid City Regional Hospital Clarkesville, Georgia
Rapid City, South Dakota
M. Lorraine Torres, MS, MT(ASCP)
Beverly A. Kirby, CLS(NCA), MT(ASCP) Program Director
Assistant Professor Clinical Laboratory Sciences
Pathology University of Texas at El Paso
West Virginia University El Paso, Texas
Morgantown, West Virginia
Meridee Van Draska, MS, CLS(NCA), MT(ASCP)
Lisa McDonnel, Ms, CLS(NCA), MT(ASCP) Program Director, Assistant Professor
Lecturer Health Sciences
Medical Technology Ihnois State University
University of Washington Normal, Illinois
Seattle, Washington
Carol Watkins, MBA, MT(ASCP)
Evelyn Paxton, MS, MT(ASCP) Program Director, Associate Professor
Program Director Fundamental and Applied Sciences
Clinical Laboratory Sciences Wayne State University
Rose State College Detroit, Michigan
Midwest City, Oklahoma
Lori A. Woeste, EDD, MT(ASCP)
Alisa J. Petree, MHSM, MT(ASCP) Assistant Professor
Program Director Clinical Laboratory Sciences
Clinical Laboratory Sciences/Medical Technology Health Sciences
Hillcrest Baptist Medical Center Illinois State University
Waco, Texas Normal, Illinois

Linda R. Riipi, MD, MT(ASCP)

Professor
Clinical Laboratory Sciences
Northern Michigan University
Marquette, Michigan
 Contents

Preface vii Chapter /hO_

Hemouiey gemiay Intracorpuscular Defects:
PART 1 Il. Hereditary Enzyme Deficiencies 196
Armand B. Glassman, MD
INTRODUCTION TO CLINICAL HEMATOLOGY 1
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Chapter 1
Chapter-H.
Morphology of Human Blood and Marrow Cells:
Hemofytic Anemias: Intracorpuscular Defects:
Hematopoiesis 1
Ann Bell, MS, SH(ASCP), CLSPH(NCA) III. The Hemoglobinopathies 207
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
David Yang, MD
Virginia C. Hughes, MS, MT(ASCP)SBB, CLS(NCA)I
Hallye Zeringer, MT(ASCP)SH
Chapter 2
Chaptef |
Bone Marrow 42
Aamir Ehsan, MD
Hemolytic Anemias: Intracorpuscular Defects:
Jennifer L. Herrick, MD IV. Thalassemia 230
Russell Aaron Higgins, MD
Chapter 3 .

Captor 13
Chantal Ricaud Harrison, MD
The Red Blood Cell: Structure and Function 64
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Hemolytic Anemias: Extracorpuscular
Chapter 4
Defects 252
Anemia: Diagnosis and Clinical Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Considerations 82 Louann W. Lawrence, DRPH, MT(ASCP)SH, CLSPH(NCA)
Armand B. Glassman, MD Ralph Green, BAPPSCI(MLS), FAIMLS
Chapter 5 Carl R. Schaub, MD

Evaluation of Cell Morphology and Introduction Chapter 14

to Platelet and White Blood Cell Morphology 95 Hypoproliferative Anemia: Anemia Associated
Kathy W. Jones, MS, MT(ASCP), CLS(NCA) with Systemic Diseases 280
Carmen J. Julius, MD
Carl R. Schaub, MD
PART 2
ANEMIAS 117
Chapter 6
PART 3
Iron Metabolism and Hypochromic WHITE BLOOD CELL DISORDERS 305
Anemias 117 Chapter 15
Kathleen Finnegan, MS, MT(ASCP)SH Cell Biology, Disorders of Neutrophils,
Chapter 7 Infectious Mononucleosis, and Reactive
Megaloblastic Anemias 158 Lymphocytosis 305
Mitra Taghizadeh, MS, MT(ASCP), CLS(NCA) Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Joe Marty, MS, MT(ASCP)
Chapter 8 Ronald G. Strauss, MD
Aplastic Anemia Including Pure Red Cell
Chapter 16
Aplasia, Congenital Dyserythropoietic Anemia,
Introduction to Leukemia and the Acute
and Paroxysmal Nocturnal Hemoglobinuria 156
Leukemias 3531
Sherrie L. Perkins, MD, PHD
Ken Gatter, MD, JD
Chapter9 Frank Cruz II, MD
laevis se to Hemolytic Anemias: Rita Braziel, MD

Intracorpuscular Defects: Chapter 17

I. Hereditary Defects of the Red Cell Membrane 176 Chronic Myeloproliferative Disorders I: Chronic
Thérésa L. Coetzer, PHD Myelogenous Leukemia 371
Stan Zail, MBBCh, MD, FRCPATH Carl R. Schaub, MD

XV
XVI Contents

Chapter 18
Chronic Myeloproliferative Disorders II: PART 5
Polycythemia Vera, Essential Thrombocythemia, LABORATORY METHODS 697
and Idiopathic Myelofibrosis 3585 Chapter 29
Kathrina Chua, MD Quality Management, Quality Assurance,
Meyer R. Heyman, MD and Quality Control 697
Chapter 19 Kim A. Przekop, BS, MT(ASCP)
Myelodysplastic Syndromes 412 Chapter 30
Giovanni D’Angelo, FCMLS Body Fluid Examination: The Qualitative,
Luigina Mollica, MD, FRCP(C), PHD
Quantitative, and Morphologic Analysis
Josée Hébert, MD, FRCP(C)
Lambert Busque, MD, FRCP(C)
of Serous, Cerebrospinal, and Synovial
Fluids 720
Chapter 20 Sharon L. Schwartz, MS, MT(ASCP)SH, CLS(NCA)
Chronic Lymphocytic Leukemia and Related
Chapter 31
Lymphoproliferative Disorders 440
Laurel D. Holmer, MEb, MT(ASCP)SH Hematology Methods 759
Virginia C. Hughes, MS, MT(ASCP)SBB, CLS(NCA)I
Carlos E. Bueso-Ramos, MD, PHD

Chapter 21 Chapter 32
The Lymphomas 466 Principles of Automated Differential
Dan M. Hyder, MD Analysis 793
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Chapter 22
Angelica Black, USAF, BSc, MS, MT(ASCP)
Multiple Myeloma and Related Plasma Cell Naomi B. Culp, DA, MT(ASCP)SH
Disorders 500 Jolanta Kunicka, PHD
AshrafZ. Badros, MD Krista M. Curcio, MT(ASCP)
Chapter 25 Tan Giles, MD
Richard Kendall, FIBMS, PHD
Lipid (Lysosomal) Storage Diseases
Rick Gooch, MBA, MT(ASCP)
and Histiocytosis 525
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA) Chapter 35
Catherine M. Spier, MD Coagulation Methods 849
Melissa Bethel, MT(ASCP)
Saas

PART 4 Chapter 34
HEMOSTASIS AND INTRODUCTION Applications of Flow Cytometry
TO THROMBOSIS 543 to Hematopathology 882
Donna M. Gandour, PHD
Chapter 24
Chapter 35
Introduction to Hemostasis 543
Denise M. Harmening, PHD, MT(ASCP), CES(NCA) Molecular Diagnostic Techniques
Claudia E. Escobar, MT(ASCP)SH in Hematopathology 911
David L. McGlasson, MS, CLS(NCA) Margaret L. Gulley, MD

Chapter 25 Chapter 36
Disorders of Primary Hemostasis: Quantitative Special Stains/Cytochemistry 929
and Qualitative Platelet Disorders and Vascular Mary Loring Perkins, MS, MT(ASCP)SH
Denise M. Harmening, PHD, MT(ASCP), CLS(NCA)
Disorders 577
Frank Xianfeng Zhao, MD, PHD
Darla K. Liles, MD
Charles L. Knupp, MD

Chapter 26 Answers to Chapter Review Questions 951

Disorders of Plasma Clotting Factors 607 Glossary 953
Sharon L. Schwartz, MS, MT(ASCP)SH, CLS(NCA) Bibliography 977
Chapter 27 Index 981
Interaction of the Fibrinolytic, Coagulation,
and Kinin Systems; Disseminated Intravascular
Coagulation; and Related Pathology 643
John Lazarchick, MD
Melanie Oswald, MHS, MT(ASCP)SH
Chapter 28
Introduction to Thrombosis and Anticoagulant
Therapy 660
Aamir Ehsan, MD
Jennifer L. Herrick, MD
PART |
INTRODUCTION TO CLINICAL HEMATOLOGY

Chapter

Morphology of Human
Blood and Marrow Cells
Hematopoiesis
Ann Bell, MS, SH(ASCP), CLSpH(NCA)
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
Virginia C. Hughes, Ms, MT(ASCP)SBB, CLS(NCA)I

Basic Morphology. and Basic OBJECTIVES

Concepts
At the end of this chapter, the learner should be able to:
Morphology of Cells on the
Normal Blood Smear 1. Describe two main differences between neutrophilic band and neutrophilic
Erythrocytes (Red Blood segmented cells.
Cells) 2. Distinguish between eosinophils and basophils.
Platelets (Thrombocytes)
Leukocytes (White Blood 3. Describe four morphological features that are helpful in identifying monocytes.

Cells) 4. List four characteristics of lymphocytes.

ee eran 5. Compare a large lymphocyte with a monocyte.

Ontogeny (Origin) of 6. Define the term hematopoiesis.

BRST 9 AUTON 7. Name organs responsible for hematopoiesis in the fetus.
Erythropoiesis peaatior
Pronormoblast (Rubriblast, 8. Define the term erythropoiesis.

Proerythroblast) 9. List the proper cell maturation sequence of the erythroid series.
Basophilic Normoblast '
10. Recognize each cell in the erythrocytic series.
(Prorubricyte, Basophilic
Erythroblast) 11. Give two or more characteristics of each nucleated red cell in the erythrocytic series.
Polychromatophilic
Normoblast (Rubricyte, 12. List the proper cell sequence for myelopoiesis (granulocytopoiesis).

Polychromatophilic 13. Recognize each cell in the myelocytic series.

Erythroblast) i : ue.
Orthochromatic Normoblast 14. Differentiate the morphological features of promyelocyte, neutrophilic myelocyte,
(Metarubricyte, neutrophilic metamyelocyte, and neutrophilic band.

Orthochromatic 15. Describe the morphology of a myeloblast.

Bear ia) 16. Describe three features of a mature plasmacyte.

Basophilic Erythrocyte, 17. State the function of megakaryocytes.

Polychromatophilic
Erythrocyte) 18. Explain the process of platelet release from the bone marrow.
2 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Erythrocyte (Red Blood Cell, 19. Distinguish between osteoblasts and osteoclasts.
Discocyte)
20. Name two differences between a megakaryocyte and an osteoclast.
Myelopoiesis
(Granulocytopoiesis)
Morphological Changes
Stages of Differentiation and
Maturation
Monopoiesis
Monoblasts and
Promonocytes
Monocytes and
Macrophages
Lymphopoiesis
Lymphoblasts and
Prolymphocytes
Lymphocytes
Plasmablasts and
Proplasmacytes
Plasmacytes (Plasma Cells)
Megakaryocytopoiesis
Bone-derived Cells
Osteoblasts
Osteoclasts
Molecular Hematology and
Advanced Concepts
Introduction to the Cell
Cycle
Specific Cell Line Ontogeny
Trends in Therapeutic
Manipulation of
Hematopoiesis
Recombinant Cytokines
Clinical Trials of
Recombinant Cytokines
CD Nomenclature
Clinical Applications of Cell
Surface Markers

Basic Morphology and Basic Concepts

Hematology is the study of blood and its related disorders. Its
birth can be traced to 1642, when Anton van Leeuwenhoek
‘Table 1-1 History of H ematology
first noted cells in the blood. More than 200 years elapsed
before other investigators expanded upon van Leeuwenhoek’s van Leeuwenhoek first notes cells in blood.
discovery. The significant milestones in the history of hema- Donne discovers platelets.
tology are outlined in Table |-1. Gulliver differentiates lymphocytes from
The average blood volume in an adult is 4 to 6 L: granulocytes by size.
women have 4 to 5 L, and men 5 to 6 L. This blood volume Malassez counts white blood cells (WBCs)
via hemocytometry.
represents about 8% of the total body weight. Blood has a pH Hayem defines methods for counting platelets.
between 7.35 and 7.45. Blood is composed of 55% plasma Ehrlich uses aniline dyes to stain WBCs.
(the fluid portion) and 45% formed elements or cells. Of the
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 3

Water (91.5%)
Plasma Proteins (7%)
Other Solutes (1.5%)

“Buffy Coat” Lymphocytes Basophils

White Blood Cells Granulocytes Neutrophils
Platelets Monocytes Eosinophils
Red Blood Cells

Figure 1-1 ® Centrifuged whole blood depicting plasma, buffy

coat, and RBC layers.

45% cellular elements, approximately 44% of the cells are

red blood cells (RBCs), whereas only 1% are white blood
cells (WBCs) and platelets (PLTs) (Fig. 1-1). Plasma is
composed of about 91.5% water and 8.5% solutes. The solutes
consist of three different kinds of proteins: albumins (55%),
globulins (38%), and fibrinogen (7%); other solutes are
electrolytes, hormones, nonprotein nitrogen compounds, nutri-
ents, and respiratory gases. The reference values for the cellular
elements are as follows: RBCs (4.2 to 5.4 x 10!/L for
females and 4.7 to 6.1 X 10'*/L for males), WBCs (4.8 to
10.8 X 10°/L and PLTs (150 to 400 X 10°/L) in adults.' These
reference values vary with age, gender, geographic ___location,
and health or disease.
Figure 1-2 ™ Cell types found in smears of peripheral blood from
normal individuals. A. Red blood cells (RBCs). B. Large lymphocyte.
Morphology of Cells on the Normal C. Segmented neutrophil. D. Eosinophil. E. Segmented neutrophil.
Blood Smear F, Monocyte. G. Platelets. H. Small lymphocyte. /. Neutrophilic
band. J. Basophil.
The primary step in assessing hematologic function and the
presence of disease is an examination of the cellular elements
in the blood. Examination of the blood frequently gives electrolytes. A normal mature erythrocyte is a biconcave disc
important information that aids in the diagnosis of hemato- that is 7 to 8 um in mean diameter and 1.5 to 2.5 um thick.
logic disease and may suggest further testing. A well made The RBC has a mean volume of 90 femtoliters (fL). After the
and well stained blood smear is vital, because the analysis of smear is stained with Wright’s stain, an erythrocyte appears as
cell morphology may be greatly hindered by poorly made and a circular cell with distinct and smooth margins and has a dull
poorly stained smears. (See Chap. 31 for smear preparation.) pinkish hue. In the central portion of the erythrocyte where
Careful examination of cell morphology on a blood the cell is thinnest, the intensity of the stain is less than at the
smear and determination of the percentage of each type of marginal area, creating an area of central pallor. Red cells
blood cell present is an important skill to master. Blood cells should be fairly uniform in size and relatively round in shape,
normally present on a blood smear are RBCs (erythrocytes), with a small area of central pallor and no nucleus or inclu-
sions (see Fig. 1-3).
white blood cells (leukocytes), and platelets (thrombocytes)
(Fig. 1-2). Morphological descriptions of each of these cellular
elements in normal blood are presented in this chapter. Platelets (Thrombocytes)
In the same area where erythrocyte morphology is being stud-
Erythrocytes (Red Blood Cells)
ied, the number of platelets per oil immersion field and
Erythrocyte morphology is evaluated in an area of the stained the morphology should be evaluated. Platelets are approxi-
smear where red cells are evenly distributed and do not over- mately | to 4 um in diameter and vary in shape.? An average of
lap (Fig. 1-3). Red cells consist of a plasma membrane sur- 7 to 15 platelets per oil immersion field is normally observed.
rounding a solution of proteins (mainly hemoglobin) and An estimate of the number of platelets in 10 oil immersion fields
4. Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Absolute Values
(per mm*)

N. segmented 2400-7500
Eosinophil 0-450
Basophil 0-200
Lymphocyte 1200-3400
Monocyte 100-900

Mature neutrophils are approximately twice the size of

Figure 1-5 ® Normal peripheral blood smear; normal erythrocytes normal erythrocytes. Granulocytes, as well as monocytes, play
and platelets.
a key role in inflammation and phagocytosis. They migrate
from the blood vessel into the tissues, where they serve as the
should be made. Platelets may be observed in small groups (see
first line of defense against infections.
Fig. 1-2G). A count of individual platelets in a group should be
BAND NEUTROPHILS.(NONSEGMENTED
made.*
In Wright’s stain a DEES contains reddish-purple gran- NEUTROPHILS, NONFILAMENTED NEUTROPHILS)
ules in a small amountof bluish cytoplasm, but there is no Peripheral blood of healthy individuals contains 2% to 6% of
nucleus. Platelets contain particular molecules needed for the band neutrophils (see Table 1-2). Band neutrophils have a
hemostasis and are able to adhere, aggregate, and supply a nucleus with a horseshoe or sausage shape in which the oppo-
surface for coagulation reactions.! site edges of the nucleus become almost parallel for an appre-
ciable distance.’ These cells do not have a nucleus separated
into lobes connected by a filament (Fig. 1-5).
Leukocytes (White Blood Cells) The nuclear chromatin is clumped, and there is usually a
dark pyknotic mass at each pole where the lobe is destined to
The morphology and the distribution of leukocytes are rou- be.* The secondary neutrophil granules are small, evenly dis-
tinely evaluated. WBCs normally observed on a blood
tributed, stain various shades of pink (on Wright’s stain), and
smear include neutrophils (neutrophil segmented and a few
contain alkaline phosphatase.' There may be an occasional
neutrophil bands), eosinophils, basophils, lymphocytes, and dark primary granule (see Fig. |—2/).
monocytes (see Fig. 1-2). Immature cells of any type are
Difficulty may arise in differentiating between band
abnormal. Cells should be examined for abnormalities in and segmented neutrophils and in deciding whether the link
nucleus or cytoplasm.
connecting the lobes is narrow enough to be called a filament
or wide enough to be identified as a band. A filamented or seg-
SEGMENTED NEUTROPHILS (FILAMENTED
mented cell has a threadlike connection between two lobes,
NEUTROPHILS, POLYMORPHONUCLEAR
and there is no visible chromatin between the two sides of the
NEUTROPHILS)
In normal peripheral blood of older children and adults, 50% to
70% of mature granulocytes called segmented neutrophils are
found (Table 1-2). The nucleus of the segmented neutrophil
is separated into two to five (usually three) lobes, with a
narrow segment or filament connecting the lobes* (Fig. 1-4).
Approximately 6% of the neutrophils have one lobe (band neu-
trophil), 35% have two lobes, 41% have three lobes, 17% have
four lobes, and 2% have five lobes.? Segmentation of the
nucleus enables these motile cells to pass through an opening
in endothelial lining cells of capillariés and to “home in” on
selected prey (such as microorganisms causing infection).*
Nuclear chromatin is heavily clumped, coarse, or pyknotic and
stains purplish-red (see Figs. 1-2C, 1-2E, and 1-4). The cyto-
plasm is light pink when stained properly and the secondary
granules (which are fine, numerous, and evenly distributed)
stain either pink or a neutral color. Neutrophil secondary
granules are lysosomes that contain alkaline phosphatase.! Figure 1-4 ™ Two segmented neutrophils.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 5

Figure 1-5 @ Neutrophilic band. Figure 1-6 ™ Eosinophil (segmented).

filament. Lobes of nuclei often touch each other or overlap, granules vary in size from 0.2 to 1.0 um.? They are coarse and
and it may be impossible to see the connecting filaments. In a unevenly distributed; vary in number, shape, and color; and
band neutrophil, there are two distinct margins with nuclear are less numerous than eosinophil granules (see Figs. 1—-2/
chromatin material visible between the margins. If the margin and 1—7). These granules have an affinity for blue or basic thi-
of a lobe can be traced as a definite and continuing line from azine dyes.' Basophil granules are algo water soluble. In cells
one side of the nucleus across the isthmus to the other side, then that are poorly fixed during staining, the center of the granule
it may be assumed that a filament is present even though it is may disappear or the entire granule may be washed away,
not visible. In attempting to differentiate between a segmented leaving a small colorless cytoplasmic area.
and a band neutrophil, identification should not be made on a Basophils show a diurnal variation similar to that of
single morphological characteristic but on combined features. eosinophils, increasing at night and decreasing in the morning.*
In case of doubt regarding a borderline cell, the questionable
cell should be placed into the mature category.’ LYMPHOCYTES
Lymphocytes are the second most numerous cells in the blood,
EOSINOPHILS comprising from 20% to 44% of the adult blood cells (see Table
Eosinophils are usually easily recognizable because of the 1-2). Most lymphocytes are small, varying from 7 to 10 um.
large, round, secondary, refractile granules that have an affin- There are also intermediate sizes and some large lymphocytes
ity for the acid eosin stain (see Figs. |-2D and 1-6). With (Fig. 1-8). Size is not a reliable basis for determining the age
Wright’s stain, normal eosinophilic granules become orange of metabolic activity of lymphocytes because their size varies
to reddish-orange. The granules are spherical, uniform in with the thickness of the smear. Lymphocytes tend to become
size, and evenly distributed. Because of the size and round- spherical and small in thick areas of the smear; in the thinnest
ness of the granules, eosinophils may be recognized in end of the smear, lymphocytes may spread out and appear
unstained moist preparations of blood on light microscopy large. Small lymphocytes are usually round with smooth
and via phase microscopy. The crystalloid core of the gran-
ule is composed mainly of major basic protein (MBP), which
binds to acid aniline dyes and which may help to explain the
staining qualities of the granule.'
Normal adult peripheral blood contains 0 to 4%
eosinophils (see Table 1-2). Normal blood eosinophils are
about the size of or slightly larger than neutrophils and have
a band or two-lobed nucleus with condensed chromatin;
rarely does an eosinophil have three lobes. There is a diurnal
variation in the percentage of circulating eosinophils, which
increases at night and decreases in the morning.

BASOPHILS
Although basophils constitute only 0 to 2% of normal blood
cells (see Table 1—2), the large, abundant, violet-blue (or
purple-black) granules aid in the immediate recognition of
this cell.! These granules are visible above the nucleus as well
Figure 1-7 @ Basophil.
as lateral to it, and they obscure most of the nucleus. The
6 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Most lymphocytes do not have granules. In some large

cells there may be a few well defined granules that vary in
size, are unevenly distributed, and can be easily counted.
These granules are a purplish-red and have been called
azurophilic, however, the term is misleading because the gran-
ules are predominantly red rather than blue* (see Figs. 1—8/,
K and 1-98).
The diameter of the nucleus of a small lymphocyte with
scant cytoplasm in peripheral blood is slightly larger than, or
the same size as, a normal erythrocyte in the same micro-
scopic field. The lymphocyte’s nucleus, in relation to its cyto-
plasm, is large (N:C ratio is 4:1 to 2:1); and the nuclei are
round or slightly indented. Chromatin structure is lumpy or
clumped and stains dark purple with lighter bluish-purple
areas between chromatin aggregates.’
Nucleoli are present in some lymphocytes but are not
visible on light microscopy because they are obscured by the
darkly stained chromatin masses. The fact that nucleoli may
be present in small lymphocytes is evidence that these cells
are capable of growth and replication. !

MONOCYTES
In the thin areas of the peripheral blood smear, a monocyte mea-
sures about 12 to 18 um* and is larger than the mature neu-
trophil.* Monocytes have abundant cytoplasm in relation to the
nucleus (N:C ratio is 1:1 or 2:1). With Wright’s stain the cyto-
plasm turns a dull gray-blue, in contrast to the pink cytoplasm
of the neutrophils. Numerous fine, small, reddish- or purplish-
stained, evenly distributed granules in the cytoplasm give the
cell a ground-glass, cloudy appearance (see Figs. 1-2F and
1-10). There may be varying numbers of prominent granules in
addition to the small granules. Some monocytes may appear
J
nongranular, suggesting rapid turnover. Digestive vacuoles may
Figure 1-8 ™ Lymphocytes. A. Small mature lymphocyte. be observed in the cytoplasm. In disease states, phagocytized
B. Lymphocyte of intermediate size. C. Lymphocyte with indented erythrocytes, nuclei, cell fragments, bacteria, fungi, and pigment
nucleus. D. Lymphocyte of intermediate size. E. Lymphocyte with may be present.*
pointed cytoplasmic projections (frayed cytoplasm); typical nucleus. The nuclei of monocytes frequently may be kidney
F. Spindle-shaped and pointed cytoplasmic projections. G. Large
lymphocyte with indented nucleus and pointed cytoplasmic projec-
shaped, deeply folded or indented, or occasionally lobular. One
tions. H. Large lymphocyte. /. Large lymphocyte with purplish- of the distinctive features of the monocyte is the appearance of
red (azurophilic) granules. J. Large lymphocyte with irregular
cytoplasmic contours. K, Large lymphocyte with purplish-red
(azurophilic) granules and with indentations caused by pressure of
erythrocytes. L. Large lymphocyte with purplish-red (azurophilic)
granules. (From Diggs, LW, et al: The Morphology of Human Blood
Cells, ed 5. Abbott Laboratories, Abbott Park, IL, 1985, pp 1-18,
25-27, with permission.)

margins (see Figs. |-2H and 1-8A). Rarely, a lymphocyte may

have a spindle form with an oval nucleus and cytoplasmic fila-
ments extending outward at each end (see Fig. 1-8F). The
margin of large lymphocytes frequently is indented by neigh-
boring erythrocytes, causing them to have a serrated (holly-
leaf) shape? (see Figs. 1-8/ through L).
With Wright’s stain, the color of the cytoplasm is blue,
varying in intensity from light to dark in different cells and
appears clear, not cloudy. The color is evenly distributed in
some cells and uneven in other cells. The intensity of the blue
stain is greater at the periphery of the cell than near the Golgi Figure 1-9 @ A. Segmented neutrophil. B. Lymphocyte with
area adjacent to the nucleus. azurophilic granules.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 7

Figure 1-10 ® Monocytes. A. Monocyte with “ground-glass” appearance, evenly distributed fine granules, occasional azurophilic granules,
and vacuoles in cytoplasm. B. Monocyte with opaque cytoplasm and granules and with lobulation of nucleus and linear chromatin.
C. Monocyte with prominent granules and deeply indented nucleus. D. Monocyte without nuclear indentations. E. Monocyte with gray-blue
color, band type of nucleus linear chromatin, blunt pseudopods, and granules. F Monocyte with gray-blue color, irregular shape, and multilob-
ulated nucleus. G. Monocyte with segmented nucleus. H. Monocyte with multiple blunt nongranular pseudopods, nuclear indentations, and
folds. |. Monocyte with vacuoles and with nongranular ectoplasm and granular endoplasm. (From Diggs, LW, et al: The Morphology of Human
Blood Cells, ed 5. Abbott Laboratories, Abbott Park, IL, 1985, pp 1-18, 25-27, with permission.)
oa

convolutions (like those in the brain) in the nucleus (see Figs. It is helpful to memorize four helpful characteristic fea-
1-10 and I-11). Another characteristic is the lacy, often deli- tures of the monocytes: nuclear convolutions; lacy, often deli-
cate chromatin network of intermingled fine strands with small cate chromatin; dull gray-blue cytoplasm; and blunt pseudopods
chromatin clumps. (see Fig. |-10H). Kinetic studies have revealed that the half-life
The shape of the monocyte is variable. Many cells are of monocytes in the circulation ranges from 8 hours to 3 days
round; other cells reveal blunt pseudopods that are manifesta- before these cells enter tissues and are transformed into
tions of their slow mobility. These ameboid cells continue to macrophages.*
move while the blood film is drying and become fixed before Monocytes account for 2% to 9% of normal blood leuko-
the cytoplasmic extensions are retracted. Pseudopods vary in cytes (see Table 1-2).
size and number; the outer portion of the outstretched cyto-
plasm may have a hyaline appearance without granules, in LARGE LYMPHOCYTES VERSUS MONOCYTES
contrast to the inner granular cytoplasm. A monocyte (see Fig. 1-10D) is often mistaken for a large
lymphocyte (see Figs. 1-8G through L) because the mono-
cytic cytoplasm may be blue, the granules may be indis-
tinct, the nucleus is round, and the blunt pseudopods and
digestive vacuoles are missing. To distinguish monocytes
from large lymphocytes, it is useful to observe the nuclear
chromatin structure, character of the cytoplasm, and shape
of the cells. The nucleus of a lymphocyte tends to be
clumped, rather than linear or lacy as it is in a monocyte
(Fig. 1-12). There is a greater tendency for the nuclear
chromatin to be condensed at the periphery of the nucleus
in the lymphocyte. The brain-like convolutions present in a
monocyte (Fig. 1-13) are not observed in a lymphocyte
(Fig. 1-14).
Large lymphocytes and monocytes may have distinct
bluish-red granules. In a monocyte the large bluish-red granules
are interspersed with numerous fine granules in the cytoplasm
and cannot be enumerated (see Figs. 1-10B and C). In a lym-
phocyte these large granules are prominent (sometimes at the
Figure 1-11 ®& Monocytes. periphery of the cytoplasm) and can be counted easily because
8 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

there are no other granules (see Figs. 1-8/ and K). Because of
the finely granular cytoplasm the monocyte has a ground-glass
appearance; the cytoplasm of the lymphocyte has a relatively
clear, nongranular background. Large lymphocytes are often
deeply indented by neighboring RBCs (see Fig. 1-8K). Mono-
cytes tend to project blunt pseudopods between cells or to
compress cells, rather than being indented by them. The mor-
phological characteristics of large lymphocytes versus mono-
cytes are summarized and compared in Table 1-3.

Hematopoiesis
Definition
Hematopoiesis is the name given to the dynamic processes of
Figure 1-12 @ A. Lymphocytes with azure granules. B. Monocyte.
blood cell production and development of the various cells of
the blood. Strong evidence exists that blood cells are the prog-
eny of a hematopoietic stem cell.* Hematopoiesis is character-
ized by a constant turnover of cells. The normal hematopoietic
system continuously maintains a cell population of erythro-
cytes, leukocytes, and platelets through a complex network
of tissues, organs, stem cells, and regulatory factors.’ This
network is responsible for the maturation and division of
hematopoietic stem cells into the lineage-committed stages that
transport oxygen and excrete carbon dioxide (RBCs), fight
infection (granulocytes), perform immune functions (lympho-
cytes), and maintain hemostasis, a process in which blood clots
and bleeding is halted (platelets) (Table 1-4).
Hematopoietic stem cells duplicate themselves during
division, as shown by the curved arrow in Figure 1—15.° The
hematopoietic stem cell has the capacity for continuous self-
replication and proliferation, together with the ability to differ-
entiate into committed progenitor cells of lymphoid and
myeloid lineages.° Under the influence of growth factors
(cytokines) such as colony-stimulating factors and interleukins,
progenitor cells divide and differentiate to form the mature cel-
Figure 1-135 & Two monocytes. lular elements of the peripheral blood (see Fig. 1—15).’
The hematopoietic system consists of the bone marrow,
liver, spleen, lymph nodes, and thymus. These tissues and
organs are involved in the production, maturation, and destruc-
tion of blood cells. The entire process of hematopoiesis evolves
from the stem cells that support hematopoiesis, the progenitor
cells that are committed to particular cell lines, and the regula-
tory factors (growth factors) to which the hematopoietic system
responds. These features enable the hematopoietic system to
respond to stimuli such as infection, bleeding, or hypoxia by
increasing hematopoiesis with emphasis on the cell type needed.

Ontogeny (Origin) of Hematopoiesis

During the first few weeks of embryonic life, hematopoiesis
begins in the mesoderm of the yolk sac (Fig. !—16) with mes-
enchymal stem cells forming large primitive nucleated erythroid
cells.* Yolk sac production of these nucleated erythroid cells
begins to decline in about 6 weeks and ends in about 2 months.
The fetal liver assumes responsibility for hematopoiesis
during the second month, with the yolk sac nucleated RBCs
Figure 1-14 @A. Lymphocyte. B. Monocyte.
migrating to the liver and remaining in the liver until the
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 9

hological Comparison of Large Lymphocytes and Monocytes

Large Lymphocyte Monocyte

Size, um 12-15 12-18

Nucleus Clumped, condensed at periphery Lacy, brain-like convolutions
Cytoplasmic granules Bluish-red, prominent granules, Bluish-red, interdispersed
easily enumerated if present with other granules, not easily enumerated
Cytoplasm Clear, nongranular background “Ground-glass” appearance (cloudy)
Cell interactions Indented by erythrocytes Projection of blunt pseudopodia

seventh month.’ From the third to the sixth month, splenic

hematopoiesis also occurs. At approximately 7 months of | Table 1-4 Hematopoietic Cell
fetal life, the responsibility for hematopoiesis shifts from the Function
liver to the bone marrow, which then becomes the major site
of blood cell development in the fetus.? The fetal marrow :unction

becomes filled with RBCs during hematopoiesis. Bones of the Granulocytes Fight infection
toes, fingers, vertebrae, ribs, pelvis, long bones, and cranium Lymphocytes Cellular and humoral immunity
are filled with erythroid cells; early lymphocytic cells also Erythrocytes Transport oxygen and excrete
may be formed during fetal life. A few megakaryocytes (pre- (red cells) carbon dioxide
Platelets Maintain hemostasis
cursors to platelets) first appear at approximately 3 months of
fetal life, and granulocytes are observed at about 5 months.”
At birth, the liver and spleen have ceased hematopoietic
cell development, and the active sites of hematopoiesis are in
e

Hematopoietic Lineage _
Commitment |
Maturation Stages
of Committed
Stem Cells
Stages - Progenitor Cells

7@---- eo
B-cell progenitors |
B-Aympnocyte

e
: —
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asrecne

Ke)
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ee > Oo om > | >

|
Megakaryocyte progenitors | Platelets

Figure 1-15 @ Diagram of hematopoietic cell differentiation. Hematopoietic stem cells can duplicate themselves during cell division (self-
replicate), as indicated by the curved arrow. Most descendants of the stem cells are committed to differentiate. This commitment process occurs
through a series of steps or stages, each of which leads to further restriction of lineage choice, until finally the descendant cells are limited to a
single lineage. After lineage commitment, the progenitor cells continue to differentiate and mature into the terminally differentiated cells found
in the blood. The diagram shows only the steps of commitment and does not depict the proliferation of cells that occurs throughout the process.
The amplification of cell numbers accompanying differentiation is very large. (From Koury, M, and Bondurant, M. News Physiol Sci 8:170-174,
1993, with permission.)
10. Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

During infancy and early childhood, hematopoiesis takes

FETUS
place in the entire medullary space, with the volume of mar-
row in the newborn infant almost equalling the hematopoietic
Yolk sac
marrow space of adults.”
Hematopoiesis gradually decreases in the shaft of the
Liver and skeleton long bones, and after the age of 4 years, fat cells begin to
spleen
appear in the long bones.’ Around age 18 to 20, hematopoietic
marrow is present exclusively in the sternum, ribs, pelvis, ver-
tebrae, and skull.t Other bones contain primary fat (yellow
Hematopoiesis marrow). After the age of 40, marrow in the sternum, ribs,
pelvis, and vertebrae is composed of equal of amounts of
hematopoietic tissue and fat.* Generally, hematopoiesis is
sustained in a steady state as production of mature cells
equals blood cell removal. When there is increased demand
10 20 30 40 50 60
for blood cells, active hematopoiesis may again occur in the
ie SShe Ney gf gs)
spleen, liver, and other tissues as a compensatory mechanism
Months Yr
known as extramedullary hematopoiesis.
Figure 1-16 ® Location of active marrow growth in the fetus and Bone marrow hematopoietic activity can be divided into
adult. During fetal development, hematopoiesis is first established in two separate pools—the stem cell pool and the bone marrow
the yolk sac mesenchyme, later moves to the liver and spleen, and
pool—with eventual release of mature cells into the periph-
finally is limited to the body skeleton. From infancy to adulthood,
there is a progressive restriction of productive marrow to the axial eral blood (Fig. 1-17). It is assumed that in the bone marrow
skeleton and proximal ends of the long bones, shown as the shaded microenvironment there is a stem cell pool where morpholog-
areas on the drawing of the skeleton. (From Hillman, RS, and Finch, ically unidentifiable multipotential stem cells (MSCs) and
CA: Red Cell Manual, ed 7. FA Davis, Philadelphia, 1996, p 2, with unipotential committed stem cells reside. In addition, there is
permission.)
a bone marrow pool, which can be divided into two distinct
pools: cells that are proliferating and maturing, and cells that
bone cavities (red marrow). Bone seems to provide a microen- are stored for later release into the peripheral blood.
vironment most appropriate for proliferation and maturation of There are also two separate granulocytic pools in the
blood cells.* Hematopoiesis occurs in the extravascular part of peripheral blood: those that are functional within the circula-
the red marrow, with a single layer of epithelial cells separating tion and those that exist in a storage form. In the granulo-
the extravascular marrow compartment from the intravascular cytic cell line in the bone marrow pool, there is a component
compartment (venous sinuses). When new blood cells produced for proliferation and maturation, as well as a storage compo-
in the marrow are almost mature and ready to circulate in the nent.' As seen in Figure 1-17, the granulocytic cells in the
peripheral blood, the migrating cells leave the marrow peripheral blood also contain 50% of circulating cells and
parenchyma by squeezing through cytoplasmic fenestrations in 50% of storage cells.'° The neutrophils that line the walls of
sinus endothelial lining cells and emerging into venous sinuses the blood vessels are sometimes referred to as the marginat-
(see Chap. 2). ing storage pool.*

HEMATOPOIESIS

Stem cell pool I Bone marrow pool Peripheral blood

I
Multi- Unipotential I Prolif. and Storage Storage Functional
potential committed | matur.
I
I
I
Granulocyte

50% 50%

Stem cell Bone marrow 100%

differentiation release
Figure 1-17 ® Hematopoiesis. (From Ersley, AJ, and Gabuzda, TG: Pathophysiology of Blood, ed 3, WB Saunders, Philadelphia, 1995, with
permission.)
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis —| |

For platelets (also known as thrombocytes), the periph- NORMAL ERYTHROCYTIC SEQUENCE
eral blood contains 70% of platelets that circulate, with 30%
being stored in the spleen.' Figure 1-17 demonstrates that the
bone marrow pool consists of only proliferating and maturat-
ing platelet precursor cells.
One hundred percent of RBCs, known as erythrocytes,
circulate in the peripheral blood in a functional state and in
the bone marrow pool. Erythrocytes in various stages of
development are a large component of proliferating and
maturing red cell precursors found in the bone marrow. At
birth the normal cellularity is 100%.° Afterwards, the cellularity
gradually decreases with age. Marrow cellularity in adults is
approximately 50% (+ 10%). The general rule to estimate age-
related normal ranges is 100 minus the age + 10.'' For exam-
ple, the estimated normal marrow cellularity for a 50-year-old Basophilic normoblast
person would be 100 — 50 + 10; representing a range from
40% to 60%. In the bone marrow, an average myeloid to ery-
throid ratio is 4:1 in terms of cellularity.'* Therefore in an adult
with 50% marrow cellularity, approximately 40% represents
granulopoiesis and 10% represents erythropoiesis.

Erythropoiesis Polychromatophilic normoblast

The term erythropoiesis identifies the entire process by which

erythrocytes are produced in the bone marrow. In response to
erythropoietin, a growth factor that stimulates the erythroid
precursors, erythropoiesis occurs in the central sinus beds of
medullary marrow over a period of about 5 days through at
least three successive reduction-divisions from pronormoblast Orthochromatic normoblast
to basophilic normoblast to polychromatophilic normoblast,
and finally to orthochromatic normoblast. With successive
developmental stages the following changes occur: reduction in
cell volume, condensation of chromatin, decrease in N:C ratio,
loss of nucleoli, decrease in ribonucleic acid (RNA) in the
cytoplasm, decrease in mitochondria, and gradual increase in Polychromatophilic erythrocyte
(reticulocyte)
synthesis of hemoglobin (Fig. 1-18). The student is urged to
memorize the following developmental stages from the “mother ett

cell” to mature erythrocyte: pronormoblast (rubriblast) to

basophilic normoblast (prorubricyte) to polychromatophilic Mature erythrocyte
normoblast (rubricyte) to orthochromatic normoblast
Figure 1-18 Erythrocytic system. Erythropoiesis. (From Diggs, LW,
(metarubricyte). The nucleus of the orthochromatic normoblast
et al: The Morphology of Human Blood Cells, ed 5. Abbott Laborato-
is eventually extruded, leaving a non-nucleated polychro- ries, Abbott Park, IL, 1985, pp 1-18, 25-27, with permission.)
matophilic (diffusely basophilic) erythrocyte (reticulocyte),
which is released into the circulating blood to mature in | to
2 days. Progressive cellular divisions of one pronormoblast
has more cytoplasm, which stains a deeper blue. Pronor-
results in production of 14 to 16 erythrocytes."
moblasts constitute 1.5% or less of the cells observed in nor-
mal bone marrow (Table 1—5). Pronormoblasts usually divide
Pronormoblast (Rubriblast, Proerythroblast) within 12 hours to make daughter cells (basophilic nor-
moblasts).* The morphological characteristics of the erythro-
The pronormoblast, the earliest recognizable cell of the erythro-
cytic series are summarized in Table 1-6.
cytic series, has a round, primitive nucleus with visible nucleoli
and chromatin strands that are distinct and dispersed. There is no
evidence of clumped chromatin. The nucleus stains reddish-blue Basophilic Normoblast (Prorubricy
with Wright’s stain. The cytoplasm stains a deep blue owing to ili Erythroblast)
Basophilic
the presence of RNA.’ The nuclear-to-cytoplasmic (N:C) ratio
in a pronormoblast is 8:1 to 6:1 (Figs. I-19A, 1-20A, 1-21, Basophilic normoblasts, the daughter cells of pronormoblasts,
1—22, and 1—23A).* require about 20 hours to develop. In normal bone marrow
Pronormoblasts range in size between 14 and 24 um.'* A there are about four times as many basophilic normoblasts as
pronormoblast is usually slightly larger than a myeloblast and pronormoblasts.* The basophilic normoblast is differentiated
12. Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

é =

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Figure 1-19 ® A. Two pronormoblasts (note the perinuclear halo). Figure 1-21 ™ Center: pronormoblasts; upper center: plasmacyte.
B. Two polychromatophilic normoblasts. C. Neutrophilic band.
D. Segmented neutrophil. £. Smudge cell.

from the pronormoblast by the coarsening of the chromatin pat- and a smaller nucleus than basophilic normoblasts.'* The
tern and the nucleoli, which are ill defined or not visible under cytoplasm contains a varying mixture of pink due to hemo-
light microscopy. As the basophilic normoblast matures, it globin and blue due to RNA; in the late polychromatophilic
accumulates more RNA and hemoglobin (Figs. 1-24, 1-25A, normoblast, the pinkish color is usually predominant.
1-26, and 1-27). The predominant color of the cytoplasm is Nuclear chromatin is thickened and irregularly con-
blue due to the staining of RNA, but there may be a pinkish densed in the polychromatophilic normoblast. Light-staining
tinge reflecting the presence of varying amounts of hemoglo- parachromatin areas are visible among the dark blue-staining,
bin. The N:C ratio in the basophilic normoblast is 6:1 to 4:1.'* irregular pyknotic masses. Nucleoli are no longer visible.
A basophilic normoblast is somewhat smaller than a pronor- The N:C ratio in a polychromatophilic normoblast is 4:1 to
moblast with a size of 12 to 17 um. Normal bone marrow 2:1'* (see Figs. 1-18, 1-19B, 1-20C, 1-25B, 1-28, and
contains 1% to 5% basophilic normoblasts (see Table 1-5). The 1-29A).
division of the basophilic normoblasts forms polychro- The maturation time for polychromatophilic normoblasts
matophilic normoblasts, which are smaller than basophilic in bone marrow is about 30 hours, and there are approximately
normoblasts but have twice the amount of hemoglobin. three times as many polychromatophilic normoblasts as
basophilic normoblasts in the bone marrow. Bone marrow in a
Polychromatophilic Normoblast (Rubricyte, normal adult contains 5% to 30% polychromatophilic nor-
Polychromatophilic Erythroblast) moblasts (see Table 1-5). Polychromatophilic normoblasts are
not present in the normal peripheral blood of adults, but they
Polychromatophilic normoblasts are smaller than basophilic may appear in small numbers in the peripheral blood of nor-
normoblasts (10 to 15 um), having relatively more cytoplasm mal newborn infants.’

Figure 1-20 @A. Pronormoblast. B. Orthochromatic normoblast.

Figure 1-22 ™ Center: pronormoblasts; lower center. lymphocyte.
C. Two polychromatophilic normoblasts.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 13

Table 1-5 Bone Marrow Cells:

Normal Adult Values

Cell Percent

Stem cell 0-0.01

Myeloblast 0-2
Promyelocyte 14
N. myelocyte 5-20
N. metamyelocyte 5-20
N. band 10-35
N. segmented 5-15
Eosinophil 0-3
Basophil 0-1
Lymphocyte 5-15
i
Plasmacyte 0-1
Monocyte 0-2
Figure 1-25 @ A. Pronormoblasts. B. Neutrophilic myelocyte.
Other cells 0-1
C. Neutrophilic metamyelocyte. D. Segmented neutrophil.
Megakaryocyte 0.1-0.5
Pronormoblast 0-1.5
Basophilic normoblast 1-5
Orthochromatic Normoblast Polychromatophilic normoblast 5-30
(Metarubricyte, Orthochromatic Orthochromatic normoblast 5-10
Erythroblast) Myeloid to Erythroid Ratio (M:E) = 4:1

Source: Percentage of total nucleated cells in bone marrow represent

Orthochromatic normoblasts are formed from polychro- normal reference ranges for adults taken from the University of
matophilic normoblasts and are recognized by the solid, blue- Texas Health Science Center and University Hospital, San Antonio,
Texas.
black, degenerated nucleus with a nonlinear clumped chromatin
pattern (see Figs. 1-18, 1-20B, 1-25C, and 1-27). The nucleus
is called pyknotic because there is no parachromatin (white
areas) present. The orthochromatic normoblast nucleus is inca- of normal newborn infants. The orthochromatic normoblast
pable of further DNA synthesis, and therefore, cannot divide. is the smallest of the nucleated erythrocyte precursors (8 to
The degenerated nucleus of the orthochromatic normoblast is 12 um). Morphological characteristics of orthochromatic nor-
destined to be extruded and will be phagocytized. The N:C ratio moblasts are given in Table 1-6.
in an orthochromatic normoblast is 1:1 to 1:2."
The cytoplasm is predominantly pink (or reddish) because
of increasing hemoglobin synthesis, but there may remain min- Reticulocyte (Diffusely Basophilic
imal amounts of blue cytoplasm due to the presence of RNA. Erythrocyte, Polychromatophilic
The maturation time for the orthochromatic normoblast is Erythrocyte)
48 hours. The number of orthochromatic normoblasts in nor-
mal bone marrow varies between 5% and 10% (see Table 1-5). The condensed, pyknotic nucleus of an orthochromatic nor-
Orthochromatic normoblasts are not observed in the normal moblast is extruded, leaving a diffusely basophilic or poly-
peripheral blood of adults, but they can be found in the blood chromatophilic cell. The membrane of the erythrocyte seals

Figure 1-25 © A. Basophilic normoblast. B. Three polychromatophilic

Figure 1-24 ™ Basophilic normoblasts. normoblasts. C. Orthochromatic normoblast.
14. Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Figure 1-26 m Left: Basophilic normoblast; center: plasmacyte. Figure 1-28 m Polychromatophilic normoblasts: early and late
stages.

itself. Some of the bluish-staining color remains because Erythrocyte (Red Blood Cell, Discocyte)
of the presence of RNA. The erythrocyte contains approxi-
mately two-thirds of its total hemoglobin content by the The morphological characteristics of a normal erythrocyte are
time the nucleus is lost. The RNA content soon begins to presented at the beginning of this chapter.
decrease. A mature erythrocyte is not able to synthesize hemoglobin,
A diffusely basophilic erythrocyte is larger than a mature because it is without a nucleus, mitochondria, or ribosomes, but
red cell (8 to 10 um).'* It is released in 2 to 3 days from the it has a unique, yet limited, metabolism to sustain itself while
marrow and circulates for 1 or 2 days before maturing into an traversing the microvasculature. The erythrocyte carries oxygen
erythrocyte. Only rarely are diffusely basophilic erythrocytes from the lungs to the tissues where it is exchanged for carbon
found in the blood of normal adults; however, polychro- dioxide. Erythrocytes are pliable or flexible and deformable,
matophilic cells are frequently seen in the blood of normal making them capable of unusual changes in shape that are nec-
newborn infants.’ essary for the passage through the microcirculation to transport
When stained with new methylene blue, these polychro- oxygen. Refer to Table 1-6 for a summary of the morphological
matophilic erythrocytes reveal ribosomes in a granulofila- characteristics of each stage of maturation of the red cell.
mentous arrangement (or network of strands and granules)
and are classified as reticulocytes (Fig. 1-30). As ribosomes
Myelopoiesis (Granulocytopoiesis)
disappear, the diffusely basophilic cell changes into a mature
erythrocyte." Myelopoiesis or granulocytopoiesis refers to the production
With anemia or hypoxia, erythropoietin stimulates mar- of neutrophils, eosinophils, and basophils (Fig. 1-31).
row erythroid precursors to proliferate and to increase the Mature neutrophils, eosinophils, and basophils have similar
number of early erythroid cells. An increased number of poly-
chromatophilic cells are delivered early from the marrow and,
therefore, the reticulocyte count is increased.

Fi er
= a .s

Figure 1-27 ™ Center: Basophilic normoblast; right: Orthochro- Figure 1-29 mA. Polychromatophilic normoblasts. B. Lymphocyte.
matic normoblast. C. Segmented neutrophil.
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Morphology of Human Blood and Marrow Cells: Hematopoiesis
15
16 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

patterns of proliferation, differentiation, division, storage in

marrow, and delivery to the blood. Maturation and division
of the myeloid series in the marrow demonstrate a contin-
uum of development from the blast to the most mature cell
(segmented neutrophil), requiring from 7 to 11 days.*
Granulocyte production proceeds after the cell lineage
commitment has determined the identity of the maturing cell
as a member of the myelocytic series. The system moves cells
throughout passages and compartments where cellular stages
occur in response to various stimuli. The proliferative or
mitotic pool contains the committed stem cells, myeloblasts,
promyelocytes, and myelocytes. These cells actively divide
and mature, taking | to 2 days for each cellular cycle.* The
maturation pool is composed of metamyelocytes and bands,
and represents the end of DNA synthesis and division. The
Figure 1-30 @ Reticulocytes. New methylene blue stain of periph- transformation of myelocyte to metamyelocyte to band takes
eral blood. Note reticulocytes with varying amounts of stained about 8 to 9 hours after entry into the maturation pool.* The
reticulum (RNA). Reticulocytosis is associated with increased erythro-
storage pool retains mature cells for release into peripheral
poietic activity reflected by polychromasia on the Wright's stain of
the peripheral blood. circulation. These mature cells leave the marrow by moving
through transiently formed pores in endothelial cells that sep-
arate marrow parenchyma from venous sinuses. When leay-
ing blood for tissue, these cells migrate between endothelial
cells (diapedesis). After release, these cells become part of the
functional pool and reside as circulating cells or as mar-
ginated cells, which line blood vessel walls. Cells are released
to enter the peripheral blood or vessel walls for a few hours,
and then leave the blood to enter the tissues and body cavities.
Under normal circumstances, the rate at which these cells en-
ter the blood and the rate at which they egress to tissue, are in
equilibrium.* As these cells exit the blood or the tissues, they
are replaced by other cells from the marrow. Once in the
blood, half of the released cells freely circulate while the
other half are in a marginating pool on the walls of blood ves-
sels, particularly those in lungs, liver, and spleen.* These
latter cells leave the peripheral vessel to be directed by
chemotactic factors to inflammatory or infectious tissue.
After these cells enter tissues, they do not re-enter the circu-
lation or the marrow.*

Morphological Changes
Many morphological changes occur during maturation of
granulocytes. These include a reduction in nuclear volume,
condensation of chromatin, change in nuclear shape,
appearance and disappearance of primary granules, appear-
ance of secondary granules, color changes in cytoplasm
from blue to pinkish-red, and change in the size of cells?
(Table 1-7).
Maturation of the granulocytic series of cells is char-
acterized by the development of primary blue-staining
Figure 1-351 ® Granulocytopoiesis: myelocytic (granulocytic) system. granules, which are replaced by secondary granules that
A. Myeloblast. B. Promyelocyte (progranulocyte). C. Basophilic myelo- differ in their affinity for various dyes. Cells with an affin-
cyte. D. Basophilic metamyelocyte. E. Basophilic band. F Segmented ity for basic dyes are basophils; the cells that stain reddish-
basophil. G. Neutrophilic myelocyte. H. Neutrophilic metamyelocyte.
orange with the acid dye eosin are eosinophils; the cells
!. Neutrophilic band. J. Segmented neutrophil. K. Eosinophilic myelocyte.
L. Eosinophilic metamyelocyte. M. Eosinophilic band. N. Segmented that do not stain intensely with either acid or basic dyes
eosinophil. (From Diggs, LW, et al: The Morphology of Human Blood are called neutrophils. As these motile cells mature, the
Cells, ed 5. Abbott Laboratories, Abbott Park, IL, 1985, pp 1-18, nucleus undergoes progressive changes from round to
25-27, with permission.)
multilobular forms.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 17

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18 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Stages of Differentiation and Maturation

NEUTROPHILS (GRANULOCYTES)
MYELOBLASTS The earliest recognizable cell in the granu-
locytic series is a myeloblast. A myeloblast usually has a
round nucleus that stains predominantly reddish-blue and has
a smooth nuclear membrane. The interlaced chromatin
strands are delicate, finely dispersed or stippled, and evenly
stained, but are not clumped. One or more nucleoli of uniform
size are usually demonstrable, but occasionally nucleoli may
be barely visible.'* A slight to moderate amount of bluish non-
granular cytoplasm stains lighter next to the nucleus than at
the periphery of the cell (see Figs. 1-31A and 1-32). The N:C
ratio in the myeloblast is 7:1 to 5:1.'* A myeloblast is smaller
and has less blue cytoplasm than a pronormoblast. After about
three to five mitotic divisions, the myeloblast matures into a Figure 1-55 = Promyelocyte.
promyelocyte as primary granules become visible. Ultrastruc-
tural studies of myeloblasts reveal numerous mitochondria, a
Golgi area, and free ribosomes.* relatively light zone adjacent to the nucleus. The periphery of the
Myeloblasts vary in size from 15 to 20 um.'* They are cytoplasm is smooth and is not indented by neighboring cells.*
not present in normal peripheral blood. Normal marrow con- The size of a promyelocyte may vary from 12 to 24 um, depend-
tains 2% or less myeloblasts (see Table 1-5). The appearance ing on the stage of a given cell in the mitotic cycle. It is often
of primary granules marks the maturation of the myeloblast 20 um and may be larger than a myeloblast.* Promyelocytes are
into a promyelocyte.’ not present in normal peripheral blood. From 1% to 4% promye-
locytes are observed in normal bone marrow (see Table 1-5).
PROMYELOCYTE (PROGRANULOCYTE) The promyelo- As the promyelocyte matures, nucleoli begin to fade, the chro-
cyte contains granules that stain dark blue or reddish-blue and matin becomes more condensed, and the granules are not as in-
may be round or irregular in shape. They appear scattered tensely stained. Specific secondary neutrophilic granules begin
throughout the cytoplasm and may overlay the nucleus. Primary to appear, and the synthesis of primary granules ceases.* A few
granules are filled with lysosomal granules, which contain primary granules remain through division and maturation and
myeloperoxidase, acid phosphatase, hydrolytic enzymes, elas- may even appear in segmented neutrophils.”
tase, beta-glucuronidase, and other basic proteins (but not alka-
line phosphatase).* NEUTROPHILIC MYELOCYTES When primary granules are
The nucleus of a promyelocyte is usually round and is large no longer synthesized and smaller, less dense secondary neu-
in relation to the cytoplasm. The chromatin of young promyelo- trophilic granules can be identified, the cell has matured into a
cytes is almost as finely granular as it is in a myeloblast. In older myelocyte. The first sign of neutrophilic differentiation has been
cells the chromatin structure is slightly coarser than that in a called the “dawn of neutrophilia” or “beginning neutrophilia,”
myeloblast. Nucleoli may be faintly visible but are not often dis- which refers to a relatively light island of ill-defined or barely
tinct (see Figs. 1-31B, 1-33, and 1—34).* The N:C ratio in a visible (pinkish) secondary lysosomal granules that develop
promyelocyte is 5:1 to 3:1.'* The cytoplasm is blue, with a adjacent to the nucleus and in proximity to the remaining

mn

aes
= .

Figure 1-352 @ Center: myeloblast: right: segmented neutrophil;

left: disintegrated neutrophil. Figure 1-54 ™ Center. promyelocyte.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 19

Figure 1-55 mA. Neutrophilic myelocyte. B. Neutrophilic Figure 1-37 ™ A. Two neutrophilic metamyelocytes. B. Three
metamyelocyte. C. Plasmacyte. D. Orthochromatic normoblasts. neutrophilic bands. C. Two segmented neutrophils.
E. Segmented neutrophils. F Nucleus of a degenerated cell.

primary granules. As myelocytes divide and age, the primary neutrophilic cell. Metamyelocytes do not divide, nor do they
granules become fewer and the secondary (specific) neu- have nucleoli.* The N:C ratio is 1:1.'*
trophilic granules predominate.* Secondary granules are consid- Many small, pinkish secondary granules fill the cytoplasm,
ered to be specific granules for neutrophils, and they contain and there may be a few primary darker granules remaining (see
collagenase, lysozyme, lactoferrin, plasminogen activators, and Figs. 1-31H, 1-35B, and 1—37A). These maturing cells remain
aminopeptidase.* in the bone marrow and represent a portion of the granulocytic
The nuclei of myelocytes may be round, oval, or flat- reserve.”
tened on one side and usually are eccentrally located? (see Metamyelocytes are somewhat smaller than myelocytes
Figs. 1-31G and 1-35A). Chromatin strands become con- (10 to 18 um) and are larger than the band neutrophil or seg-
densed, partly clumped, and thickened, and are unevenly mented cell.'* These cells are usually absent in normal periph-
stained. Nucleoli are absent or indistinct in myelocytes. The eral blood. There are approximately 5% to 20% metamyelocytes
neutrophilic myelocyte is the last myeloid precursor capable in normal bone marrow.
of division.*
Neutrophilic myelocytes are often smaller than promye- BAND NEUTROPHILS When the stage is reached in which
locytes (10 to 18 um) and have relatively large amounts of the nuclear indentation in the early granulocyte is greater than
cytoplasm (N:C ratio is 2:1 to 1:1),’* which gradually half the width of the nucleus (see Fig. 1—36), the cell is iden-
becomes less basophilic and more pinkish. The normal tified as a band neutrophil.’
peripheral blood does not contain neutrophilic myelocytes. The opposite edges of the nucleus become almost paral-
There are 5% to 20% myelocytes in normal bone marrow. lel, giving the appearance of a horseshoe, hot dog, or a curved
link of sausage. The shape of the nucleus of a band neutrophil
NEUTROPHILIC METAMYELOCYTES As maturation pro- is often folded or twisted, giving rise to difficulty in distin-
ceeds, the nucleus becomes slightly indented (bean- or kidney- guishing a band from a segmented neutrophil. The nuclear
shaped), and this shape serves to identify the cell as a chromatin is pyknotic, and there is usually a dark condensed
metamyelocyte. The indentation is less than half the width of mass at each end where the lobe is destined to be.* The small
an arbitrary round nucleus (Fig. 1—36).* There is noticeable secondary neutrophilic granules are evenly distributed and
condensation with clumping of the chromatin, but the chro- stain various shades of pink (see Figs. 1-31/ and 1—37B). An
matin structure is not as dense as that of the segmented occasional dark primary granule may be observed. The N:C
ratio of a neutrophilic band is 1:1 to 1:2."
Neutrophilic band cells are often slightly smaller than
TERMINOLOGY BASED ON INDENTATION OF NUCLEI metamyelocytes. Band forms constitute from 10% to 35% of
the nucleated cells in the bone marrow.

SEGMENTED NEUTROPHILS As stated earlier in this chap-

ter, the nucleus of the segmented neutrophil is divided into two
to five (often three) lobes that are connected by a thin filament
or strand (see Figs. 1-31/, 1-32, 1-35E, 1-36, and 1—37C).
Myelocyte Metamyelocyte Band Segmented
The N:C ratio of a segmented neutrophil is 1:3.'* Approxi-
Figure 1-36 wiTerminology based on indentation of nuclei: (/eft to mately 5% to 15% segmented neutrophils are noted in normal
right) myelocyte, metamyelocyte, band, segmented. bone marrow of older children and adults.
20. Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Because there is a gradual transition between the various

stages of granulocytes, the division of neutrophils into devel-
opmental stages is somewhat arbitrary. This division is, how-
ever, necessary for morphological evaluation. Borderline
cells that are difficult to distinguish from each other are often
present. In this dilemma, the borderline cell should be classi-
fied as the more mature cell.* Differentiation of band neu-
trophil and segmented cells is described in the first part of this
chapter on peripheral blood cells. Table 1—7 lists the morpho-
logical characteristics of the neutrophilic series.
Figure 1-59 @ Center: eosinophilic myelocyte.
TISSUE NEUTROPHILS Tissue neutrophils are large mar-
row cells with ample cytoplasm having irregular, blunt
pseudopods that are often multipointed and may have nebu- EOSINOPHILS Eosinophils pass through the same develop-
lous cytoplasmic streamers (Fig. 1-38). These cells are mental stages as neutrophils: myelocyte, metamyelocyte,
readily indented by adjacent marrow cells or may be band, and segmented stages (see Figs. |-31K through NV). The
squeezed between adjacent cells. Often there are long and earliest eosinophil (eosinophilic myelocyte) has a few dark
tenuous cytoplasmic extensions that seem to wrap around bluish primary granules intermingled with the few specific,
other cells. These cells are not phagocytic and seldom have reddish-orange granules (Fig. 1-39). During development the
cytoplasmic vacuoles. The cytoplasm stains light blue and bluish granules become less visible and disappear, and the
has a fine lattice-like structure. Granules vary in number and round, specific, or secondary bright red refractile eosinophilic
stain varying shades of red to blue, but the majority have a granules fill the cytoplasm (Figs. 140A, 141A, and 1-42A).
reddish-purple stain. Many of the granules tend to be Production of eosinophils in the marrow takes 3 to 6
arranged in chains. The beadlike granular aggregates extend days before the eosinophils appear in the peripheral blood.
into the cytoplasmic projections.* Bone marrow provides a storage area for eosinophils so that
The large, round, or oval nucleus has a coarse chromatin they can be rapidly mobilized when needed. The factors that
structure with a distinct linear pattern. Nucleoli are usually regulate production and release of eosinophils into blood are
conspicuous and stain light blue.* probably different from those that regulate neutrophils. The
Tissue neutrophils are fixed or semifixed tissue cells. mean transit time of these cells in the circulatory system of
They are immobile end-stage cells that are probably derived humans has been reported to be about 8 hours, but in some
from the same progenitor cells as neutrophils. Tissue neu- disease states with eosinophilia, the time may be longer.*
trophils occur infrequently in normal bone marrow. However, Much less is known about the stem cell kinetics of the
they are found in increased numbers in bone marrow smears eosinophil than of the neutrophil.
of patients who have conditions in which there is a prolifera- Eosinophils migrate from blood to tissue, such as
tion of neutrophilic cells. These conditions include chronic bronchial mucosa, skin, gastrointestinal tract, and vagina in
myelocytic leukemia, myelocytic/monocytic leukemia, and about 12 days.* Eosinophils may migrate from tissue back
myelofibrosis, as well as neutropenic states in which there is into blood and marrow. Eosinophils, which are motile, can
an arrest in the maturation and delivery of cells into the circu- migrate between endothelial cells into the tissue or into an
lating blood.* area of inflammation in the same manner as neutrophils.*

Figure 1-38 m Tissue neutrophil (large center cell). Figure 1-40 @ A. Eosinophilic metamyelocyte. B. Neutrophilic
band. C. Polychromatophilic normoblast.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 21

O% i el

Figure 1-45 i Arrow: tissue eosinophil; A. binucleated orthochro-

matic normoblast. B. Orthochromatic normoblast.
a f'% £ Al WN)

Figure 1-41 @ A. Eosinophilic band. B. Neutrophilic band. BASOPHILS Basophils may be identified as basophilic
C. Lymphocyte. D. NRBC (orthochromatic normoblast). £. Seg- myelocytes, metamyelocytes, bands, and segmented cells
mented neutrophil. based upon the shape of their nuclei. The shape of the
nucleus is, however, often masked by large basophilic gran-
The granules of eosinophils contain various hydrolytic ules (see Figs. 1-31C through F). The specific violet-blue
enzymes, including peroxidase, acid phosphatase, aryl sulfa- granules of basophils are formed in the myelocytic stage and
tase, beta-glucuronidase, phospholipase, cathepsin, and ribonu- continue to be produced throughout all later maturation
clease, but they lack lysosome, cationic proteins, and alkaline stages (see Figs. 1-31C through F).
phosphatase.* Maturation of basophils in the bone marrow takes place
Normal adult bone marrow contains 0 to 3% eosinophils. over 7 days.* Mature basophils rarely have more than two
The morphological characteristics of the eosinophilic series segments. Basophils circulate for a few hours in blood, then
are summarized in Table 1-8. migrate into skin, mucosa, and other serous membranes.*
Basophils in all stages of maturation are smaller than
Tissue Eosinophils promyelocytes and neutrophil myelocytes; their size approx1-
In smears of bone marrow, occasionally there may be a large mates that of neutrophils. The morphological characteristics
cell with elongated and tapering cytoplasmic extensions, con- of the basophilic series are summarized in Table 1-9. Normal
taining typical reddish-orange granules of the type seen in the bone marrow has 0 to 1% basophils.
eosinophils of the circulating blood? (Fig. 1-43). The nucleus of
such cells, instead of being indented or lobulated, is round or Tissue Basophil (Mast Cell)
oval and has a well-defined reticular chromatin and, often, Tissue basophils (mast cells) (Fig. 1-44) and blood basophils
nucleoli. Such cells are identified as tissue eosinophils and are are closely related in their functions and biochemical character-
thought to be fixed tissue variants of the more motile eosinophils istics, but the relationship between them is still being studied.
of the circulating blood.* Tissue eosinophils arise from the same Both cells participate in a similar manner in acute and delayed
progenitor cells as eosinophils in the marrow and blood.’ allergic reactions. The granules of both cells have similar
morphological characteristics, and each cell contains hista-
mine and heparin and is water soluble.*

Figure 1-42 m A. Segmented eosinophil. B. Lymphocyte.

ae

C. Neutrophilic band. D. Neutrophilic metamyelocyte.

Figure 1-44 @ Tissue basophil (arrow).
E. Plasmacyte. F. Two diffusely basophilic red cells.
 9
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 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 23

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24 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Tissue basophils are derived from the pluripotential stem and sometimes contain a few peroxidase-positive granules.
cell and are fixed tissue cells. The cytoplasm of the tissue The N:C ratio in promonocytes is 4:1 to 2:1 (see Fig. |-45£)."*
basophils is filled with large, prominent, intensely stained Promonocytes are slightly motile and may infrequently take
violet-blue granules. The granules are usually round and about part in phagocytosis.
the same size (0.1 to 0.3 um). They may overlay the margins of Promonocytes and monoblasts are not easily identifiable
the palely stained nucleus or obscure the nucleus completely. in bone marrow or peripheral blood smears except in disor-
The nucleus is small, round or oval, and not segmented.* ders in which there is marked proliferation of monocytic cells.
Tissue basophils are widely scattered in the connective The identification of early monocytic cells is based on slightly
tissue of various organs, bone marrow, and the mucosal area indented, or folded, large nuclei and on association with more
of serous membranes. In bone marrow tissue basophils are mature cells that have pseudopods and brain-like convolu-
usually observed in the hypercellular area and can be located tions in the nucleus.”
in the “squashed” smear made from a marrow particle. Some
tissue basophils have spindle shapes and jagged margins
resulting from trauma in the process of aspiration.’
Monocytes and Macrophages
Promonocytes develop into monocytes. Monocytes enter the
circulation for a short time and then migrate into tissue to
Monopoiesis
transform into tissue macrophages.’
The mononuclear phagocyte system (MPS) is composed of The characteristic features of monocytes in normal periph-
monocytes, macrophages, and their precursors—monoblasts eral blood are given in the earlier part of this chapter (see
and promonocytes.* The cells composing this system arise in Fig. 1-10). The scanning electron microscope shows the mono-
the bone marrow from progenitor cells that are committed to cyte to have a ruffled plasma membrane with long, thin
monocyté-macrophage production.* microvilli.’
As monocytes mature, they become too large to pass read-
Monoblasts and Promonocytes ily through capillaries, and so they move into tissue and convert
into macrophages in many organs (e.g., pulmonary alveolar
Monoblasts are large and have an eccentrically placed nucleus macrophages, peritoneal macrophages, splenic macrophages,
that may be minimally indented, one or two large prominent Kupffer cells in the liver, and connective tissue macrophages). '”
nucleoli, a fine, lacy nuclear chromatin, and a nongranular cyto- This transformation involves rapid growth, enlargement, and
plasm that stains a deep blue (Fig. 1458). The N:C ratio in intensified phagocytic activity. Macrophages do not normally
these cells is 7:1 to 4:1 (Table 1—10).'* Monoblasts are non- re-enter the bloodstream but may re-enter the circulation during
motile and nonphagocytic cells. Monoblasts divide and give rise inflammation.*
to promonocytes and then to monocytes. Macrophages are large, irregularly shaped tissue cells (25
Promonocytes also are large and have indented or folded to 80 um) with a round or reniform nucleus; and contain one
nuclei and fine chromatin. They often have a visible nucleolus or two nucleoli, clumped chromatin, abundant cytoplasm with

Figure 1-45 = Lymphocytic, monocytic, and plasmacytic systems. A. Lymphoblast. B. Monoblast. C. Plasmablast. D. Prolymphocyte.
E. Promonocyte. Ff. Proplasmacyte. G. Lymphocyte with clumped chromatin. H. Monocyte. /. Plasmacyte. (From Diggs, LW, et al: The
Morphology of Human Blood Cells, ed 5. Abbott Laboratories. Abbott Park, IL, 1985, pp 1-18, 25-27, with permission.)
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 2

vacuoles, and numerous azurophilic granules.* Macrophages nucleolus'® (see Fig. 1-45C). The cytoplasm is blue.
are also called histiocytes (histio = tissue; cyte = cell). The Plasmablasts are identified primarily in the presence of
morphological characteristics of the monocytic macrophage proplasmacytes and plasmacytes but cannot be easily differen-
series are summarized in Table 1-10. tiated from other blasts. The plasmablast appears slightly
larger than the more mature plasmacyte. The plasmablast is 16
to 25 um and the mature plasma cell is 10 to 20 um.'°
Lymphopoiesis Proplasmacytes and plasmacytes differ from plas-
The lymphoid progenitor cell is derived from the hematopoietic mablasts in that the color of the cytoplasm is deep blue, the
stem cell. The common lymphoid progenitor cell can differen- juxtanuclear light areas are prominent, and the nuclei are
tiate into either T or B cells, depending on the microenviron- eccentric.* The chromatin structure of the nuclei in proplas-
ment. T cells differentiate in the thymus, B cells in adult bone macytes is intermediate between that of plasmablasts and
marrow. Null cells, or third-population cells, originate in the plasmacytes. In proplasmacytes the nucleolus may be ill
bone marrow, although the maturation sequence is unknown.'” defined or absent.* The N:C ratio in proplasmacytes is 4:1 to
T, B, and null cells cannot be separately identified morpholog- 3:1_(seé Fig. 1-45 F).*°
ically but can be distinguished functionally and by immuno- Plasmablasts and proplasmacytes, although not observed
logic marker studies. (See the section on CD Nomenclature in normal bone marrow, are seen in diseases associated with
Jater in this chapter.) abnormal immunoglobulin production, especially multiple
In primary lymphoid organs stich as the thymus and bone myeloma.*
marrow, lymphocytes differentiate, proliferate, and mature into
fully functional immune cells. In secondary lymphoid organs Plasmacytes (Plasma Cells)
such as lymph nodes, spleen, and mucosal tissues (tonsils,
Peyer’s patches), lymphocytes communicate and interact with Plasmacytes represent the end stage of B-lymphocyte lineage.
antigen-presenting cells (APCs), phagocytes, and macrophages They are not observed in the peripheral blood smears of normal
in an active immune response.* individuals but constitute about 1% of the nucleated cells in
normal marrow.’ Mature plasmacytes range in size from 10 to
20 um.'® They may be round or oval, with slightly irregular
Lymphoblasts and Prolymphocytes margins.
The cytoplasm is nongranular and usually stains a deep or
The earliest lymphocytes are identified as lymphoblasts vibrant blue. The cytoplasm adjacent to the nucleus is pale, with
and prolymphocytes. Lymphoblasts contain a large, round a perinuclear clear zone containing the Golgi apparatus, and at
nucleus with a small or moderate amount of basophilic the cell periphery there are secretory vesicles. Fibrillar structures
cytoplasm. The nuclear chromatin strands in lymphoblasts that stain blue may be demonstrable in the cytoplasm. One or
are thin, loose, evenly stained, and not clumped. One or sev-
several small vacuoles may be observed. There is no evidence of
eral nucleoli are usually demonstrable.* These cells measure phagocytosis of visible particles.*
10 to 20 um in diameter and have a N:C ratio of 7:1 to 4:1" The nucleus of a plasmacyte is relatively small, round, or
(see Fig. 1-45A). oval, and eccentrically placed in the cell. The nuclear chro-
Prolymphocytes have an intermediate chromatin pattern matin is clumped or coarse and lumpy, similar to that of a
that has clumps in some areas of the nucleus but does not appear lymphocyte (see Figs. 1-45/, 1-46, and 1-47). The N:C ratio
as clumped as in mature lymphocytes.’ Parachromatin, which of a plasma cell is 1:1 to 1:2.'°
appears reddish-purple, may be present in the nucleus. Nucleoli
are less distinct than in lymphoblasts. Prolymphocytes are
slightly smaller than lymphoblasts, approximately 9 to 18 um,
and have a N:C ratio of 5:1 to 3:1'* (see Fig. 145D). Differ-
ences are subtle, and in case of doubt the cell should be called a
lymphocyte.” The morphological characteristics of the lympho-
cytic series are summarized in Table 1-11.

Lymphocytes
The morphological description of lymphocytes may be found
in the first part of this chapter.

Plasmablasts and Proplasmacytes

Cells designated as plasmablasts are similar to blast cells of
other series. The nuclei are large in relation to the cytoplasm
(N:C ratio is 5:1 to 4:1; see Table 1-12), appear round Figure 1-46 @ Center: plasmacyte; upper right: segmented
with fine, linear chromatin strands, and have a clearly visible neutrophil; lower left: resting monocyte.
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 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis PAT

Pathologic alterations in megakaryoblasts are observed in

myeloproliferative disease. The presence of micromegakary-
oblasts is typical of acute megakaryocytic leukemia (classified
as M7 in the French-American-British [FAB] classification of
acute leukemia) (see Chap. 16). They are also present in the
blast crisis of chronic granulocytic leukemia, in myelofibrosis,
and in other acute leukemias. Micromegakaryoblasts are small
and difficult to distinguish from myeloblasts, but cytoplasmic
blebs or budding (suggesting early platelet formation) helps to
identify micromegakaryoblasts.*
As the megakaryoblast matures into a promegakary-
ocyte, it increases both the amount of nuclear material and the
amount of cytoplasm itself (N:C ratio is 3:1 to 1:1).
A promegakaryocyte not only increases the size of the
nucleus but also becomes lobulated, with each lobe having a
Figure 1-47 mm Plasmacyte. 2n complement of DNA. The size of the promegakaryoblast
ranges from 20 to 80 um.'* Reddish granules appear in the
enlarging bluish cytoplasm. Electron micrographs reveal that
demarcation membranes are beginning to develop as invagi-
Plasmacytes in bone marrow are semifixed cells that
nations from the plasma membrane of the megakaryocyte.
may be torn in the process of aspiration and appear in
The demarcation membrane system establishes an outer limit
marrow smears with irregular spiculate margins. Plasma-
of each platelet, which is released as a cytoplasmic fragment?
cytes may cluster around large nongranular or finely granular
(see Fig. 1-48).
tissue cells. This contact of plasmacytes with tissue cells is
As endomitosis and DNA synthesis cease and maximum
probably a manifestation of the immune response in which
nuclear number (ploidy) is attained, the megakaryocyte has
antigenic materials, processed by macrophages, are trans-
increased in volume with an abundant amount of pinkish cyto-
ferred to the plasmacytes, which in turn will manufacture
plasm and a multilobulated nucleus (Figs. 149 through 1-53).
immune globulins.*
The size of the megakaryocyte ranges from 30 to 100 um.'* The
Immune globulins manufactured by plasmacytes pro-
majority of the cells are of the 8n, 16n, and 32n ploidy classes
duce unusual morphological variants. The proteinaceous
(16n average ploidy represents eight lobes).* The chromatin is
material appears in the form of round globules that are often
linear and coarse. Numerous small, uniformly distributed, dense
red or pink, but may be blue or almost colorless, and are
granules that stain reddish-blue are present. The demarcation
called Russell bodies.* The globules may fill the cytoplasm,
membrane system is uniform and its lumen open; the cytoplasm
giving the appearance of a bunch of grapes (berry, grape, or
is divided into partitions that define platelet limits. The N:C
morula cells).* The morphological characteristics of the plas-
ratio at the megakaryocyte stage of development ranges from
macytic series are summarized in Table 1-12.
1:1 to 1:2.'* The morphological characteristics of the megakary-
ocytic series are given in Table I-13.
Megakaryocytopoiesis After maturation is completed, the megakaryocyte
membrane ruptures, the entire megakaryocyte cytoplasm
The megakaryocyte is the largest hematopoietic cell in the fragments, and thrombopoiesis occurs. The polyploid naked
bone marrow and descends from the same multipotential stem nucleus (Fig. 1-54) is soon to be engulfed by a macrophage.”
cell as do the other blood cells. The mission of megakary- Some mature megakaryocytes are located adjacent to
ocytes is to proliferate and then fragment their cytoplasm into marrow sinuses and extend portions of their cytoplasm
platelets, when needed, in order to maintain a normal number through the basement membrane and between endothelial
of platelets (150,000 to 400,000/uL).* The maturation of the cells of the marrow sinusoids in order to put platelets into the
megakaryocyte involves endoreduplication (or endomitosis), sinus. Membrane-bound platelets are released and swept into
which is a process whereby the nuclear material reduplicates the bloodstream from these cytoplasmic projections. Further
but the nucleus does not divide. The result of endoreduplica- fragmentation to form individual platelets occurs after release
tion is a polyploid nucleus. Each nuclear reduplication causes into the sinus. One megakaryocyte can release several thou-
a doubling of the nuclear material. The cytoplasm increases in sand platelets.*
amount and in number of granules, but it does not divide. In the past, megakaryocytes were believed to shed
Megakaryoblasts are moderately sized cells in the range of 20 platelets from their outer surface. However, transmission elec-
to 45 um with a single, round (or slightly oval), primitive tron micrographs demonstrate that mature megakaryocytes
nucleus; one or two nucleoli; and blunt protrusions that stain have a well defined marginal zone and that there are only a few
blue and that may contain chromophobic globules.'* The channels of the demarcation membrane system in which
scanty cytoplasm is nongranular and basophilic. The N:C platelets could be shed from the surface.* Therefore, during
ratio is 5:1 to 3:1 (Table 1-13).'* The megakaryocytic thrombocytopoiesis, the entire megakaryocyte cytoplasm frag-
series is shown in Figure 1-48. ments to form platelets.
 28
Chapter 1

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Morphology of Human Blood and Marrow Cells: Hematopoiesis

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 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 29

Figure 1-48 @ Megakaryocytic system. A. Megakaryoblast with single oval nucleus, nucleoli, and bluish foamy marginal cytoplasmic struc-
tures. B. Promegakaryocyte with two nuclei, granular blue cytoplasm, and marginal bubbly cytoplasmic structures. C. Megakaryocyte with
granular cytoplasm and without discrete thrombocytes (platelets). D. Megakaryocyte with multiple nuclei and with thrombocytes (platelets).
E. Megakaryocyte nucleus with attached thrombocytes. F Thrombocytes (platelets). (From Diggs, LW, et al: The Morphology of Human Blood
Cells, ed 5. Abbott Laboratories, Abbott Park, IL, 1985, pp 33-34, 48-50, 85, with permission.)

Figure 1-49 ®@ Center: early megakaryocyte; top left: segmented BSUS lw Center: early megakaryouyte.
neutrophil; bottom center: neutrophilic metamyelocyte.
30 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Ss
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Pigure 1-51 ™ Megakaryocytes without platelets. Figure 1-53 ™ Megakaryocyte with platelets.

Ultrastructural features and marker studies on megakary- Studies have shown that bone formation and hematopoiesis are
ocytes aid in identification of all stages. Ultrastructural fea- closely linked, in that the degree of hematopoiesis correlates
tures are the demarcation membrane, alpha (u) granules, and with the rate of bone turnover. Two normal, nonhematopoietic
platelet peroxidase activity. Monoclonal antibodies for cells that exhibit different functions, yet play essential roles in
platelet glycoproteins Ib, Ib, and IIa; factor VIII antigen; the formation of the bone cavity, are the osteoblast and the
beta-thromboglobulin, and factor V are markers for all stages osteoclast.
of megakaryocytes.'’ In marrow smears of normal individu-
als, there are approximately | to 4 megakaryocytes per 100
Osteoblasts
nucleated cells, and these cells are in the late stage of matura-
tion.2 The morphological characteristics of the megakary- An osteoblast is a large cell that can measure up to 30 um, with
ocytic series are summarized in Table 1-13. ample cytoplasm and a small, round, eccentrically placed
nucleus. These cells may be traumatized in the process of mar-
row aspiration and smearing and often have irregular shapes and
Bone-Derived Cells
cytoplasmic streamers. The cells may have comet or tadpole
The formation of the bone marrow cavity results from a com- shapes. The nucleus may be partially extruded, similar to a
plex process in which hematopoietic cells migrate and colonize small, round head on a round body. The nuclear chromatin
spaces originally occupied by cartilage and bone. This process strands and nuclear margins are well defined and stain purple-
occurs both in the long bones of the limbs and in the membra- red. Usually there is a distinct blue nucleolus? (Fig. 1-55).
nous bones of the skull, which develop directly into bone. Throughout the blue cytoplasm there are small spherical
bodies that are colorless and give a bubbly appearance to the
cytoplasm. Within the cytoplasm there is a prominent round
or oval chromophobic zone that stains lighter than the rest of
the cytoplasm. This area is usually away from the nucleus but
may be adjacent to it.”
Osteoblasts, more often seen in marrow from young
children, are responsible for the formation, calcification, and
maintenance of trabeculae and cancellous bone. Osteoblasts
secrete large amounts of collagen and proteoglycans, con-
tributing to structure of the bone and stromal matrix.4
Osteoblasts morphologically resemble plasmacytes,
both having irregular shapes, eccentric nuclei, cytoplasmic
protrusions, blue cytoplasmic fibrils, and vacuoles. The rel-
atively unstained zone of the plasmacyte is adjacent to the

~ae nucleus and partially surrounds the nucleus like a collar,

whereas the chromophobic zone of the osteoblast is often
distinctly separate from the nuclear margin and, when adja-
Figure |-52 ™ Megakaryocytes tend to be in small groups with cent to the nucleus, does not surround or enclose the
multilobulated single nuclei. Mature megakaryocytes have numer- nucleus.*
ous fine cytoplasmic granules, and occasionally platelet units can be The protein secretions of the plasmacytes impart a red-
seen at their periphery. (magnification x 640) dish background color to the cells that is not demonstrable in
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 3]

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1S)iw) Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Figure 1-54 ™ Naked nuclei, megakaryocyte. Figure 1-56 @ Group of osteoblasts (center) aspirated from the
marrow of a child. (magnification x 400)

absorption of bone.'* Osteoclasts have from 2 to 50 nuclei,

which are separate, usually round or oval, uniform in size, and
haphazardly distributed within the cytoplasm.” Osteoclasts
have visible nucleoli (Fig. 1-57).
The abundant cytoplasm with ragged margins is
bluish, with numerous reddish lysosomal granules contain-
ing acid phosphatase. In thin marrow smears it may be pos-
sible to demonstrate a ruffled cytoplasmic fringe consisting
of diaphanous veils, fingerlike protrusions, and saccular
invaginations.”
Osteoclasts and megakaryocytes are sometimes difficult to
differentiate. Both cells are large with granular cytoplasm, irreg-
ular shapes, and multiple nuclei. The nuclei of megakaryocytes
are connected by a nuclear strand, have irregular nuclear shapes,
and may be superimposed, whereas the nuclei of osteoclasts are
separated, uniform in size, and have no visible connections to
Figure 1-55 m Three osteoblasts.
each other (see Figs. 1-57 and 1-58). The morphological char-
acteristics of the multinucleated osteoclast and the multilobu-
lated megakaryocyte are compared in Table 1-14.”
osteoblasts. Osteoblasts that occur in clusters or aggregates Osteoclasts secrete enzymes that aid in dissolution of
may be misinterpreted as malignant cells (Fig. 1-56). Malig- osteoid tissue and calcific bone. Osteoclast-activating factor
nant cells in a cluster are crowded and distorted, with indis-
tinct margins, rendering it impossible to identify individual
cells. Individual osteoblasts in a cluster can usually be identi-
fied. The size, shape, structure, and color of malignant cells
are variable, whereas osteoblasts are more orderly and uni-
form. Chromophobic areas in the cytoplasm of osteoblasts are
seldom demonstrable in malignant cells.
It is thought that the mature osteoblast arises from the
immature preosteoblast exhibiting mitotic activity. Other the-
ories have placed osteoblast formation at the mesenchymal
stem cells residing in the periosteum. It is probable, however,
that there exist many intermediate stages in the development
of the mature osteoblast as described by studies using mono-
clonal antibodies specific for bone phenotypes.*

Osteoclasts
Figure 1-57 @ The osteoclast is usually seen as a single giant cell
Osteoclasts are giant (greater than 100 um), multinucleated, with multiple and separated nuclei and basophilic granular
irregularly shaped marrow phagocytes that are capable of cytoplasm (center). (magnification 640)
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis eS)ioe)

MEGAKARYOCYTE OSTEOCLAST (Go) after division. From the Gp state the resting cell
enters the G, phase, which is the postmitotic rest phase and
which directly precedes the DNA synthesis phase. The cell
proceeds into the synthesis (S) phase of active DNA synthe-

ag
sis, where the DNA content is doubled. The next phase is the
premitotic rest period (G;) as the cell prepares to enter the
mitotic period (M). During the final, or M, phase, there is cel-
lular division of the chromosomes in the nucleus and the
cytoplasm, resulting in two daughter cells.' T, is the cycle of
Figure 1-58 m Osteoclast versus a megakaryocyte. one complete mitotic division. After final differentiation, the
cell leaves the cycle as a nondividing cell (Gy) (see Fig. 1-59).
Differentiation is associated with cell-cycle arrest.*? In
(OAF), secreted by plasmacytes in myeloma, is a lymphokine erythropoiesis, terminal differentiation is coupled to prolifer-
that stimulates osteoclastic activity in the endosteum near ation.*? Erythroid precursors can undergo “renewal divisions”
groups of myeloma cells.‘ The presence of OAF helps to which is defined as divisions that double the number of
explain the development of osteolytic bone lesions observed erythroid precursors (progenitors), while maintaining the
in myeloma.* differentiation potential.*? This occurs when the expansion of
These cells are involved in the degradation (or reabsorp- erythroid compartment is required during fetal erythropoiesis
tion) of bone, which is essential for the formation of the bone or following hypoxic stress. Renewal divisons comform to
marrow cavity and bone remodeling. Osteoclasts are derived standard cell cycle characteristics.”° Several specific regula-
from the monocyte/macrophage hematopoietic cell lineage.'* tory proteins have been recently shown to regulate genes
Osteoclasts adhere to the bone matrix and secrete lytic involved in development and differentiation.*'! This provides
enzymes that degrade it. Osteoclast proliferation and survival a critical link between the cell cycle and differentiation
of their precursors depend on the cytokine, macrophage during development?!
colony-stimulating factor (M-CSF).'? The morphological
characteristics of the osteoblast and osteoclast are compared
in Table 1-15.
Specific Cell Line Ontogeny
MULTIPOTENTIAL STEM CELLS—COLONY-FORMING
UNITS
Molecular Hematology and Advanced The morphology of the recognizable stages of peripheral
Concepts blood and marrow cells has been described. As stated ear-
Introduction to the Cell Cycle lier, these cells come from an unrecognizable pluripotential
stem cell. The pluripotential stem cell has the capacity for
THE GENERATIVE (G) CELL CYCLE KINETICS continuous self-replication and also for differentiation into
When stimulated by hematopoietic growth factors (see the the multipotential stem cell and the lymphoid stem cell
discussion of colony-stimulating factors and interleukins later without capacity for self-renewal.*? The multipotential stem
in this section), hematopoietic cells undergo a continuous cell becomes committed to support progenitor cells for
generative (G) cycle in which the cells divide, differentiate, or myelopoiesis, erythropoiesis, monopoiesis, and megakary-
remain dormant”? (Fig. 1-59). The bone marrow contains cell opoiesis. The lymphoid stem cell line supports lym-
populations in all phases of cell development. The generative phopoiesis. The multipotential stem cell was shown to exist
cell cycle is divided into five phases: Gp, G,, S, Gs, and M.' in a classic experiment in 1961 by Till and McCullock,?*
The cells able to proliferate enter a resting or dormant phase who irradiated mice to empty the hematopoietic organs and

Cell
Size, pm Shape Cytoplasm Nuclei

Irregular Granular Multiple, uniform in size,

unconnected by nuclear
strands

Megakaryocyte Irregular Granular Multilobed, connected

by nuclear strands, not
uniform in size
34 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

Name Nucleolus Cytoplasm

Osteoblast Chromophobic area, Resemble

uninuclear usually away from plasmacytes
nucleus

Osteoclast Multinucleated, Present Bluish, reddish lysosomal May be confused

uniform size granules, cytoplasmic with megakaryo-
protrusions cytes

then injected a suspension of marrow cells intravenously. other immunocompetent cells. B cells differentiate into plas-
About a week later, nodules of injected marrow could be macytes, which secrete specific immunoglobulins important in
observed on the cut surface of the spleen colonies. All cell the host’s defense against infection. Another population of
lines found in normal marrow were generated from the lymphocytes, called null cells, has none of the characteristics
multipotential stem cells in the marrow suspension. The mul- of either the T or the B cells. The null cell category includes
tipotential stem cell giving rise to several cell lines was killer (K) cells, which interact with antibody to cause destruc-
called the colony-forming unit—granulocyte-erythrocyte- tion of antibody coated targets, and natural killer (NK) cells,
monocyte-macrophage-megakaryocyte (CFU-GEMM).** which can lyse target cells through direct cytotoxic activity.
The CFU-GEMM in a colony assay forms a series of The different types of lymphocytes and their functions are
progenitor cells (CFU-GM, CFU-Eo, CFU-Bas, CFU-Meg, listed in Table 1-17.
BFU-E, CFU-E) under appropriate growth conditions (see
Table 1-16 for acronyms). CFU-GM makes colonies of gran- COLONY-STIMULATING FACTORS AND INTERLEUKINS
ulocytes and monocytes and/or macrophages (Fig. 1-60). Each cell line is dependent on cytokines, which are soluble
CFU-Eo forms colonies of eosinophils. CFU-Bas makes early mediators secreted by cells for the purpose of cell-to-cell com-
basophils and mast cells. CFU-Meg forms megakaryocyte munication. The different cell types and the cytokines they pro-
colonies. There are two colonies of erythroid progenitor cells: duce are listed in Table 1-18. The characteristics of cytokines
the early burst-forming unit-erythroid (BFU-E) (Fig. 1-61) are listed in Table 1-19. Cytokines act on multipotential stem
and the more mature colony-forming unit-erythroid (CFU-E). cells to stimulate their proliferation and differentiation to
The lymphoid stem cell is also derived from the pluripo- committed cell lines (Fig. 1-62).™ For a cell to develop from
tential stem cell. The lymphoid stem cell has the potential to a multipotential cell to a myeloblast, monoblast, erythroblast,
differentiate into a T or B cell. T cells participate in immune or megakaryoblast, cytokines are necessary. Colony-stimulat-
functions of a cellular nature, either directly cytotoxic, or help- ing factors (CSFs) or growth factors and interleukins (ILs) are
ing or suppressing immune activities through interaction with two types of cytokines. The cytokines involved in hematopoi-
etic blood cell development are summarized in Table 1—20,
DNA
synthesis

Synthesis ----- <2) Dividing

Abbreviation Full Name

——f -~-- Division

fe)
CFU-GEMM Colony-forming unit—granulocyte,
erythrocyte, macrophage—
Interphase” Nondividing monocyte, megakaryocyte
cell CFU-GM Colony-forming unit—granulocyte,
macrophage—monocyte
Postmitotic CFU-Eo Colony-forming unit-eosinophil
rest period CFU-Bas Colony-forming unit—basophil
CFU-Meg Colony-forming unit—
Figure 1-59 m Cell cycle kinetics. Tg = one complete mitotic megakaryocyte
cycle; Gy = resting or dormant phase; G, = postmitotic rest period; BFU-E Burst-forming unit-erythrocyte
S = active DNA synthesis phases; G, = premitotic rest period; M = CFU-E Colony-forming unit-erythrocyte
mitotic period; Gy, = nondividing cell.
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis ios)Nn

/Functions
of Lymphocytes —
Lymphocyte Function

T cells Cell-mediated immunity

B cell Humoral immunity
Null cells (killer) Antibody-dependent
cell-mediated lysis
Natural killer (NK) cells Direct cytotoxic activity

recombinant DNA technology. Many growth factors includ-

ing G-CSF, GM-CSF, Epo, and the interleukins have been
used for clinical application.*? In vivo, the regulation of
hematopoiesis is under the control of cytokine production in
the basal state, maintaining normal blood counts and the anti-
Figure 1-60 @ CFU-GM at 14 days (x50 magnification). Colony-
gen stimulus state, eliciting cytokine stimuli above the basal
forming unit that makes colonies of granulocytes, monocytes,
and/or macrophages under appropriate growth conditions. state to combat infection. CSFs have been used to strengthen
patients with cancer and acquired immunodeficiency syn-
drome (AIDS) and to guard against infection in bone marrow
which also lists their sources and the target cells that they
transplantation recipients. These factors have also been used
stimulate.
to treat patients with anemia caused by either surgery or kid-
CSFs and interleukins regulate blood cell development
ney failure. The blood counts of autologous donors can be
by mediating proliferation, differentiation, and maturation of
raised for donation before surgical procedures. Interleukins
hematopoietic progenitor cells (see Fig. 1-62).*
are used clinically for wound healing, activating lympho-
cytes, and assisting in the growth of transplanted or damaged
Trends in Therapeutic Manipulation bone marrow.”°
of Hematopoiesis
Clinical Trials of Recombinant Cytokines
Recombinant Cytokines
Clinical trials of recombinant cytokines using biologic sub-
Many growth factors have been isolated, biochemically char-
stances similar to those in the human body have provided
acterized, purified, genetically cloned, and produced through
new opportunities for evaluating their clinical usefulness in
the treatment of hematologic and oncologic disorders.*°
Investigations have shown that recombinant human granulo-
cyte CSF (rHuG-CSF) accelerates recovery from neutropenia
induced by myelotoxic chemotherapy for different types of
carcinoma.*’ This recombinant CSF has been given to
patients receiving myelosuppressive chemotherapy and under-
going autologous bone marrow transplantation to accelerate
the rate of neutrophil recovery.** Clinical trials are being con-
ducted to determine whether rHuG-CSF is effective in cor-
recting severe neutropenia in hematopoietic malignancies,
such as hairy-cell leukemia, and also in non-neoplastic
hematopoietic diseases, such as aplastic anemia and cyclic
neutropenia.”
Recombinant human GM-CSF appears to offer useful
therapy in accelerating myeloid recovery with graft failure
after bone marrow transplantation.*° Hematopoietic growth
factors can be combined with chemotherapy in treating both
patients with advanced malignancies and bone marrow
transplantation patients by increasing production of granulo-
cytes and platelets.*” However, these increases do not neces-
Figure 1-61 m BFU-E at 18 days (x75 magnification). Early burst-
sarily correlate with an improvement in the outcome of the
forming unit, an erythroid progenitor committed to making
colonies of erythroid cells. patient.*°
36 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

IL-1
IL-6 Phitinotent IL-1
IL-3 Stem Cell IL-6

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IL-1
IL-2 rg:
BFU-E CFU-Meg
IL-4 IL-4
GM-CSF GM-CSF GM-CSF iL-5
GM-CSF
IL-3 IL-3 IL-3 act IL-6

IL-3/IL-4

CFU-G
GM-CSF
G-CSF

Megakaryoblast Myeloblast Monoblast Myeloblast B Lymphobiast T Lymphoblast

GM-CSF GM-CSF GM-CSF IL-B/L-4

IL-3 G-CSF M-CSF

GS antigen antigen
q , driven driven

Proerythroblast ideo yosyie Neutrophilic Promonocyte Eosinophilic Basophilic

Myelocyte Myelocyte Myelocyte
Thrombo- GM-CSF GM-CSF GM-CSF -3AL-4
|
sro poietin |G-CSF M-CSF |IL-5 ee

7 3% & @ @
a a>
=

a i y 4
Erythrocyte Thrombocytes Polymorpho- Monocyte Eosinophil Basophil B Cell T Cell
nucleated GM-CSF
Neutrophil |M-CSF IL-3/IL-4

Macrophage Mast Cell

@
Plasma Cell

Figure 1-62 ® Regulation of hematopoiesis by cytokines. BFU-E = burst-forming unit-erythroid; CFU-Bas = colony-forming unit-basophil; CFU-E
= colony-forming unit-erythroid; CFU-Eo = colony-forming unit-eosinophil; CFU-G = colony-forming unit-granulocyte; CFU-GEMM = colony-
forming unit-granulocyte, erythroid, monocyte macrophage, megakaryocyte; CFU-M = colony-forming unit-monocyte; CFU-Meg = colony-
forming unit-megakaryocyte; EPO = erythropoietin; G-CSF = granulocyte colony-stimulating factor; M-CSF = monocyte-colony-stimulating
factor. (Reprinted from Sandoz Pharmaceuticals Corporation and Schering-Plough, with permission.)
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis = 37

antibodies and the development of multiple commercial

sources under a variety of trade names and designations led
to the development of a standardized nomenclature for
human leukocyte differentiation antigens termed the Clus-
Cell Type Cytokine* ters of Differentiation (CD) nomenclature. In 1989, at a
Endothelial cell GM-CSF, G-CSF, M-CSF, IL-6 series of international workshops on human leukocyte dif-
Monocyte-macrophage GM-CSF, G-CSF, M-CSF, IL-3, ferentiation antigens (HLDA) sponsored by the World
IL-6, EPO, SCF Health Organization, monoclonal antibodies, produced in
T cell GM-CSF, IL-2, IL-3, IL-4, IL-5 many laboratories, having similar reactive patterns with
Fibroblasts GM-CSF, G-CSF, M-CSF, IL-6,
tissue, cells, or molecules were evaluated and compared.
SCF
NK cells GM-CSF These similar reacting antibodies were assigned to a “Clus-
Osteoblasts IL-6, GM-CSF ter of Differentiation” and given a (CD) number for the
PMN G-CSF, GM-CSF antigen it reacts with. CD numbers with a “w” indicate a
B cells GM-CSF, M-CSF, IL-2, IL-5, IL-6 provisional cluster that may or may not be promoted to full
Marrow stroma GM-CSF, G-CSF, M-CSF, IL-6,
CD status at subsequent workshops. Now, 339 CD antigens
IL-3, SCF, IL-11
Renal parenchyma, EPO have been classified by these workshops.*? The eighth
liver, and marrow HLDA workshop was held in Adelaide, Australia in
cells 2004.*+*° The CD antibodies which define these leukocyte
antigens are widely used in research, differential diagnosis,
*mRNA, protein, or specific biologic activity detected at baseline
levels or immediately after induction. monitoring, and treatment of disease.*’7 The human leuko-
cyte differentiation antigens (HLDA) have now been suc-
ceeded by “Human Cell Differentiation Molecules.”** This
is because the HLDA Workshops have recognized that
Interleukin-3 (IL-3), together with M-CSF, stimulates leukocytes do not act alone and, therefore, have included
myelocytes, erythrocytes, and platelet production in aplas- studies on other cell types such as endothelial, stromal,
tic anemia, myelodysplastic syndrome, and prolonged dendritic, and stem cells.
chemotherapy for malignancy.*' Among patients with With the exception of lymphocytes, the current CD anti-
chronic anemia, treatment with recombinant human ery- gens for each cell lineage as determined by the Eighth Inter-
thropoietin (rHuEPO) has increased RBC production and national Workshop on HLDA can be found at the Web site
alleviated anemia in the majority of patients.** The rise in www.hlda8.org
hematocrit is dose-dependent and is in proportion to the The lymphocyte surface markers currently available
increase in RBC mass.* Clinical trials using other synthe- can be found in the references listed at the end of this
sized cytokines are currently in progress to determine activ- chapter2
ity in controlling hernatopoiesis. Each factor needs to be
purified and its function determined before the interactions
Clinical Applications of Cell Surface
between hematopoietic growth factors in the marrow
microenvironment can be completely understood.
Markers
The use of monoclonal antibodies specific to cell surface
markers (CDs) allows phenotypic characterization of cells in
CD Nomenclature
disease states.*' By using flow cytometry (see Chap. 34), cells
The classification of cell surface antigens on hematopoietic labeled with monoclonal antibodies are sorted and enumer-
cells has been aided by the development of monoclonal ated to identify a specific population of cells.”
antibody technology. The rapid production of monoclonal Certain cell markers have been identified as being pre-
sent on the cell surface in disease states such as the acute
leukemias, autoimmune disease, and thromboembolytic
disease.*' Cell markers have also been identified in the man-
4 “Table 1-19 Cytokine Characteristics agement of renal, cardiac, and bone marrow transplantation.
Although diagnosis of disease states is dependent on clinical
Glycoproteins
Produced by many cell types presentation, cytochemistry, and examination of morphol-
Usually act on multiple cell lineages ogy, flow-cytometry characterization of cells has added
Interact synergistically with one another another dimension to disease classification.*! Monoclonal
Activate receptors at very low concentrations antibodies are used to characterize cells in the acute
Usually act on the neoplastic counterpart of normal target cells
leukemias. Such markers allow for the differentiation of
Usually act throughout the maturation hierarchy from stem
cell to the terminally differentiated cell myeloblasts, lymphoblasts, monoblasts, megakaryoblasts,
and erythroid ontogeny.
38 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

table 1-20 Cytokines Involved in Hematopoietic Cytokines Inver in

Hematopoietic Blood Cell Development pear iy)

SyHODES BOUrces, Target Gene Location

Growth Factors
Epo Erythropoietin Kidney, liver Erythroid progenitors
G-CSF Granulocyte colony- Macrophages, Stem cells, neutrophil
stimulating factor endothelial cells, precursors
fibroblasts
GM-CSF Granulocyte-macrophage- T lymphocytes, Progenitors for neutrophils,
colony-stimulating factor macrophages, eosinophils, monocytes
endothelial cells,
fibroblasts
CSF-1, Monocyte- Endothelial cells, Mononuclear phagocytes
macrophage CSF fibroblasts, B cells,
monocytes-macrophages,
stromal cells
SCF Stem cell factor, Fibroblasts Stem cells
c-kit ligand

Interleukins
mei Hematopoietic-1; Macrophages, fibroblasts, Mononuclear phagocytes
response modulator endothelial cells progenitor cells
IL-2 T-cell growth factor T lymphocytes, T cells, B cells
macrophages
IL-3 Multi-CSF T lymphocytes Precursors of neutrophils,
platelets, monocytes,
eosinophils, basophils,
stem cells
IL-4 B-cell stimulatory factor I T lymphocytes B cells, mast cells, T cells
IL-5 B-cell growth factor II, T lymphocytes B cells, eosinophils
eosinophil differentiation
factor
IL-6 Interferon 8 hybridoma T lymphocytes, Stem cells, B cells
growth factor macrophages
IL-7 Lymphopoietin- 1 Stromal cells Pre-B cells, T cells,
early granulocytes
IL-8 Granulocyte Monocytes, T cells, Neutrophils, T cells,
chemotactic factor fibroblasts basophils
IL-9 T-cell growth factor II T cells BFU-E, T cells, mast cells
IL-10 Cytokine synthesis T cells, macrophages, B cells, macrophage,
inhibitory factor B cells T cells, mast cells
IL-11 Adipogenesis Stromal, fibroblasts Megakaryocyte, B cells,
inhibitory factor mast cells
IL-12 NK cell stimulatory factor B cells, macrophages T cells, NK cells Not reported
IL-13 IL-13 T cells B cells 5q
IL-14 High-molecular-weight T cells Activated B cells Not reported
B- cell growth factor

BFU-E = burst- foanine unit-erythroid; NK = natural killer cell.

 Chapter 1 Morphology of Human Blood and Marrow Celis: Hematopoiesis 39

De alocytic ay ies a kidney-shaped nucleus

“ hematopoiesis in 1 es “with clumped chromatin and small, pink, secondary
a. Liver granules with a few primary dark granules?
b. Spleen a. Band
c. Bone marrow b. Myelocyte
d. All of the above c. Promyelocyte
2. Which listing represents the proper cell sequence of d. Metamyelocyte
erythropoiesis? Th Which granulocytic cell has large, abundant violet-blue
a. Rubriblast, prorubricyte, rubricyte, metarubricyte, or purple-black granules?
reticulocyte, erythrocyte a. Eosinophil
b. Rubriblast, rubricyte, prorubricyte, metarubricyte, b. Basophil
reticulocyte, erythrocyte c. Neutrophil
c. Rubriblast, prorubricyte, metarubricyte, rubricyte, 8. What is the proper cell sequence for the monocyte-
reticulocyte, erythrocyte macrophage phagocytic system?
d. Rubriblast, reticulocyte, prorubricyte, rubricyte, a. Monoblast, macrophage, promonocyte, monocyte
metarubricyte, erythrocyte b. Monoblast, monocyte, promonocyte, macrophage
3. What is the best description of a metarubricyte? c. Monoblast, promonocyte, monocyte, macrophage
a. Solid, blue-black degenerated nucleus with nonlinear d. Monoblast, promonocyte, macrophage, monocyte
clumped chromatin pattern; no nucleoli; pink cytoplasm U), Which cell classification is described by the following
b. Round nucleus with visible nucleoli; indistinct and statements: second most numerous cell in the blood;
dispersed chromatin; blue cytoplasm usually small and round; intensely blue cytoplasm; and
c. Coarse chromatin; ill-defined or absent nucleoli; pre- nucleus with clumped dark purple chromatin?
dominantly blue cytoplasm with pink tinge a. Monocyte
d. Small nucleus; thick and condensed nuclear chromatin; b. Lymphocyte
no nucleoli; mixture of pink and blue cytoplasm c. Null cell
4, What is the sequence for the maturation pools of granu- d. Plasmacyte
locyte production? 10. What is the average blood volume?
a. Maturation, proliferation, storage, functional (or mar- @aSutO. Ons
ginated) pool b.4to6L
b.)Proliferation, maturation, storage, functional (or mar- exoito J 2
ginated) pool de sito SE
c. Storage, maturation, proliferation, functional (or mar-
11. Which of the following is a plasma protein?
ginated) poo!
a. Fibrinogen
d. Functional (or marginated) pool, storage, proliferation,
b. Globulin
maturation
c. Albumin
5. Which listing represents the proper cell sequence of d. All of the above
granulocytopoiesis?
. What percent of the blood volume represents the formed
a. Myeloblast, myelocyte, promyelocyte, metamyelocyte,
elements?
band, segmented cell
al S17
b. Myeloblast, metamyelocyte, myelocyte, promyelo-
b. 50%
cyte, segmented cell, band
c. 60%
c. Myeloblast, promyelocyte, myelocyte, metamyelocyte,
d. 45%
band, segmented cell
_ d. Myeloblast, band, promyelocyte, myelocyte,
See answers at the back of this book.
_ metamyelocyte, eed cell
40 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis

where it remains until the seventh month, before finally

shifting to the bone marrow, which becomes the major site
of blood cell development in the fetus and after birth.
# Blood is composed of 55% plasma, the liquid portion, and
m Erythropoiesis identifies the entire process by which ery-
45% cells, the formed elements (RBCs, WBCs, and
throcytes are produced in the marrow and develop from
platelets).
rubriblasts to diffusely basophilic cells and finally into a
a The average blood volume in an adult is 4 to 6 L. mature erythrocyte.
m= Plasma contains mainly water (91.5%), proteins (7%), w Myelopoiesis refers to the production, proliferation, dif-
other solutes (1.5%). ferentiation, division, storage, and delivery to the blood of
g The plasma proteins are albumin, globulin, and fibrinogen. neutrophils, eosinophils, basophils, and monopoiesis.
g Erythrocyte morphology is evaluated in the thin area of w A rubriblast differs little from a myeloblast: both have a
every stained smear where red cells are evenly distrib- round primitive nucleus, visible nucleoli, and chromatin
uted, do not overlap, and are close together. strands that are distinct and dispersed; however, the rubri-
m Platelets should be evaluated in the same thin area of blast is slightly larger than a myeloblast and has more
every stained blood smear where red cells are described cytoplasm, which stains a deeper blue.
by counting the number of platelets in 10 or more oil mwA metarubricyte is recognized by the solid, blue-black,
immersion fields. The normal finding is 7 to 15 platelets degenerated nucleus with nonlinear clumped chromatin
per oil-immersion field. and cytoplasm that is predominately pinkish because of
@ Blood smears should be well made and well stained in increasing hemoglobin synthesis; however, there may
order to properly differentiate leukocytes, platelets, and remain a minimal amount of bluish cytoplasm due to RNA.
erythrocytes. mA promyelocyte has dark blue granules throughout the
gw Normal neutrophil segmented cells contain from two to cytoplasm and sometimes lying over the nucleus, a large
five lobes (usually three) connected by a threadlike fila- nucleus with slightly condensed chromatin, and often
ment(s) and constitute 50% to 70% of mature neutrophils faintly visible nucleoli.
in an adult. w A neutrophilic myelocyte is identified by a round or oval
mw Normal neutrophil band cells have a horseshoe-shaped nucleus with condensed and unevenly stained chromatin
nucleus without evidence of a filament and make up only strands and secondary pinkish-staining (neutrophilic)
2% to 6% of the blood cells in an adult. granules.

w Normal eosinophil granules are large, round, and stain # A neutrophilic metamyelocyte has a bean-shaped nucleus
orange to reddish-orange; eosinophils are found in 0 to with the indentation less than half the width of the arbi-
4% of the blood cells in an adult. trary round nucleus, noticeable chromatin clumping, and
cytoplasm filled with pinkish (neutrophilic) secondary
a The majority of lymphocytes on an adult blood smear are
granules.
small, have a relatively round nucleus with clumped chro-
matin and a small amount of pale blue cytoplasm; lympho- = Lymphoblasts contain a large, round nucleus with thin,
cytes comprise 20% to 44% of the blood cells in an adult. evenly stained, nonclumped chromatin strands; one or
more nucleoli; and a small amount of blue cytoplasm.
mA monocyte is larger than the mature neutrophil; has
abundant gray blue cytoplasm with fine, reddish or pur- m= Monoblasts are large and demonstrate a large nucleus
plish, evenly distributed granules; and has a nucleus with (sometimes minimally indented), one or two prominent
folds or brain-like convolutions, and lacy, often delicate, nucleoli, fine lacy nuclear chromatin, and nongranular,
chromatin. Monocytes comprise 2% to 9% of blood cells often deep blue cytoplasm.
in an adult. mPlasmacytes are characterized by an _ eccentrically
m Large lymphocytes may reveal a few well defined placed, round, small nucleus with lumpy chromatin;
purplish-red granules that can be easily counted, whereas nongranular deep or vibrant blue cytoplasm; perinuclear
numerous fine indistinct granules that cannot be enumer- clear area; occasional vacuoles; and slightly irregular
ated are present in a monocyte. margins.
m Hematopoiesis is defined as the dynamic processes of pro- a The mature megakaryocyte, the largest of the hematopoi-
duction and development of the various blood and mar- etic cells (range is 30 to 100 um) in the bone marrow, has
row cells. a multilobulated nucleus with coarse linear chromatin
and bluish cytoplasm, containing numerous small, dense,
m Hematopoiesis begins in the mesoderm of the yolk sac
reddish-blue granules, which fragment to form platelets.
and, after 2 months, migrates to the liver and spleen,
 Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis 4]

REFERENCES . Anderson, SC, and Poulsen, KG: Ander- 30. Ringden, OT, et al: Granulocyte and
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2006. entiation antigens workshop: The evolving
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—. Bryder, D, and Jacobsen, SE: Interleukin-3
measurement of the radiation sensitivity HLDAS8 blind panel of flow cytometry
supports expansion of long-term multilin-
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Radiat Res 14:213, 1961. 2005.
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96(5):1748, 2000.
STATSA facilitates the generation of findings and conclusions. J Immunol
Majumdar, MK, et al: Human marrow-
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support long-term hematopoiesis when
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. Surveys & Educational Anatomic Pathol. Oncol 12(5):344, 2005. man cell differentiation molecules.
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 Chapter

Bone Marrow
Aamir Ehsan, MD
Jennifer L. Herrick, MD

Introduction OBJECTIVES
Bone Marrow Structure At the end of this chapter, the learner should be able to:
Erythropoiesis
Granulopoiesis Le Describe the process of hematopoiesis.
Megakaryopoiesis 2. List indications for bone marrow studies.
Lymphopoiesis
Stem Cells 3. Name the most common skeletal sites for hematologic studies.
Hematogones 4. Explain the role of the clinical laboratory scientist during a bone marrow procedure.
Marrow Stromal Cells
Mast Cells 5. Describe the preparation of bone marrow aspirate for laboratory examination.
Bone-Forming Cells 6. Explain the effective use of different bone marrow preparations and specimen
Bone Marrow Function distribution.

Indications for Bone Marrow — . List complications and contraindications of the bone marrow procedure.
Studies
. Explain how to calculate the myeloid-to-erythroid (M:E) cell ratio.
Obtaining and Preparing
Bone Marrow for . State the difference between bone marrow aspirate and biopsy.
Hematologic Studies . Explain how to estimate bone marrow cellularity.
Equipment
Aspiration . List the essential components of a bone marrow report.
Preparation of Bone Marrow
Aspirate
Histologic Marrow Particle
Preparation
Bone Marrow Core Biopsy
Preparation of Trephine
Biopsy
Bone Marrow Examination
Estimation of Bone Marrow
Cellularity
Bone Marrow Differential
Count
Bone Marrow and Peripheral
Blood Interpretation
Based on Cellularity and
M:E Ratio Changes
Bone Marrow Iron Stores
Bone Marrow Report
Case Study 1
Case Study 2
 Chapter 2 Bone Marrow 43

Introduction about 1500 grams in adults.' The hematopoietic marrow is orga-

nized around the bone vasculature.** An artery entering the
The hematopoietic system consists of the bone marrow, liver, bone branches out toward the periphery to specialized vascu-
spleen, lymph nodes, and thymus. Hematopoiesis (blood cell lar spaces called sinuses (Fig. 2-1). Several sinuses combine
production and maturation) can be seen at different anatomic in a collecting sinus, forming a central vein that returns into
locations (yolk sac, liver, spleen, axial and radial bones), the systemic circulation. Hematopoietic cords, in which
depending on the gestational and postnatal period. In normal hematopoiesis takes place, lie just outside of the sinuses.
adults, hematopoiesis is seen mainly in the bone marrow. The After maturation in the cords, the hematopoietic cells cross
bone marrow-derived pluripotential hematopoietic stem cells, the walls of the sinuses and enter the blood.*° Hematopoietic
under the influence of various cytokines or growth factors, or cell colonies are compartmentalized in the cords. Structurally,
both, differentiate into myeloid (granulocytes, monocytes, bone marrow consists of hematopoietic cells (erythroid,
megakaryocytes, erythrocytes) and lymphoid cell lineages. myeloid, lymphoid, and megakaryocyte), adipose tissue, bone
Benign conditions and malignant diseases related to these cells and its cells (osteoblasts and osteoclasts), and stroma.
are called hematolymphoid disorders. Nonhematolymphoid
diseases may also involve the bone marrow; therefore, exami-
nation of the bone marrow has a wide application in clinical
Erythropoiesis
medicine. Because hematologic diseases involving the bone Erythropoiesis takes place in distinct anatomic units called
marrow can result in morphological abnormalities of the erythropoietic islands’ (Fig. 2-2). Each island consists of a
peripheral blood cells, the bone marrow examination should macrophage surrounded by a cluster of maturing erythrob-
be interpreted in conjunction with a peripheral smear examina- lasts. Hemoglobin synthesis occurs as early as the pronor-
tion. Severe thrombocytopenia is generally not a contraindica- moblastic stage, but most hemoglobin synthesis occurs in the
tion to the procedure. In experienced hands and with the polychromatophilic stage. The average life span of circulating
needles currently available, the bone marrow aspiration and red cells is 120 days.
biopsy carry minimal risk.

Granulopoiesis
Bone Marrow Structure
Granulopoiesis is less conspicuously oriented toward a distinct
The bone marrow is one of the body’s largest organs, repre- reticulum cell, yet may be recognized as a unit* (Fig. 2-3).
senting 3.4 to 6 percent of total body weight and averaging Early granulocytic precursors are located deep in the cords and

i C) =~ >. & >.

= NO) @ @J@) © = =

>” G @ _G
(BX
sinus (CV). The venous
Figure 2-1 m Graphic presentation of hematopoietic tissue. The vascular compartment consists of arteriole (A) and central
of the
sinusoids are lined by endothelial cells (End), and their wall outside is supported by adventitial-reticulum cells (Adv). Fat tissue (F) is part
(RCP), and erythro-
marrow. The compartmentalization of the hematopoiesis is represented by areas of granulopoiesis (GP), areas of erythropoiesis
poietic islands (El) with their nutrient histiocyte (Hist). The megakaryocytes protrude with small cytoplasmic projections through the vascular
the
wall (Meg). Lymphocytes (Lym) are randomly scattered among the hemapoietic cells, whereas plasma cells (Pla) are usually situated along
vascular wall.
44 Chapter 2. Bone Marrow

Megakaryopoiesis
Megakaryopoiesis occurs adjacent to the sinus endothelium.
The megakaryocytes protrude as small cytoplasmic processes
through the vascular wall, delivering platelets directly into the
sinusoidal blood.’ Megakaryocytes are situated close to marrow
sinuses and, through a mechanism that is not entirely under-
stood, shed platelets into the circulation (Fig. 2-4). Approxi-
mately 5 days are required for the megakaryocytes to produce
platelets, which are released in small groups called proplatelets.
Two-thirds of shed platelets are present in the circulation,
whereas one-third are sequestered in the spleen. The average life
span of platelets in the circulation is approximately 10 days.

Lymphopoiesis
Figure 2-2 ™ Erythropoietic island composed mainly of polychro-
matophilic normoblasts. The nutrient-histiocyte (arrow) is slightly Lymphocytes and plasma cells are minor components of nucle-
displaced off its central position by smearing of the particle. Its cyto-
ated cells in normal bone marrow. Lymphocyte production is
plasmic slender processes envelop a basophilic normoblast, establish-
ing intimate contact with the maturing red cell precursor. compartmentalized in lymphoid follicles, and lymphocytes
(Wright-Giemsa, magnification x 600) are randomly dispersed throughout the cords. The lymphoid
follicles (most often seen in elderly individuals) are unevenly
distributed and tend to influence the variability of the lympho-
around the bone trabeculae. Neutrophils in the marrow can be cyte count in aspirated bone marrow samples (Fig. 2-5).
divided into the proliferating pool and the maturation storage Plasma cells are situated along the vascular wall.
pool. The proliferating pool includes myeloblasts, promyelo-
cytes, and myelocytes. These cells, which spend 3 to 6 days in
this pool, are capable of DNA synthesis and undergo cell divi- Stem Cells
sion. The maturation storage pool consists of nondividing
metamyelocytes, bands, and segmented neutrophils. The cells The marrow stem cells have two unique biologic characteristics:
typically spend 5 to 7 days in this pool before entering into the self-renewal and multilineage differentiation. These stem
circulation. Depending on the demand, the cells from the stor- cells can be further subcategorized as pluripotential stem cells
age pool (representing 15 to 20 times as many cells as in the (which give rise to many different cell lines) and committed
blood) can be released into the peripheral blood, thus increas- stem cells. Because committed stem cells are dedicated by
ing the total white blood cell (WBC) count in minutes or lineage to one cell type and do not have the potential of self-
hours. The average life span of circulating neutrophils is 6 to renewal, they are also called progenitor cells. A progenitor
10 hours. cell is a cell committed to a single line of proliferation and
differentiation. The marrow stem cells on Wright’s stained
smears are morphologically indistinguishable from small
lymphocytes. In the presence of appropriate growth factors,

Figure 2-5 @ Compartment of granulopoiesis. A reticulum cell

(arrow) with open reticulated chromatin and light blue cytoplasm
containing dustlike fine granules is situated among numerous granu-
locytic precursors, especially myelocytes. (Wright-Giemsa, magnifica- Figure 2-4 ™ Mature megakaryocytes releasing proplatelets (pack-
tion x600) ages of platelets). (Wright-Giemsa, magnification x 600)
 Chapter 2 Bone Marrow 45

Figure 2-5 ™A lymphocytic nodule (follicle) in bone marrow as Figure 2-6 m An arrow points toward a hematogone showing high
shown here may alter very significantly the marrow differential nuclear-to-cytoplasmic ratio, homogeneous chromatin, and scant
count when aspirated and give a false impression of lymphocytic cytoplasm. It can be confused with a blast of lymphoblastic
malignancy. (H&E, magnification x200) leukemia. (Wright-Giemsa, magnification 1000)

the stem cells differentiate into myeloid and lymphoid cells Marrow Stromal Cells
that carry tissue and humoral immune functions, respec-
tively. Some lymphoid progenitor cells produced in the mar- The meshwork of stromal cells in which the hematopoietic
row mature in the thymus as T lymphocytes'®; others are cells are suspended is in a delicate semifluid state and is com-
produced and continue their maturation and differentiation posed of reticulum cells, histiocytes, fat cells, and endothelial
in bone marrow as B lymphocytes from the 12th gestational cells. The reticulum cells are associated with fibers that can be
week throughout life.'' Therefore, the bone marrow and thy- visualized after silver staining. They are adjacent to the sinus
mus are primary lymphoid organs of antigen-independent endothelial cells, forming the outer part of the wall as an adven-
progenitor lymphoid cell proliferation and differentiation, titial reticulum cell. Their fine cytoplasmic projections extend
which gives rise to new lymphocytes. These new lympho- deep into the cords, making contact with similar projections of
cytes may then populate the secondary lymphoid organs such other cells. Occasionally, the nuclear region of these cells
as lymph nodes, spleen, and lymphoid apparatus of the gas- can be seen deep in the cords surrounded by granulopoiesis.
trointestinal tract. Under appropriate stimulation, the mature Cytochemically, these cells are alkaline-phosphatase positive
lymphocytes of the peripheral lymphoid organs undergo (Fig. 2-7). Histiocytes or macrophages are seen as perisinu-
antigen-dependent effector cell proliferation, resulting in soidal cells related to the bone marrow—blood barrier, and as
cytokines and antibody production from T and B lympho- central storage macrophages of the erythropoietic islands. As
cytes, respectively. storage nutrient cells that deliver iron to the growing immature
erythroblasts, the storage macrophages send out long, slender
cytoplasmic processes that envelop the erythroid precursors.
This extensive and intimate contact with the maturing ery-
Hematogones
thropoietic cells is necessary in transferring iron from the
Hematogones are normal cellular constituents of bone marrow macrophage to the red cell precursors. As phagocytic cells,
that resemble small- to intermediate-sized lymphocytes. They the macrophages also undergo hyperplasia when there is
range in size from 10 to 20 um and have a high nuclear-to- increased destruction of hematopoietic cells. Histochemically,
cytoplasmic ratio and smooth, smudged homogeneous chro- the macrophages are acid-phosphatase positive (Fig. 2—8).
matin. Their cytoplasm is deeply basophilic and devoid of any The stromal cells produce an extracellular matrix'® com-
granules or vacuoles (Fig. 2-6). Hematogones are thought to posed of collagens, glycoproteins, proteoglycans, and other
be committed progenitor cells of lymphoid lineage. Their proteins. This extracellular matrix is essential in maintaining
numbers are increased in normal infants, older children, and normal renewal and differentiation of marrow cells. The bone
sometimes in adults (in regenerative marrow after chemother- marrow consists of red marrow (hematopoietic active) and
apy and bone marrow transplantation), as well as in marrows fatty yellow marrow (hematopoietic inactive). The marrow
of children with neuroblastoma with or without metastasis, cellularity is estimated as a percentage of red marrow to total
iron-deficiency anemia, and idiopathic thrombocytopenic marrow. The fatty yellow marrow (adipose cells) can vary
purpura.'2!5 These cells closely resemble blasts of lym- in amount according to the age of the individual patient and
phoblastic leukemia, and laboratory personnel should be the skeletal ___location from where the marrow is obtained. In
aware of the conditions in which their numbers may be young children, most of the marrow is composed of red,
increased. hematopoietic active marrow with only a few fat cells present.
46 Chapter 2. Bone Marrow

Figure 2-7 ® An alkaline phosphatase-stained reticulum cell Figure 2-9 @A string of endothelial cells aspirated from hypocellu-
extends its slender cytoplasmic projections deep in the hemopoietic lar marrow. The nuclei are elongated and slightly tapered. The
cord, maintaining an intimate contact with granulopoiesis. The cytoplasm is transparent and barely visible. (Wright-Giemsa,
background cells are stained with neutral red. (magnification x 600) magnification < 600)

The adipose tissue gradually increases after 4 years of age. In oval reticular nucleus and abundant blue-purple granules that
adults, fat cells average about 50% of the total marrow obscure the nucleus. Their granules, in addition to all other
volume in the vertebrae and flat bones of the pelvis. The substances that are present in the granules of basophils, con-
marrow fat and the extracellular matrix are dynamic tissues tain serotonin and proteolytic enzymes. The numbers of mast
similar to the hematopoietic tissue, and these may be altered cells can be increased in chronic infections, autoimmune dis-
rapidly in disease states. eases, chronic lymphoproliferative disorders, and especially
The bone marrow is a highly vascularized tissue from in systemic mastocytosis.!7'*
which endothelial cells can occasionally be aspirated.
Endothelial cells are more visible in hypoplastic marrows and
should not be mistaken for metastatic tumors (Fig. 2-9). Bone-Forming Cells
In marrow aspirates, cells are occasionally seen originating
Mast Cells from bone tissue. Osteoblasts are bone matrix—synthesizing
cells usually found in groups. They are up to 30 um in diame-
Tissue mast cells (Fig. 2-10), 6 to 12 um in diameter, are con- ter and resemble plasma cells. The osteoblast nucleus has a fine
nective tissue cells of mesenchymal origin, normally present chromatin pattern with a prominent nucleolus. A perinuclear
in the bone marrow in varying numbers. They have a round or halo, detached from the nuclear membrane with a cytoplasmic

Figure 2-8 ® Two acid phosphatase-positive macrophages in bone

marrow of a patient treated with chemotherapeutic agents. Figure 2-10 Three mast cells, known also as tissue basophils, are
Macrophages are also scavengers and cleaners of the hematopoietic shown in this marrow aspirate in a background of erythroid hyper-
tissue, so they increase in number during massive destruction of plasia. Numerous regular round granules fill their cytoplasm and
hematopoietic cells. (magnification x 600) obscure the nuclear details. (Wright-Giemsa, magnification x 600)
 Chapter 2. Bone Marrow 47

bridge, represents the Golgi apparatus area. Osteoblasts are limitations. Bone marrow aspiration and bone marrow biopsy
alkaline-phosphatase positive. They are characteristically seen are usually performed concurrently.
in bone marrow aspirates of children and patients with meta- Although obtaining the bone marrow for examination
bolic bone diseases. carries little procedural risk for the patient, the procedure is
Osteoclasts, or bone remodeling cells, are multinucle- costly and can be quite painful. For this reason, bone marrow
ated giant cells more than 100 um in diameter, resembling studies should be performed only when clearly indicated or
megakaryocytes. The nuclei of the osteoclasts are separate whenever the physician expects a beneficial diagnostic result
from each other and may have nucleoli (compared with the for his or her patient (Table 2-1). Hematologic diseases
megakaryocyte nucleus, which is multilobed). Their cyto- affecting primarily the bone marrow and causing a decrease
plasm is well delineated and finely granular. or increase of any cellular blood elements are among the most
common indications. It is not unusual for more than one blood
element to be increased or decreased, as occurs in leukemias
Bone Marrow Function and some refractory anemias. In these situations, bone
marrow study affords specific information, and it usually
The main function of the marrow is to supply mature
precedes any other diagnostic procedure.
hematopoietic cells into the peripheral blood in a steady-
Systemic diseases may affect the bone marrow secondar-
state condition as well as to respond to increased demands. A
ily and require bone marrow studies for diagnosis or monitor-
semidormant pool of pluripotential stem cells maintains a
ing patients’ conditions. Patients having any of the solid
self-renewal property. Granulocytic, monocytic, eosinophilic,
malignant tumors may undergo bone marrow studies when
erythroid, and megakaryocyte progenitor cells are influ-
the initial diagnosis is established for evaluation of the degree
enced in their differentiation by colony-stimulating factors
of tumor spread and staging of the disease. On occasion, a
(CSFs)."°*! CSFs are produced by T lymphocytes—as
bone marrow study may result in a diagnosis of unsuspected
well as stromal cells, fibroblasts, endothelial cells, and
metastatic malignant tumor. During the course of malignant
macrophages—when stimulated by monocyte interleukin- |
disease, additional marrow studies may be performed period-
(IL-1) and tumor necrosis factor (TNF). Some CSFs, such as
ically to monitor the status of tumor burden and its therapeu-
IL-3 and granulocyte-monocyte CSF, have a broad influence
tic response.
and are required throughout proliferation and differentiation
Infections manifesting clinically as “fever of unknown
of progenitor cells. Others, which include granulocyte,
origin” may exhibit granulomas, focal necrosis, or histiocytic
monocyte, and eosinophil CSFs, are lineage specific, and
proliferations. Intracytoplasmic organisms may be seen in the
regulate division and differentiation only of correspond-
marrow. Material for morphologic studies and bacterial cultures
ing, committed progenitor cells. In addition, erythropoiesis
may be collected simultaneously during a single procedure. The
is influenced by erythropoietin produced in the kidney.****
suspected diagnoses of disseminated tuberculosis, fungal infec-
In the process of cell egression from the cords to the circula-
tions (particularly histoplasmosis and cryptococcosis), and
tion, a number of releasing factors are identified. The best
some protozoan infections are frequently confirmed through
characterized of these are granulocyte colony—stimulating fac-
such studies. Hereditary and acquired conditions occasionally
tor (G-CSF) and granulocyte—macrophage colony—stimulating
involve the bone marrow histiocytes (e.g., Gaucher’s disease as
factor (GM-CSF), but other factors, such as components of
shown in Fig. 2-11, sea blue histiocytosis, hemophagocytic
a complement system, androgenic steroids, and endotoxins,
may play a role.** The endothelial lining of the sinusoids
forms a continuous, veil-like wall through which the
mature cells migrate from extravascular sites into the cir-
culation.*> This is accomplished by close contact between
mature hematopoietic cells and endothelial cells. A tran-
sient migration pore is formed during such contact through
which the mature cells pass into the circulation without Hematologic
loss of plasma to the extravascular pool.’° It is evident that
e Anemias, polycythemia
the bone marrow is subjected to a complex regulation by
¢ Unexplained leukopenia or leukocytosis
many cellular and humoral systems of the body, and any ¢ Presence of blasts, immature, or abnormal cells in the
disease that affects these systems is likely to affect circulation
hematopoiesis. ¢ Unexplained thrombocytopenia or thrombocytosis
e Evaluation of plasma cell disorders
Systemic Diseases
Indications for Bone Marrow Studies ¢ Staging and management of solid malignant tumors arising
studies in the elsewhere in the body, such as lymphomas, carcinoma, and
In 1929, Arinkin2’ introduced bone marrow
sarcomas
diagnosis of hematopoietic disorders. Once a formidable ¢ Infections or fever of unknown origin, granulomas
task, obtaining bone marrow tissue has become, with current ¢ Hereditary or acquired metabolic disorders
improved techniques, a standard procedure. Several tech- e Systemic mast cell disease
niques have been devised, each having its own merits and
48 Chapter 2. Bone Marrow

$2 —
ifC i> Sternum

Spinal :
rocesses ([&
P a] \
Posterior i
superior Bp \
iliaccrest (/,

Anterior \\\\\
: id \Y
superior \
iliac crest
Figure 2-1 1 @ Bone marrow biopsy showing a collection of
Gaucher cells. (H&E, magnification 600)

ey; )
syndrome, and others). A simple procedure such as bone
marrow aspiration or biopsy may establish the diagnosis.

Obtaining and Preparing Bone Marrow

for Hematologic Studies
The sites for bone marrow studies in adults are most com- i
monly the posterior superior iliac crest, occasionally the CS
sternum, and very rarely the anterior superior iliac crest and
Figure 2-12 ™ Common sites from which bone marrow is obtained
spinal processes or vertebral bodies (Fig. 2-12). Sternal for studies.
aspiration should be avoided in children, as well as in
patients with multiple myeloma and metastatic carcinoma,
because these diseases can cause thinning and erosion of flow cytometric, cytogenetic, microbiologic, electron micro-
bone that may increase the chance of perforation and can scopic, molecular, and other studies as indicated in a partic-
cause potentially fatal cardiac complications. Occasionally, ular case.
when a localized bone lesion is visualized on roentgenogram In experienced hands, complications of bone marrow
or computed tomographic (CT) scan, a directed or “open” biopsy and aspirate are very rare (0.1%). These rare compli-
bone marrow biopsy of the lesion may be performed by a cations include bleeding or infection at the biopsy site, tran-
radiologist or surgeon in an operating room with the patient sient neuropathy, and osteomyelitis. After the procedure, the
under anesthesia. In newborns and infants, a bone marrow patient is advised to lie on the biopsy site, which should be
sample can be obtained from the upper end of the tibial re-evaluated in 15 to 30 minutes for any bleeding or oozing.
bone. Bone marrow aspiration and biopsy can be performed
Before performing the procedure, the physician should on patients with severe thrombocytopenia, coagulation fac-
inform the adult patient or the parent or guardian of a child of tor deficiencies (more than 50% activity is required), and in
the procedure, its risks, and its expected benefits for the diagnos- those receiving anticoagulant therapy. The only valid con-
tic process. The bone marrow procedure cannot be performed traindication is failure to meet the indication criteria (see
until a consent form is signed and witnessed by a second person, Table 2-1);
commonly the patient’s nurse. The actual procedure is often per-
formed with the assistance of a clinical laboratory scientist.
Equipment
Although the physician performs the procedure, and the nurse
attends to the patient, the clinical laboratory scientist gives full The instrument tray used to perform a bone marrow procedure
attention to the processing of the specimens. It is the clinical lab- should contain enough equipment to complete the procedure
oratory scientist’s responsibility to ensure that the samples are and to prepare the tissues obtained for the appropriate studies
adequate. If they are not, the physician is informed immediately (Table 2-2). Complete bone marrow trays are sold as dispos-
so that the procedure can be repeated before the patient is able equipment, which is convenient, and also avoids the risk
discharged. Samples are preserved appropriately for histologic, of transmitting infectious diseases. Because of potential
 Chapter 2 Bone Marrow 49

Required Materials
. 30-mL syringes
. 20-mL syringes
. 10-mL syringes
. 5-mL syringes
. 2% Lidocaine
. Prepodyne prep
. Alcohol (70%) or prep
. 23-Gauge needles
. 21-Gauge needles
. Bone marrow biopsy/aspiration needle 11-gauge X 4 in.
. Filter papers
. Buffered formalin 10% with a pH of about 6.8 or other
fixative for histologic processing of bone biopsy and
marrow particles
. Tube containing liquid EDTA anticoagulant
. One box of slides
. One slide folder
. One rubber bulb
7, Pasteur pipet Figure 2-13 ™ Adult-sized Jamshidi 11-gauge x 4-inch biopsy/
. Petri dish aspiration needle showing stylet (/eft), biopsy needle (center), and
. Sterile blades probe (right).
. Gloves (several pairs of different sizes)
. Sterile gauze and cotton balls
. Applicator sticks (Xylocaine) is infiltrated into the skin, in the intervening
. Bandage tissues between the skin and bone, and in the periosteum of the
. Culture bottles for bacterial culture. (Note: Save some bone from which the marrow is to be obtained. A cut of about
bone marrow specimen in syringe for tuberculosis and 3 mm is made through the skin with a Bard-Parker blade to
fungal cultures, when indicated.)
facilitate piercing skin and subcutaneous tissue.
. Pencil to label slides
. No. 11 Bard Parker blades The physician penetrates the bone cavity with an aspira-
tion needle, assembled with guard and stylet locked in place.
When the marrow cavity is penetrated, the stylet is removed,
a syringe is attached to the free end of the needle, and the
plunger is quickly pulled, drawing 1.0 to 1.5 mL of marrow
disease transmission, nondisposable bone marrow trays are particles and sinusoidal blood into the syringe. Because the
rarely used today. vacuum created in the syringe is important for rapid and
Several different styles of aspiration and trephine bone efficient suctioning of the cells and particles, the syringe
biopsy needles or instruments are commonly used. Most should be 10 mL or larger with a well-fitting plunger. Despite
instruments used today are patterned on the needle intro- the use of local anesthesia, the patient normally experiences
duced by Jamshidi.** These instruments are produced in discomfort during the aspiration process (aspiration pain).
several sizes for both adult and pediatric patients. An exam- Accomplishing the aspiration with a quick and continuous
ple of the Jamshidi bone marrow biopsy/aspiration needle pull on the plunger diminishes the patient’s discomfort and
for adults is shown in Figure 2-13. Modifications of the decreases the chance of clotting the specimen. A clotted
original aspiration and trephine needles have been devel- specimen is useless for smear preparation because the fibrin
oped by different companies and are manufactured as threads strip the cytoplasm off of the cells and hamper their
disposable equipment. spreading.
Keeping the volume of the initial aspirate small also pre-
vents dilution of the sample with large amounts of sinusoidal
Aspiration
blood, thus improving the quality of the aspirate. This first-
A bone marrow aspiration may be performed as an indepen- aspirated material is used immediately for preparing smears.
dent procedure or in conjunction with a bone marrow biopsy. Additional aspirate may be obtained in separate syringes if
The procedure can be performed in the outpatient setting in needed for flow cytometry, chromosome studies, bacterial
clinics or in the physician’s office. As a rule, children and very cultures, and other tests (Fig. 2-14). Once an adequate aspi-
apprehensive patients receive a mild sedative before the pro- rate is obtained, the quality of the smear depends entirely on
cedure. The site selected is shaved, if needed, and washed with the clinical laboratory scientist’s skill and speed in preparing
soap. Then an antiseptic is applied, and the area is draped with the smears and preserving the morphology of the marrow
sterile towels. A local anesthetic such as 1% to 2% lidocaine cells. Part of the first aspirate is used for the preparation of
50. Chapter 2. Bone Marrow

direct and marrow particle smears. Another portion is placed dropper or Oxford pipette and transferred to a clean glass
in an ethylene diaminetetraacetic acid (EDTA) anticoagulant- slide, which is covered gently with another slide. The two
containing tube for use as a particle preparation. If some aspi- slides are pulled in opposite and parallel directions to smear
rate still remains, it can be left to clot. The clot may be fixed the particles without crushing the cells. Some people recom-
in 10% buffered formalin or another chosen fixative and mend an alternate technique using two cover glasses. In this
processed for histologic examination. The preferred anticoag- process, the marrow particles are squashed between two
ulants for ancillary studies performed on the bone marrow cover slips, which are then gently pulled apart.
aspirate are EDTA for flow cytometry and sodium heparin for Techniques for preparing particle smears vary from person
cytogenetics. If the aspiration attempt is unsuccessful (a “dry to person and from laboratory to laboratory. The aspirate may be
tap”), an additional core biopsy may be obtained (placed in transferred into a watch glass and the particles collected with a
saline or RPMI) for flow cytometric studies. In these situa- capillary pipette or the broken end of a wooden stick applicator.
tions, touch preparations of the core biopsy are useful for With experience, one usually adapts a technique that facilitates
Wright-Giemsa stained morphologic evaluation, as well as production of high-quality slides. The clinical laboratory scien-
cytochemical and fluorescence in situ hybridization (FISH) tist should prepare an adequate number of slides of smeared
studies, if needed. marrow particles. In cases of newly diagnosed acute leukemia,
no fewer than 10 slides should be prepared. These are needed
Preparation of Bone Marrow Aspirate for histochemical stains such as myeloperoxidase, Sudan black
B, naphthyl AS-D chloroacetate esterase, alpha-naphthyl
All necessary materials, preservatives, and slides should be butyrate esterase, and others.
meticulously clean and in readiness to avoid any delay. The Marrow particle smears are used in the evaluation of cel-
aspirate in the first syringe contains mostly blood admixed lularity (usually marrow biopsy is ideal) and the relationship
with fat, marrow cells, and particles of marrow tissue, which of the cells to each other. Well-prepared smears have the
should be used for smears. Several direct smears can be pre- added advantage of excellent cell morphology, allowing
pared immediately, using the technique for blood film prepara- subtle changes in cell maturation and cytoplasmic inclusions
tion.” A small drop is placed on a glass slide, and the blood to be recognized easily
and the particles are dragged behind a spreading slide with a All direct and particle smears should be labeled at the
technique similar to that for preparing blood films. Although bedside with the patient’s name, identification number, and
this method of preparation preserves the cell morphology well, the date and then air dried.
it is inadequate for the evaluation of the cells in relationship to
each other and for the estimation of marrow cellularity. Histologic Marrow Particle Preparation
Smears of marrow particles are prepared by pouring a
small amount of the aspirate on a glass slide. The marrow The leftover marrow particles obtained during the aspiration
tissue is seen as gray particles floating in blood and fat procedure can be processed for histologic examination. The
droplets. The particles are aspirated selectively with a plastic tissue particles, admixed with blood, may be left to clot, then

BONE MARROW SAMPLING METHODS

Direct aspirate smears Marrow particles Clot sections Ancillary studies

To assess cytomorphology (in EDTA) (allowed to clot) * Flow cytometry (in EDTA)
* Perform differential count * Cytogenetics (in sodium heparin)
and ME ratio exe -¢ Molecular studies (in EDTA)
* Perform cytochemical stains * Cultures (in culture media)

Figure 2-148 Distribution of bone marrow sample. EDTA = ethylene diaminetetraacetic acid; M:E = myeloid-to-eryt
hroid ratio. (Note:
Fluorescence in situ hybridization (FISH) studies can be performed on aspirate smears, touch preparations, and all
of the tissue sections.)
 Chapter 2 Bone Marrow 51

fixed in 10% buffered formalin and processed for histologic sec- Park Memorial Institute) solution. Adequate cell suspensions
tioning. However, better results are obtained if the blood and can then be made from this biopsy sample and processed for
particles are transferred to an EDTA anticoagulant—containing flow cytometric studies.
tube before clotting sets in. The blood and particles are then fil-
tered through histo-wrap filter paper, and the concentrated par-
ticles enfolded in the paper are fixed in 10% buffered formalin. Preparation of Trephine Biopsy
In the histology laboratory, these particles are collected by
TOUCH PREPARATION
scraping the paper and then embedding the particles in paraffin
The bone marrow core biopsy sample is supported lightly
for further processing.*°
without pressure between the blades of forceps and touched
several times on two or three clean slide surfaces. The biopsy
Bone Marrow Core Biopsy core sample should not be rubbed on the slide, because rub-
bing destroys the cells. The slides are air dried. The touch
A bone marrow core biopsy is especially indicated when the preparations are fixed in absolute methanol and stained with
marrow cannot be aspirated (“dry tap”), owing to pathologic Wright-Giemsa stain. In the absence of a good aspirate smear,
alterations encountered in acute leukemias, myelofibrosis, the touch preparations may be the only source for studying
hairy cell leukemia, and other disorders. A trephine bone cellular details and the maturation sequence of the bone
marrow core biopsy is also performed for the diagnosis of marrow biopsy sample. For example, in a case of hairy cell
neoplastic and granulomatous diseases. In multiple myeloma leukemia, dry taps are common and cellular morphology by
and for staging of lymphomas or solid tumors, bilateral pos- Wright-Giemsa stained touch preparation may be a useful
terior superior iliac crest biopsies are recommended, as clue to the diagnosis and allow cytochemical staining proce-
increased sampling size enhances the likelihood of capturing dures, such as tartrate-resistant acid phosphatase (TRAP)
a focal process. An adequate biopsy sample is at least 15 mm (Figs. 2-16 and 2-17). Sometimes the touch preparations
in length.*! contain enough cells to obtain differential counts and blast
When a bone marrow biopsy is performed in conjunction evaluation, and to perform histochemical studies.*”
with a marrow aspiration, customarily the biopsy sample is
obtained after the aspirate. This sequence is usually achieved by HISTOLOGIC BONE MARROW BIOPSY PREPARATION
changing the direction of the needle to avoid the aspiration arti- The biopsy specimen is immersed without delay in B-5 or
fact. However, this technique may result in an aspiration artifact 10% buffered formalin fixative. Histology laboratories may
with hemorrhage into the area of the biopsy site, leading to dif- have a choice of other preferred fixatives such as Zenker’s
ficulties in evaluating cellularity and morphology (Fig. 2-15). solution, Carnoy’s solution, and others.**** After fixation, the
Therefore, a core biopsy sample should be obtained before the biopsy specimen undergoes standard histologic processing of
aspiration or the marrow biopsy procedure is to be performed decalcification, dehydration, embedding in paraffin blocks,
through a new puncture site in the anesthetized area. In some sectioning of 2- to 3-um thick sections, and histologic staining.
cases when flow cytometry is indicated, and the aspirate is dif- The advantage of the bone marrow biopsy is that it represents
ficult to obtain (e.g., in patients with hypercellular marrow, hairy a large sample of marrow and bone structures in their natural
cell leukemia, or marrow fibrosis) an additional core marrow relationships. A variety of different stains can be used to demon-
biopsy may be obtained in normal saline or RPMI (Roswell strate marrow iron, reticulum, and collagen. However, because

Figure 2-16 ® Wright-stained bone marrow touch preparation

Figure 2-15 m Bone marrow biopsy specimen showing aspiration from a patient with hairy cell leukemia. Aspirate was a dry tap. A
artifact that can affect the cellularity and alter the relationship of few diagnostic hairy cells are seen with abundant, fluffy, light blue
cells to each other. (H&E, magnification x 200) cytoplasm and inconspicuous nucleoli. (magnification x 1000)
nNbo Chapter 2. Bone Marrow

Figure 2-17 ™ Tartrate-resistant acid phosphatase (TRAP) stain Figure 2-19 ™ Acid-fast stain on a bone marrow biopsy specimen
showing a strong positive reaction in neoplastic cells which is useful from the same patient as in Figure 2-18 shows acid-fast organisms,
in the diagnosis of hairy cell leukemia. (magnification x 1000) suggesting infection with Mycobacterium. (magnification x 1000)

of decalcification, the core biopsy may not be a good method for Wright-Giemsa stained aspirates or core biopsy touch pre-
studying marrow iron stores, as the processing leaches iron from parations may supply the missing morphologic details
the tissue which may be underrepresented in the iron stain. (Figs. 2-24 and 2-25). Multiple touch preparations also
Acid-fast organisms and fungi in granulomatous diseases may offer an opportunity for histochemical stains (myeloperoxi-
be detected quickly with specific stains, offering great advan- dase, Sudan black B, naphthyl AS-D chloroacetate esterase,
tages in diagnosing these infections (Figs. 2-18, 2-19, and a-naphthy] butyrate esterase, etc.), which are essential in the
2-20). For instance, mycobacterial cultures may require weeks classification of leukemias. Polymerase chain reaction
of incubation to show growth of organisms, whereas on tissue (PCR) and FISH can be performed on paraffin-embedded,
sections, the histologic and etiologic diagnosis may be made formalin-fixed marrow biopsy specimens. PCR technology
within 10 to 12 hours. When metastatic tumors and lymphomas may be used to evaluate B-cell or T-cell lymphomas, various
are found in the bone marrow, immunohistochemical stains can leukemias, and minimal residual disease.
be used on histologic sections to demonstrate specific tumor Trephine bone marrow biopsy specimens may be
markers (Figs. 2-21, 2-22, and 2-23). Thus, a very precise embedded in methyl methacrylate, a synthetic plastic
diagnosis of the origin of a tumor can be made without elabo- medium, and sectioned into | to 2um thin sections without
rate, expensive, and invasive techniques. decalcification. The morphological quality of the cells is
A disadvantage of the bone marrow biopsy is that fine extremely well preserved, and a differential count can be done
cellular details are lost in the processing; therefore, it is of on hematoxylin—-eosin (H&E) or Giemsa-stained _ slides.*
little value in the diagnosis of myelodysplastic syndromes However, this technique requires specially trained personnel,
and subtyping of acute leukemias. In these situations, the equipment, and separate handling in the histology laboratory,

Figure 2-18 ® Bone marrow biopsy specimen from an HIV-positive Figure 2-20 ™ GMS (Gomori’s methenamine silver) stain on bone
patient with tuberculosis shows a well-formed granuloma. (H&E, marrow biopsy specimen from an HIV-positive patient shows multiple
magnification x 200) budding yeasts consistent with histoplasmosis. (magnification 1000)
 Chapter 2 Bone Marrow 53

see Seas
oe

Figure 2-21 ™ Bone marrow biopsy specimen from a patient with

metastatic carcinoma shows glandular formation, a morphological
feature of adenocarcinoma. (H&E, magnification x 600)

which increases the cost of the procedure. The processing time of

the tissue also increases, which may not be acceptable if rapid
diagnoses are required. In addition, tissue embedded in plastic
media, instead of paraffin, may not be suitable for immunohisto-
chemical studies of bone marrow.

eA Ie PRT LS A ;
Bone Marrow Examination
The examination of the bone marrow aspirate smears should
start at low magnification with a dry objective of 10. Scan-
ning the slide permits selection of a suitable area for exami-
nation and the differential count. “Bare nuclei” should be
avoided; such nuclei result from destruction of the marrow
cells by squashing or stripping of their cytoplasm by fibrin
threads. An area is selected in which the cells are well spread,
intact, and not diluted by sinusoidal blood. When marrow par- Figure 2-25 ™ Aspirate smear from a patient with metastatic
ticles are examined, such areas are found at the periphery of prostate carcinoma to the bone marrow. Note the cohesive crowded
groups of large neoplastic cells (A) and giandular formation (B) sug-
gesting adenocarcinoma. (Wright-Giemsa, magnification < 600)

Figure 2-22 ™ Immunohistochemical stain for prostate-specific Figure 2-24 @ H&E stained bone marrow biopsy specimen from a
antigen (PSA) performed on a marrow biopsy specimen from the patient with acute lymphoblastic leukemia shows an increased number
same patient as in Figure 2-21. The specimen shows positive stain- of blasts. Note that the blasts are not cohesive and are individually scat-
ing with PSA, thus confirming that the metastatic tumor is from tered. This is a morphological feature of hematolymphoid malignancy.
prostate. (magnification 600) Fine cellular details are lost in biopsy sections. (magnification x 600)
54 Chapter 2. Bone Marrow

Figure 2-26 ™ Smear of normal cellular marrow with normal mat-

uration of erythropoietic, granulocytic, and megakaryocytic cells.
(Wright-Giemsa, magnification 200)
Figure 2-25 ® Wright-Giemsa-stained aspirate smear of acute
lymphoblastic leukemia showing cytoplasmic and nuclear details
useful in the diagnosis of acute leukemias. As compared to the The immediate subcortical region of adult bone marrow is usu-
metastatic tumor cells seen in Figure 2-23, the blasts are not ally hypocellular as compared with the deeper medullary area.
cohesive and scattered. Therefore, sections that contain predominantly subcortical bone
are frequently suboptimal for assessing true marrow cellularity.
the particles. At this low magnification, marrow cellularity is The bone marrow biopsy specimen is most reliable for
also evaluated. The megakaryocytes are usually noted adja- assessment of cellularity, because it offers a large amount of
cent to a spicule, about five to ten per low-power field. Non- tissue for evaluation. However, the evaluation of cellularity
hematopoietic tumor cells infiltrating the bone marrow may can also be done on well-prepared aspirate smears or marrow
also be seen at this magnification. These are usually larger particles. The best area for examination of cellularity in
than the granulocytic or erythropoietic precursors and are smears is the area between two uncrushed particles. The ratio
scattered in small groups and crowded clusters. Some show a of cells to fat is evaluated at low magnification (objective
glandular configuration (see Fig. 2-23). 10X), so that larger areas are included in the field of observa-
After the initial scan, immersion oil is applied to the tion. The empty spaces that result from the spreading of the
slide and the examination continues on high magnification cells but are not occupied by fat cells are disregarded and
(oil immersion objective 50X or 100X). The high magnifica- treated as an artifact. The terms decreased or increased cellu-
tion provides details of the nuclear and cytoplasmic matura- larity are used when fewer or more than the expected normal
tion process (Fig. 2-26). The iron in histiocytes is visualized number of cells are found. Precise evaluation can be achieved
as brown-blue granules. Cytoplasmic inclusions of a diagnos- with experience, and good reproducibility can be attained
tic nature can be seen in histiocytes and granulocytes. Differ- among several observers. The marrow cellularity can be
ential counts of bone marrow are performed with the oil expressed in percentages, but this is best done on histologic
immersion objective. sections of biopsy specimens (Figs. 2-27, 2-28, and 2-29).
Marrow cellularity has diagnostic value when it is related to
Estimation of Bone Marrow Cellularity the M:E ratio, which is calculated after a differential count is
performed.
Cellularity is reflected in the ratio of nucleated hematopoietic It is always important to look for any abnormal changes
cells to fat cells. Bone marrow cellularity normally varies with in the bony trabeculae. Various conditions can alter the mor-
age, and the estimated cellularity must be compared to age- phological appearance of these trabeculae (Fig. 2-30). Marked
related normal ranges. At birth the normal marrow cellularity is thickening of trabeculae (Fig. 2-31) can be seen in myelopro-
100%. Thereafter, the cellularity gradually decreases. Overall liferative disorders (myelofibrosis with myeloid metaplasia),
marrow cellularity in adults is about 50% (+ 10%). The gen- whereas thinning of trabeculae (Fig. 2-32) can be seen in older
eral rule to estimate age-related normal ranges is 100 minus adults, in patients with acquired immunodeficiency syndrome
age + 10. For example, the estimated normal marrow cellular- (AIDS) or other cachectic conditions, and after chronic steroid
ity of a 40-year-old person would be 100-40 = 10 (..e., a range administration. Various metabolic disorders can also alter the
from 50% to 70%). morphology of bony trabeculae. Examples include a mosaic
Cellularity may vary from area to area, and, therefore, pattern, seen in patients with Paget’s disease, and resorption
estimated cellularity should represent the average percentage. If and cyst formation, in persons with hyperparathyroidism and
hypercellular (90%) and hypocellular (10%) areas are seen, this chronic renal failure.
finding should be mentioned descriptively in the report because When lymphoid aggregates are seen in the bone marrow
in such cases an average cellularity may be difficult to estimate. biopsy specimen, the differential diagnosis includes benign
 Chapter 2 Bone Marrow 55

Gee
Sd

Bigs
OF
Yi

sf 2 Steg)
se 52
foe
i
gets&
oad
le
da Masa
— Si ae Ve fad

Figure 2-27 ® Normal bone marrow biopsy specimen from a Figure 2-29 m Hypercellular bone marrow biopsy specimen from
50-year-old patient shows approximately 50% cellularity. (H&E, low a 60-year-old patient with 80% cellular marrow. This patient was
magnification) receiving growth factor therapy. (H&E, low magnification)
.

lymphoid aggregates and malignant lymphoma. Usually benign The fibrosis can be graded as mild, moderate, or severe. In ad-
aggregates are small and well demarcated, nonparatrabecular, dition, a description of the fibrosis as fine or coarse, and
and composed predominantly of small, round lymphocytes with focal or diffuse, may be helpful, especially when following the
plasma cells at the periphery and blood vessels present within improvement of a patient with myelofibrosis after treatment or
the aggregate (see Figs. 2-5 and 2-33). Conversely, malignant bone marrow transplantation. Trichrome stain is usually per-
follicles are usually large with ill-defined borders, paratrabecu- formed to detect any collagenous fibrosis, which if present may
lar, composed of atypical lymphocytes, and lack plasma cells at indicate irreversible fibrosis. Fibrosis and other bone marrow
the periphery (Fig. 2-34). However, a neoplastic lymphoid infil- conditions such as marked hypercellularity in acute leukemias
trate can also be interstitial, diffuse and patchy. In some cases, may result in a “dry tap” when attempting the aspiration proce-
immunohistochemical stains can be performed on the marrow dure. However, if flow cytometry is needed, it is a good practice
core biopsy to differentiate between benign lymphoid aggre- to obtain two bone marrow biopsy samples. One biopsy sample
gates and malignant lymphoma. can be put in formalin and processed for morphological exami-
Bone marrow fibrosis may be found in patients with hairy nation. The other core biopsy sample can be put in saline or
cell leukemia, in myeloproliferative and myelodysplastic syn- RPMI medium; a cell suspension is then made for flow cytomet-
dromes, sometimes in acute leukemia, after radiation, and after ric studies.
toxic injury to the marrow. On routine H&E sections, streaming
of marrow stroma and dilated sinusoids suggests marrow fibro- Bone Marrow Differential Count
sis; this finding can be confirmed and graded by performing reti-
culin and trichrome stains (Fig. 2-35). Normally, occasional A bone marrow differential count is an excellent tool for train-
reticulin-positive fibers may be seen around the blood vessels. ing a novice in bone marrow morphology and is widely used in

2 ‘a
& =

q
:

aN-- ys

Figure 2-28 @ Markedly hypocellular bone marrow biopsy speci- Figure 2-50 ® Bone marrow biopsy specimen showing normal
men from a 20-year-old patient. (H&E, low magnification) bony trabeculae. (H&E, low magnification)
56 Chapter 2 Bone Marrow

Figure 2-35 ™ High-power view of the bone marrow biopsy from a

60-year-old patient shows lymphoid aggregate (low-power view is
seen in Fig. 2-5). Note the presence of blood vessel and plasma cells
Figure 2-51 ® Bone marrow biopsy specimen shows marked thick- at the periphery of the lymphoid aggregate. (H&E, magnification
ening of bony trabeculae (osteosclerosis). (H&E, magnification x 200) x 400)

may be misinterpreted as blasts if the observer is unfamiliar

diagnosing and following up patients with leukemias, refrac- with these characteristics (see Fig. 2-6). In children up to
tory anemias, and myelodysplastic and myeloproliferative 3 years old, one-third or more of the marrow cellularity is made
syndromes. Because of the compartmentalization of the up of lymphocytes. The lymphocyte number gradually declines
hematopoietic cells and high cellularity of marrow, at least to the normal adult level thereafter.
500 to 1000 nucleated cells need to be classified for a repre- In adult marrow the lymphocytes are distributed ran-
sentative differential count. domly among the hematopoietic cells and within lymphoid
In infants during the first month after birth, dramatic alter- follicles. This can introduce significant variation in the dif-
ations occur in the distribution of the different marrow com- ferential count from sample to sample in the same patient.
partments.*° At birth there is a predominance of granulocyte The majority of adult marrow is composed of granulopoietic
precursors, which switches within a month to a predominance and erythropoietic precursors. For the purpose of the differ-
of lymphoid elements. In early infancy many lymphocytes ential count, these are enumerated into different categories
have fine chromatin and a high nuclear-to-cytoplasmic ratio, according to their stage of maturation. When adequate num-
and lack distinct nucleoli. They are called hematogones and bers of cells are tabulated, the percentage of each category is
represent normal lymphoid progenitor cells.'* Hematogones calculated. The ratio between all granulocytes and _ their

Figure 2-34 ™ Bone marrow biopsy specimen shows a paratrabec-

ular lymphoid aggregate. This pattern of infiltration is most likely
Figure 2-52 ™ Bone marrow biopsy specimen shows thinning of indicative of involvement of the marrow by malignant lymphoma.
bony trabeculae (osteopenia). (H&E, magnification « 200) (H&E, magnification « 200)
 Chapter 2 Bone Marrow 57

Bone Marrow and Peripheral Blood

Interpretation Based on Cellularity
and M:E Ratio Changes
A bone marrow aspirate or biopsy sample represents a minute
part of a very large and dynamic organ. Its activity and
responses are reflected in blood changes; therefore, evalua-
tion of the bone marrow should always be done in conjunc-
tion with evaluation of the peripheral blood. In adults with
50% marrow cellularity, about 30% to 40% represents granu-
lopoiesis and 10% to 15% erythropoiesis, with an average
M:E ratio of 4:1. An increase or a decrease in marrow cellu-
larity with the normal M:E ratio usually indicates a balanced
granulocytic and erythrocytic hyperplasia or hypoplasia,
respectively. However, if cellularity changes occur simultane-
Figure 2-355 m Reticulin stain on a marrow biopsy specimen from
a patient with hairy cell leukemia (same patient as Figs. 2-16 and ously with the M:E ratio change, the interpretation requires a
2-17) showing diffuse and severe fibrosis. Dilated sinusoids are broader understanding of hematopoietic tissue physiology
present. As expected, the aspirate was a dry tap. (magnification and its reactions during disease.
x 200) Cell morphology and the M:E ratio are well represented
in random bone marrow specimens. The variations are not
significant even when samples are compared from sternal and
precursors and all nucleated red cell precursors represents iliac crest aspirates.*’ However, marrow cellularity is poorly
the M:E ratio. represented in random smears; thus, this interpretation should
Some hematologists prefer to exclude the segmented be considered with some degree of reservation. Even large
neutrophils from the differential count as being part of the biopsy specimens may have a great degree of variation in
neutrophil storage pool of the marrow. The normal M:E cellularity.** For these reasons, in diseases in which marrow
ratio in this case is between 1.5 and 3. However, patholo- cellularity is crucial for the diagnosis (aplastic anemia, mar-
gists and hematologists who interpret the bone marrow row hypoplasia), more than one bone core biopsy may be
histologic sections of particle clot and biopsies in conjunc- required.
tion with marrow smears include the segmented neutrophils Table 2—4 has been included to provide both a simple
in the differential counts, because these cannot be excluded guide and some basic information to the reader. It cannot
in the evaluation of histologic specimens and are part of the serve as a diagnostic tool without the addition of the
marrow cellularity. The normal M:E ratio then is slightly patient’s clinical history and a clinical evaluation of the dis-
higher and ranges between 2 and 4. The granulopoietic ease. The reader is also cautioned that the variety of prob-
tissue occupies two to four times greater marrow space than lems frequently presented by different patients with the
the erythropoietic precursors, owing to the shorter survival same disease may not fit within such a simple schematic
of the granulocytes in the circulation (1.e., neutrophils, 6 to concept.
10 hours, versus erythrocytes, 120 days). Changes in the
survival of granulocytes and erythrocytes are reflected in Bone Marrow Iron Stores
changes in the M:E ratio.
Megakaryocytes are not included in the differential The storage iron of the bone marrow is in the form of hemo-
count. Megakaryocytes are unevenly distributed, and a siderin. The iron content of hemosiderin is higher than that of
differential count is a poor means for their evaluation. Usu- ferritin. Other components of hemosiderin are protein, ferritin
ally five to ten megakaryocytes are seen per microscopic aggregates, some lipids, and membranes of cellular organelles.
field at low magnification (objective 10). When clusters Hemosiderin can be seen on unstained smears as golden-yellow
of megakaryocytes and promegakaryocytes are seen in granules. On Wright-Giemsa-stained smears it appears as
every field, it is an indication of megakaryocytic hyperpla- brownish-blue granules. However, for more precise evaluation,
sia. In a normocellular marrow, finding fewer than two Prussian blue reaction is used to demonstrate the intracytoplas-
megakaryocytes per field on screening may indicate mic iron of histiocytes and red cell precursors. The evaluation of
megakaryocytic hypoplasia. A marked increase or decrease marrow iron stores is essential in the diagnosis of anemias and
in the number of megakaryocytes is easy to evaluate, especially in refractory and dyserythropoietic anemias. When
whereas slight to moderate changes are difficult to judge the morphological characteristic of the iron particles in the stor-
and are better estimated on histologic sections of biopsy age nutrient histiocyte and erythroblastic precursors is an impor-
and particle specimens. tant diagnostic consideration (e.g., in sideroblastic anemias), an
Table 2-3 represents the data of normal marrow refer- iron stain is performed on a particle smear. If the overall distri-
ence ranges used by the University Hospital and University of bution of the amount of iron is of clinical importance
Texas Health Science Center at San Antonio, Texas. (e.g., iron-deficiency anemia, anemia of chronic diseases,
58 Chapter 2 Bone Marrow

Birth to 1 Month Children Adults

Undifferentiated cells qe 0-1 0-1

Myeloblasts 0-2 0-2
Promyelocytes 0-4 0-4
Myelocytes
Neutrophilic
Eosinophilic
Basophilic
Metamyelocytes and bands
Neutrophilic
Eosinophilic
Basophilic
Segmented neutrophils
Pronormoblasts
Basophilic normoblasts
Polychromatophilic normoblasts
Orthochromatic normoblasts
Lymphocytes
Plasma cells
Monocytes _

*Normal eee ranges on the agian of Texas Health ieee Center and PCa es Hospital, San Antonio, TX.

hemochromatosis, and others), then histologic sections of bone (Fig. 2-36). Hemoglobin iron and dispersed ferritin do not
marrow biopsy sample, and/or marrow aspirate, and marrow stain. Normal marrow iron is seen as fine cytoplasmic gran-
clotted particles are stained for iron. The biopsy sample and the ules within histiocytes, and 30% to 50% of marrow erythrob-
particles are a more reliable source of information, because they lasts contain iron specks within mitochondria and are called
represent a large sample of hematopoietic tissue. Bone marrow sideroblasts. Clumps of iron easily seen at scanning magnifi-
biopsy samples for iron studies should be decalcified by the cation (10) indicate increased iron storage, whereas only a
EDTA chelating method, which does not affect the storage few specks of iron found after searching several microscopic
iron.’ Rapid-acid decalcifying solutions extract iron and must fields (SOX or 100 magnification) indicates decreased iron
not be used in these cases. storage. When no stainable iron is detected on the bone
After Prussian blue staining, hemosiderin and some fer- marrow smear or tissue sections, this indicates iron storage
ritin aggregates are seen as bright blue specks and granules depletion or absence.*” The storage iron may be reported as

Complete VE Count Bone DTATrOW Collalanity | M: E Ratio” Bone Marrow Interpretation

Normal Increased or decreased* Neat Normal

Neutropenia Decreased Decreased Granulocytic hypoplasia
Neutropenia Normal or increased Increased Decreased neutrophilic
survival or ineffective
granulopoiesis
Neutrophilia Normal or increased Increased Granulocytic hyperplasia
Anemia Normal or decreased Increased Red cell hypoplasia
Anemia Normal or increased Decreased Erythrocytic hyperplasia or
ineffective erythropoiesis‘
Erythrocytosis Normal or increased Decreased Erythrocytic hyperplasia
(polycythemia)
Pancytopenia Decreased Normal Marrow hypoplasia
Pancytopenia Increased Normal, increased, Ineffective myelopoiesis or
or decreased hypersplenism
*Because of poor representation of cellularity in random specimen.
‘Reticulocyte count is necessary to differentiate between erythrocytic hyperplasia and ineffective erythropoiesis.
 Chapter 2 Bone Marrow 59

” ® i
8. The diagnostic conclusion. This should encompass sepa-
rate diagnoses of blood and bone marrow even where the
~~ same diagnosis is applicable to both; for example: Blood—
oe pancytopenia, Bone marrow, left posterior iliac spine aspi-
" / x * rate and biopsy—myelodysplastic syndrome, refractory
anemia with ringed sideroblasts; or Blood—acute myeloid
er. leukemia minimally differentiated (WHO classification),
‘ i}
‘ Bone marrow, left posterior iliac crest aspirate and
biopsy—acute myeloid leukemia minimally differentiated
ae
hee (WHO classification).
a % 3
ae % : *
In summary, bone marrow can provide a representative pic-
ture of disease processes and has wide application in clini-
cal medicine.*°*! Marrow examination has a significant role
Sao woe A, in the evaluation of leukemias, lymphomas, plasma cell
Figure 2-56 @ Iron stain of a bone marrow smear shows a marked disorders, myeloproliferative disorders, myelodysplastic
increase in marrow storage iron, thus ruling out the possibility of disorders, myelofibrosis, metastatic tumors, various ane-
iron-deficiency anemia. (magnification <x200) mias, granulomatous diseases, infectious diseases, meta-
bolic diseases, in evaluating the status of engraftment after
bone marrow transplantation, and in assessing chemotherapy
99 66
effects. The clinical laboratory scientist’s contribution in
“absent,” “decreased,” “adequate,” “moderately increased,”
this phase consists of preparing the optimum blood and
and “markedly increased,” or it can be given corresponding bone marrow slides and performing the differential count.
numerical values from 0 to 4, where 2 represents the normal Examination of the blood and bone marrow, correlation
or adequate iron stored in an adult. In children; however, iron
with the clinical presentation, and diagnostic conclusions
is stored mainly in ferritin and does not stain with Prussian on each specimen are the responsibility of a physician who
blue in normal iron states. Some chronic derangements in iron has adequate training and experience to integrate all of the
metabolism (i.e., myelodysplastic syndromes) may result in available clinical and laboratory information in reaching
aberrant ringing of sideroblastic iron around erythroblast the correct diagnosis.
nuclei; these cells are named ringed sideroblasts.

Bone Marrow Report

The bone marrow report usually encompasses the following
(see the case study that follows):

1. The name of the laboratory or physician’s office from

Case Study 1
which the report originates. Patient Name: John Doe
2. The patient’s data, including age and relevant clinical sum- Pathology Services, University Health System
mary or clinical diagnosis. Hospital Number: XXXXXX
3.A description of material received for studies, such as Surgical Pathology Accession Number: SOO—00000
smears of aspirate, marrow particles, and bone biopsy (or Physician: Dr. Z
biopsies).
4. Data from the complete blood count (CBC) and WBC BONE MARROW EXAMINATION GROSS
differential count, and a description of the blood smear, DESCRIPTION
preferably from the day on which the bone marrow speci- Specimen A: Specimen label, patient’s name, and right poste-
men is obtained. A platelet count should be included, as rior iliac crest biopsy. Received in formalin is a 1.2 cm X
well as a reticulocyte count, if available. 0.2 cm X 0.2 cm bone marrow biopsy sample. Entirely
Nn . The bone marrow differential count. submitted after decalcification: block A.
6. A description of cellularity, M:E ratio, granulopoiesis, ery- Specimen B: Specimen label, patient’s name, and right pos-
thropoiesis, and megakaryocytopoiesis. Any change in the terior iliac crest aspirate. Received in EDTA are marrow
nonhematopoietic elements of marrow, such as hemo- particles that aggregate to 0.8 cm X 0.5 cm X 0.2 cm.
Entirely submitted: block B.
phagocytosis, granulomas, microorganisms, metastatic tu-
Aspirate smears: 14 unstained and 4 Wright-stained
mor cells, histiocytic hyperplasia, or the appearance of
samples from the right posterior iliac crest, all labeled with
bony trabeculae, is included in this section of the report. the patient’s name. Touch preparations: 3 unstained and
The status of iron stores and special staining procedures 2 Wright-stained samples from the right posterior iliac crest,
performed are reported. all labeled with patient’s name.
7. A description of histologic sections of marrow particles or continued
bone marrow biopsy.
60 Chapter 2 Bone Marrow

DIAGNOSIS
Case Study Ve tonca
Blood:
Acute lymphoblastic leukemia, precursor B-cell
Also received is bone marrow aspirate in one EDTA tube
(circulating blasts 35%)
for flow cytometry and one sodium heparin tube for cytoge-
Normochromic normocytic anemia, moderate
netic studies.
Thrombocytopenia, marked
Bone Marrow, Right Iliac Crest Aspirate Smears, Aspirate
MICROSCOPIC DESCRIPTION Particle Preparation, and Biopsy:
Peripheral Blood Acute lymphoblastic leukemia, precursor B-cell (blasts 65%,
CBC obtained from University Hospital on 02/22/06 cellularity 95%) (see comment).
reveals:
RBEP See 10Z/5 WBC: 20 X 107/L COMMENT
Hgb: 7.5 g/dL Manual differential
Flow cytometry performed on the bone marrow aspirate re-
Het: 22% Segmented neutrophils: 31%
veals a precursor B-cell phenotype. The blasts have the fol-
MCY: 94.0 fL Bands: 1%
lowing phenotype: CD19, CD10, CD34, CD22, HLA-DR
MCH: 28 pg Lymphocytes: 20%
positive; and CD20, kappa (k), lambda (A) light chain neg-
MCHC: 32 g/dL Monocytes: 2%
ative. Other T-cell and myelomonocytic markers are nega-
RDW: 19.0 Eosinophils: 1%
tive. Cytogenetic studies are pending and an amended report
Platelet count Basophils: 1%
will follow. A call was made and results were given to the
OPO 710 Metamyelocytes: 4%
patient’s physician.
Myelocytes: 3%
Promyelocytes: 2%
Blasts: 35%
Anisocytosis: moderate with occasional microcytes and few
macrocytes.
Poikilocytosis: mild with a few teardrop cells and rare schisto-
cytes.
Polychromasia: mild. NRBCs are present 2/100 WBCs Patient Name: Jane Doe
The white blood cells display a left shift with increased Pathology Services, University Health System
number of circulating blasts. The blasts are of small size, show Hospital Number: XXXXXX
minimal variation, and have high nuclear-to-cytoplasmic ratio Surgical Pathology Accession Number: SOO—00000
and fine and lacy chromatin with occasional small nucleoli. No Physician: Dr. Y
Auer rods are seen. History: 65-year-old woman with persistent pancytopenia and
The platelet count is 5.0 < 10°/L. Few giant platelets are normal vitamin B,, and folate levels.
seen. No platelet clumps are noted.

Bone Marrow BONE MARROW EXAMINATION GROSS

Examination of the bone marrow aspirate smears reveal DESCRIPTION
hypercellular marrow with decreased megakaryocytes. Specimen A: Specimen labeled with patient’s name and
Differential count performed on aspirate smears reveals: right posterior iliac crest biopsy. Received in formalin is a
Blasts: 65% Pronormoblasts: 1% 1.2 cm X 0.2 cm X 0.2 cm bone marrow biopsy sample.
Promyelocytes: 6% Basophilic normoblasts: 4% Entirely submitted after decalcification: block A.
Myelocytes: 5% Polychromatic normoblasts: 4% Specimen B: Specimen labeled with patient’s name and right
Metamyelocytes Orthochromatic
posterior iliac crest aspirate. Received in EDTA are marrow
and bands: 4% normoblasts: 7%
particles that aggregate to 0.8 cm X 0.5 cm X 0.2 cm.
Segmented Monocytes: 1% Entirely submitted: block B.
neutrophils: 2% Lymphocytes: 1%
Aspirate smears: 14 unstained and 4 Wright-stained
Blasts are markedly increased. The morphology of blasts is samples from the right posterior iliac crest, all labeled with
similar to that described in the peripheral smear. Residual the patient’s name. Touch preparations: 3 unstained and
myeloid and erythroid precursors showing progressive mat- 2 Wright-stained samples from the right posterior iliac crest,
uration are seen in the background. The M:E ratio is 5:1. all labelled with patient’s name.
Cytochemical stains performed on the aspirate smears Also received is additional bone marrow aspirate in one
show that the blasts are negative for Sudan black B, sodium heparin tube for cytogenetic studies.
myeloperoxidase, specific and nonspecific esterase, and
periodic acid—Schiff (PAS).
Sections from the right iliac crest biopsy and right parti-
MICROSCOPIC DESCRIPTION
cle preparation reveal hypercellular marrow (cellularity Peripheral Blood
90%) with complete involvement by leukemic blasts. Scant CBC obtained from University Hospital on 06/22/06 reveals:
residual trilineage hematopoiesis is seen in the background. RBC: 3.0 X 10!°/L WBC22 x 107
Bony trabeculae are unremarkable. Hgb: 9.0 g/dL Manual differential
continued
continued
 Chapter 2 Bone Marrow 61]

Het: 27% Segmented neutrophils: 55% progressive maturation to the neutrophil stage. Only few
MCV: 102 fL Blasts 2% blasts are present and no Auer rods are seen in their
MCH: 28 pg Lymphocytes: 30% cytoplasm.
MCHC: 32 g/dL Monocytes: 10% An iron stain performed on the aspirate smear shows
RDW: 19.0 Eosinophils: 2% increased storage iron with many ringed sideroblasts.
Platelet count Basophils: 1% Sections from the right iliac crest biopsy and right parti-
10.0 X 10°/L cle preparation reveal hypercellular marrow (cellularity
There is moderate aniso-poikilocytosis with many macro- 90%) with increased erythroid precursors. Aggregates of
cytic red cells and few target cells. Coarse basophilic stip- blasts are not seen. No lymphoid aggregates or granulomata
pling is present. are seen. Bony trabeculae are unremarkable. Reticulin stain
Polychromasia: minimal of the core biopsy did not show increased fibrosis.
The white blood cells display few circulating blasts (2%).
No hypersegmented neutrophils are seen. The platelet count DIAGNOSIS
is 10.0 x 10°/L. Few large and hypogranular platelets are
seen. No platelet clumps are noted. Blood:
Pancytopenia with macrocytic anemia (circulating
Bone Marrow blasts 2%)
Examination of the bone marrow aspirate smear reveals
hypercellular marrow. Differential count performed on aspi- Bone Marrow, Right Iliac Crest Aspirate Smears, Aspirate
rate smears: Particle Preparation, and Biopsy:
Blasts: 1% Pronormoblasts: 12% Myelodysplastic syndrome, refractory anemia with ringed
Promyelocytes: 8% Basophilic normoblasts: 25% sideroblasts (cellularity 90%; blasts 1%) (see comment).
Myelocytes: 9% Polychromatic normoblasts: 16%
Metamyelocytes Orthochromatic COMMENT
and bands: 4% normoblasts: 7%
The patient’s serum B,, and red cell/serum folate levels are
Segmented Monocytes: 4%
normal. The presence of pancytopenia in conjunction with a
neutrophils: 10% Lymphocytes: 4%
hypercellular marrow, red cell dysplasia, and many ringed
The M:E ratio is 0.51. There is an increase in erythroid pre-
sideroblasts is supportive of a diagnosis of a myelodysplastic
cursors with moderate dyserythropoesis characterized by
syndrome, refractory anemia with ringed sideroblasts subtype.
megaloblastoid changes, nuclear irregularity, binucleation,
Preliminary cytogenetic studies performed on the marrow
and nuclear fragmentation. Megakaryocytes are increased.
aspirate reveal clonal chromosomal abnormalities including
Myeloid precursors are decreased in number and show
monosomy 7, del(7q). Results called to Dr. Y at ————.
continued

Cj axenows
1. The average life spans of red cells, neutrophils, and 4. Occasionally, nonhematopoietic cells are seen in marrow
platelets in the circulation are: aspirates. Which of the following is most likely to be
a. 120 days, 7 days, and 10 days misidentified as a megakaryocyte?
b. 30 days, 7 days, and 10 days a. Reticular cell
c. 120 days, 6-10 hours, and 10 days b. Storage histiocyte
d. 30 days, 6-10 hours, and 10 hours c. Osteoclast
. Bone marrow aspirate smears are more informative than
d. Endothelial cell
tw

the bone core biopsy in the diagnosis and classification of: . If the aspirate is unsuccessful (1.e., a “dry tap”), which
a. Granulomatous diseases alternative processing steps are possible?
b. Acute leukemias a. An extra core biopsy can be obtained and placed in
c. Metastatic carcinomas saline or RPMI for flow cytometric studies.
d. Gaucher’s disease b. Touch preparations of the core biopsy can be used for
Wright—Giemsa morphologic evaluation.
3. The least reliable specimen on which to perform an iron
c. Touch preparations of the core biopsy can be used for
stain 1S:
FISH studies and/or cytochemical stains.
a. Core biopsy
d. All of the above.
b. Clot section
c. Aspirate smears
d. Particle preparation
 Chapter 2. Bone Marrow

6. In early childhood up to one third of the cellularity can 9, Which is/are true for lymphoid aggregates?
be comprised of the following cells: a. Paratrabecular lymphoid aggregates are usually
a. Erythroblasts malignant.
b. Granulocytic precursors b. Benign aggregates are usually nonparatrabecular.
c. Lymphocytes c. Reactive lymphoid aggregates commonly have a
d. Histiocytes—monocytes blood vessel and plasma cells at the periphery.
. When is a bone marrow study not indicated?
d. All of the above.
a. When circulating blasts are seen on the peripheral smear 10.QO The most appropriate site for bone marrow studies in
b. For pancytopenia adults is:
c. In thrombocytopenia with no apparent cause a. Anterior superior iliac crest
d. In iron deficiency anemia b. Posterior superior iliac crest
. Which is true for hematogones? c. Sternum
a. They are malignant cells. d. Tibia
b. They belong to the granulocytic series.
c. Morphologically they mimic blasts of lymphoblastic See answers at the back of this book.
leukemia.
_d. They are most commonly seen in elderly individuals.

~} SUMMARY CHAR mw Among the most common indicators for bone marrow
studies are diseases that affect the bone marrow, causing a
decrease or increase in any of the cellular blood elements.
a The hematopoietic system consists of bone marrow, liver,
spleen, lymph nodes, and thymus. In normal adults, g In adults, the most common site for bone marrow aspira-
hematopoiesis occurs mainly in the bone marrow. tion or biopsy is the posterior superior iliac crest; in new-
borns and infants, bone marrow is obtained from the
g The bone marrow is one of the body’s largest organs, rep-
upper end of the tibia.
resenting 3.4% to 6% of total body weight and averaging
about 1500 g in adults. g Ina bone marrow aspiration, 1.0 to 1.5 mL of marrow par-
ticles and sinusoidal blood is drawn into a syringe; all
w The structure of the bone marrow consists of hematopoi-
direct smears should be prepared quickly from unclotted
etic cells (erythroid, myeloid, lymphoid, and megakary-
specimens and labeled at the bedside with the patient’s
ocytes), adipose tissue, bone and its cells (osteoblasts and
name, identification number, and date.
osteoclasts), and stroma.
# When marrow cannot be aspirated (“dry tap’’), the com-
w Erythropoiesis takes place in distinct anatomic units
mon differential diagnosis includes acute leukemia,
called erythropoietic islands.
myelofibrosis, and hairy cell leukemia.
mGranulocytic precursors are located deep in the
@ In the estimation of marrow cellularity (ratio of nucleated
hematopoietic cords and around the bone trabeculae.
hematopoietic cells to fat cells) low-power magnification
w Megakaryopoiesis occurs adjacent to the sinus endothelium; is used. Overall marrow cellularity in adults is about
megakaryocytes protrude as small cytoplasmic processes 50% + 10%.
through the vascular wall, delivering platelets directly to the
m= When performing the marrow differential count, at least
sinusoidal blood.
500 to 1000 nucleated cells are classified; the oil immer-
mu Lymphocyte production is compartmentalized in lym- sion objective is used, and megakaryocytes are not
phoid follicles, and lymphocytes are randomly dispersed included in the differential count.
throughout the hematopoietic cords.
m The normal M:E (myeloid-to-erythroid) ratio for adults is
m Hematogones are normal cellular constituents of bone 4:1; granulopoietic tissue occupies a marrow space that is
marrow that resemble small-to-intermediate sized lym- two to four times greater than that occupied by the ery-
phocytes; they range in size from 10 to 20 um, have a high thropoietic precursors.
N:C ratio and deeply basophilic cytoplasm, and are
@ The storage form of iron in the bone marrow is hemosiderin.
devoid of any granules or vacuoles.
On Wright-stained smears, iron appears as brownish-blue
m= The meshwork of stromal cells in which the hematopoi- granules. Decalcification leaches iron from the tissue and
etic cells are suspended is in a delicate semifluid state and may result in an erroneous interpretation of absent iron
is composed of reticulum cells, histiocytes, fat cells, and stores in the core biopsy.
endothelial cells.
 Chapter 2 Bone Marrow 63

REFERENCES . Kobayashi, SD, et al: The transient Sul, Brynes, RK, et al: Bone marrow aspira-
i. Bloom, W, and Fawcett, DW: A Text- appearance of small blastoid cells in the tion and trephine biopsy, an approach to
marrow after bone marrow transplanta- a thorough study. Am J Clin Pathol
book of Histology, ed 10. WB Saunders,
tion. Am J Clin Pathol 96:191, 1991. LOTOS OVS:
Philadelphia, 1975, pp 204-232.
16. Gordon, MY: Annotation. Extracellular
2) By. Humphries, JE: Dry tap bone marrow
. Tavassoli, M, and Jossey, JM: Bone
matrix of the marrow microenvironment. aspiration: Clinical significance.
marrow, structure and function.
Br J Haematol 70:1, 1988. Am J Hematol 35:247, 1990.
Alan R. Liss, New York, 1978, p 43.
. Hutchinson, RM: Mastocytosis and WwWWw. Carson, FL: Histotechnology—A Self
. Lichtman, MA: The ultrastructure of the
co-existent non-Hodgkin’s lymphoma Instructional Text. ASCP Press, Chicago,
hemopoietic environment of the marrow:
and myeloproliferative disorders. Leuk 1990, pp 10, 38.
A review. Exp Hematol 9:391, 1981.
Lymphoma 7:29, 1992. . Wilkins, BS, and O’Brien, CJ: Tech-
. Tavassoli, M, and Shaklai, M: Absence of
18. Travis, WD, et al: Systemic mast cell niques for obtaining differential cell
tight junctions in endothelium of marrow
diseases. Analysis of 58 cases and litera- counts from bone marrow trephine
sinuses: Possible significance for marrow
ture review. Medicine 67:345, 1988. biopsy specimens. J Clin Pathol 41:558,
cell egress. Erythropoiesis in bone
. Clark, SC, and Kamen, R: The human 1988.
marrow. Br JHaematol 41:303, 1979.
hematopoietic colony-stimulating . Williams, WJ, and Douglas, NA: Exami-
. Aoki, M, and Tavassoli, M: Dynamics of
factors. Science 236:1229, 1987. nation of the marrow. In Beutler, E, et al
red cell egress from bone marrow after
. Neinhuis, AW: Hematopoietic growth (Eds): Williams Hematology, ed 5.
blood letting. Br JHaematol 49:337,
factors. Biologic complexity and clinical McGraw-Hill, New York, 1995, p 18.
1981.
practice. N Engl J Med 318:916, 1988. . Rosse, C, et al: Bone marrow cell popu-
. Aoki, M, and Tavassoli, M: Red cell
. Ogawa, M: Differentiation and prolifera- lation of normal infants: The predomi-
egress from bone marrow in state of
tion of hematopoietic stem cells. Blood nance of lymphocytes. J Lab Clin Med
transfusion plethora. Exp Hematol 9:231,
81:2844, 1993. SENS), WONT.
1981.
. Erslev, AJ: Humoral regulation of red ove Rubinstein, MA: Aspiration of bone
. Gulati, GL, et al: Structure and function
cell production. Blood 8:349, 1953. marrow from iliac crest comparison of
of the bone marrow and hematopoiesis. iliac crest and sternal bone marrow
. Erslev, AJ: Erythropoietin coming of
Hematol Oncol Clin North Am 2:495, age. N Engl J Med 316:101, 1987. studies. JAMA 137:1821, 1948.
1988. . Hartsock, RJ, et al: Normal variations
. Lichtman, MA, et al: Williams Hematol-
oe). Western, H, and Bainton, DF: Association
ogy, ed 7, McGraw-Hill, New York, 2006 with aging of the amount of hematopoi-
of alkaline-phosphatase-positive reticu- etic issue in bone marrow from anterior
. Becker, RP, and de Bruyn, PPH: The
lum cells in bone marrow with granulo-
transmural passage of blood cells into iliac crest. Am J Clin Pathol 43:326,
cytic precursors. J Exp Med 150:919,
myeloid sinusoids and the entry of 1965.
1979. platelets into the sinusoidal circulation. 3). Lee, GR, et al (Eds): Microcytosis and
. Lichtman, MA, et al: Parasinusoidal
A scanning electron microscopic investi- the anemias associated with impaired
___location of megakaryocytes in marrow: gation. Am J Anat 145:183, 1976. hemoglobin synthesis. In Wintrobe’s
A determinant of platelet release. 26. de Bruyn, P, et al: The migration of blood Clinical Hematology, ed 9, vol I, Lea &
Am J Hematol 4:303, 1978. cells of the bone marrow through the Febiger, Philadelphia, 1993, pp 799-800.
. Claman, HN, et al: Immunocompetence sinusoidal wall. J Morphol 133:417, 1971. A0. Hyun, BH, et al: Bone marrow examina-
of transferred thymus-marrow cell com- . Arinkin, MJ: Intravitale Unter- tion: Techniques and interpretation.
binations. J Immunol 97:828, 1966. suchungsmethodik der Knochenmarks. Hematol Oncol Clin North Am 2:513,
. Hassett, JM: Humoral immunodefi- Folia Haematol (Leipz) 38:233, 1929. 1988.
ciency: A review. Pediatr Ann 16:404, . Jamshidi, K, and Swaim, WR: Bone 4]. De Wolf Peeters, C: Bone marrow
1987. marrow biopsy with unaltered architec- trephine interpretation: Diagnostic utility
. Caldwell, CW, et al: B-cell precursors ture. A new biopsy device. J Lab and potential pitfalls. Histopathology
in normal pediatric bone marrow. Clin Med 77:335, 1971. 18:489, 1991.
Am J Clin Pathol 95:816, 1991. I), Izadi, P, et al: Comparison of buffy coat
. Longacre, TA, et al: Hematogones: A preparation to direct method for the See Bibliography at the back of this book.
multiparameter analysis of bone marrow evaluation and interpretation of bone
precursor cells. Blood 73:432, 1989. marrow aspirates. Am J Hematol
. Van den Doel, LJ, et al: Immunological 43:107, 1993.
phenotype of lymphoid cells in regenerat . Rywlin, AM, et al: A simple technique
ing marrow of children after treatment for for the preparation of bone marrow
acute lymphoblastic leukemia. smears and sections. Am J Clin Pathol
Eur J Haematol 41:170, 1988. 53:389, 1970.
 Chapter |

The Red Blood Cell

Structure and Function
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)

Introduction OBJECTIVES
The Red Blood Cell At the end of this chapter, the learner should be able to:
Membrane
Red Blood Cell Membrane Le List the areas of red cell metabolism that are crucial to normal red cell survival and
Proteins function.
Deformability . Describe the chemical composition of the red cell membrane in terms of percentage
in)
Permeability of lipids, proteins, and carbohydrates.
Red Blood Cell Membrane
Lipids . List the two most important red blood cell (RBC) membrane proteins and describe
their function and the characteristics of deformability and permeability.
Hemoglobin Structure and
Function . List the abnormalities that may lead to a change in RBC structure and name the asso-
Hemoglobin Synthesis ciated RBC morphology.
Abnormal Hemoglobins of
. List the major lipid components of the red cell membrane.
Clinical Importance
Maintenance of Hemoglobin . Define the primary function of hemoglobin.
Function: Active Red Blood . List the criteria for normal hemoglobin synthesis.
Cell Metabolic Pathways
. List the structural components of normal hemoglobin.
Erythrocyte Senescence and
Hemolysis Wa
en
S). Describe hemoglobin function.
(e9
Ney

Extravascular Hemolysis . Describe the assembly of the protoporphyrin ring.

Intravascular Hemolysis
. List the globin chains found in HbA, HbA,, and HbF, and their respective concentra-
tions (%) found in vivo.

. Describe hemoglobin function in terms of the oxygen dissociation curve.

. Define “shift to the left” in relation to the hemoglobin—oxygen dissociation curve.

. Define “shift to the right” in relation to the hemoglobin—oxygen dissociation curve.

. Define P;, and state normal in vivo levels.

. Define the toxic levels for abnormal hemoglobins of clinical importance.

. List the various metabolic pathways involved in red cell metabolism, stating the
specific function of each one.
. List the steps in the extravascular breakdown of senescent RBCs.

. List the steps in the intravascular breakdown of senescent RBCs.

. List the proteins that carry the following components in circulation: iron, hemoelo-
bin dimers, metheme, and bilirubin.

64
 Chapter 3 The Red Blood Cell: Structure and Function 65

Introduction
Three areas of red blood cell (RBC) metabolism are crucial
for normal erythrocyte survival and function: the RBC mem-
brane, hemoglobin structure and function, and metabolic
pathways (Table 3-1). Defects or problems associated with
any of these areas will result in impaired RBC survival. A
thorough working knowledge of these areas of RBC physiol-
ogy will ensure basic understanding of the various complex
erythrocyte functions.

The Red Blood Cell Membrane

The RBC membrane viewed by transmission electron
microscopy (TEM) appears as a trilaminar structure consist-
ing of a dark—light-dark band arrangement of layers
(Fig. 3-1). These layers represent:

1. An outer hydrophilic portion chemically composed of gly-

colipid, glycoprotein, and protein
2. A central hydrophobic layer containing protein, choles-
terol, and phospholipid
3. An inner hydrophilic layer containing protein

The RBC membrane is highly elastic, responds rapidly to Figure 5-1 ™ Transmission electron microscopy (TEM) of plasma
membrane.
applied stresses of fluid forces, and is capable of undergoing
large membrane extensions without fragmentation.
The RBC membrane represents a highly complex struc- Red Blood Cell Membrane Proteins
ture consisting of a semipermeable lipid bilayer supported by
a meshlike cytoskeleton structure’ (Fig. 3-2). The RBC In early studies the RBC membrane proteins’ nomenclature was
membrane cytoskeleton is a network of proteins on the inner based on their migration rate on sodium dodecyl] sulfate (SDS)
surface of the plasma membrane that is responsible for main- gels and labeled, for example, as bands | through 8.° Today,
taining the shape, stability, and deformability or flexibility of with the use of the high-performance mass spectrometers and
the RBC.' The fluid lipid matrix contains equal amounts of sophisticated data analysis methods, a total of 340 membrane
cholesterol! and phospholipids with a mosaic of proteins proteins and 252 soluble proteins have been identified.° These
interdispersed throughout at various intervals.* The proteins proteins have been categorized on the basis of their subcellular
that extend from the outer surface and traverse the entire localization, protein family they belong to, and their function.°
membrane to the inner cytoplasmic side of the RBC are Of the 340 membrane proteins identified, 31% were
termed integral membrane proteins.* The other major class of classified as integral membrane proteins, 16% as membrane-
RBC membrane proteins, called peripheral membrane pro- associated or bound, 1% as glycosyl phosphatidylinositol
teins, is limited to the cytoplasmic surface of the membrane, (GPI)-anchored, 12% as cytoskeletal, 6% as organelle pro-
which is beneath the lipid bilayer and forms the RBC teins, 12% as cytoplasmic, and 6% as extracellular. For 16%
cytoskeleton.* Both the protein and the lipids are organized of the proteins, the subcellular localization was not available
asymmetrically within the RBC membrane.’ The chemical com- (Table 3-2).
position of the membrane mass is approximately 40% lipids, Many proteins are associated with the outer layer of the
52% proteins, and 8% carbohydrates. ! RBC membrane through a posttranslationally added GPI
anchor.’ The functional significance of this type of protein link-
age is unclear. It is known that this linkage can increase lateral
mobility of proteins migrating to the cell surface and reinsert-
Table - ‘Areas ofRedCell =
if
ing into the RBC membrane.’ It has been postulated that this
linkage may also be involved in cell signaling.’ The membrane-
: _ Metabolism Important. in
associated, GPI-anchored and cytoskeletal proteins are consid-
Normal RBC Survival and ered “peripheral” proteins. Extracellular proteins are outside
n ‘Function : e the RBC (extracellular in origin) but remain associated with the
RBC membrane. Proteins have also been recently categorized
¢ RBC membrane
according to molecular function and biological process.° The
¢ Hemoglobin structure and function
* RBC metabolic pathways majority of the proteins (34%) are involved in binding, or pos-
sess a catalytic activity (29%). Fourteen percent are transporter
66 Chapter 3. The Red Blood Cell: Structure and Function

| = integral proteins
Spectrin P = peripheral proteins
ankyrin-band 3
Phospholipids interaction

Fatty acid
chains Membrane
surface

Lipid
bilayer

Membrane
eS
>. ‘‘
L
cytoskeleton
Adducin Protein 4.1
)Spectrin dimer-dimer
Al Spectrin- | Ankyrin > interaction
Spectrin, Beta chain actin-4.1-adducin *----=-==<==
interaction

Figure 3-2 ® Schematic illustration of red blood cell membrane depicting the composition and arrangement of red cell membrane proteins.
GP-A = glycophorin A; GP-B = glycophorin B; GP-C = glycophorin C. Numbers refer to pattern of migration of sodium dodecyl-polyacrylamide
gel pattern stained with Coomassie brilliant blue. Relations of proteins to each other and to lipids are purely hypothetical positions of the
proteins relative to the inside or outside of the lipid bilayer are accurate. (Note: proteins are not drawn to scale, and are omitted.)

proteins, 9% are signal-transducer proteins, and 7% of the pro- Glycophorin is the principal RBC glycoprotein, repre-
teins are involved in structural activity.° senting approximately 20% of the total membrane protein.°
In terms of biological processes, 51% of the proteins The molecule contains approximately 60% carbohydrate and
identified are involved in cellular processes, 50% in physio- accounts for most of the membrane sialic acid, which gives the
logic, and 11% in regulatory processes. Six percent of the pro- erythrocytes their negative charge. As a result, RBCs repel
teins identified were found to be involved in development of each other as they move through the circulation. Glycophorin,
the RBC and possibly differentiation.° Regulatory mecha- similar to other integral membrane proteins, spans the entire
nisms in the RBC include regulation of cellular and physio- thickness of the lipid bilayer and appears on the external sur-
logic processes as well as enzyme activities. Because RBCs face of the RBC membrane, accounting for the ___location of
differentiate and mature during development, not all proteins many RBC antigens.*” Most of these proteins, as mentioned
are present in their active form.® Degradation of organelles previously, carry RBC antigens and are receptors (such as the
occurs during certain stages of maturation. In addition, during glycophorins) or transport proteins (such as the anion-
the 120-day life span of the mature RBC, chemical and enzy- exchange channel glycoprotein). The anion-exchange channel
matic modifications occur.® comprises approximately 30% of the RBC membrane pro-
Two of the most important protein constituents of the RBC tein.© The plasma membrane envelope is anchored to the RBC
membrane include glycophorin, an integral membrane protein, cytoskeleton network of proteins through tethering sites of in-
and spectrin, a peripheral membrane protein. Table 3—3 lists just tegral (transmembrane) proteins located in the lipid bilayer.*
a few of the integral and peripheral membrane proteins. The condensed fluid lipid bilayer plus integral (transmembrane)
proteins chemically isolates and regulates the cell interior.
The RBC cytoskeleton network of proteins provides rigid

Integral membrane proteins

Membrane-associated or bound
Cytoskeletal proteins Integral Proteins Peripheral Proteins
Cytoplasmic proteins
Organelle proteins Glycophorin A
GPI-anchored proteins Glycophorin B Actin (band 5)
Extracellular proteins Glycophorin C Ankyrin (band 2.1)
Localization unavailable Glycophorin D Band 4.1 and 4.2
Anion exchange-channel Adducin
GPI = glycosyl phosphatidylinositol. Protein (band 3) p55
 Chapter 3 The Red Blood Cell: Structure and Function 67

support and stability to the lipid bilayer and is responsible for by a protein kinase present in the membrane, which is energy-
the deformability properties of the RBC membrane. It is spec- dependent, being catalyzed by adenosine triphosphate (ATP).'*
ulated that the anion-exchange channel and the glycophorins A database of all RBC proteins identified to date can be found
play a major role in anchoring the RBC membrane cytoskele- at http://proteins.biochem.mpg.de/RBC.
ton to the lipid bilayer.*'° As a result, lateral mobility of these As mentioned earlier, the normal chemical composition,
integral proteins within the lipid bilayer is relatively restricted.’ structural arrangement, and molecular interactions of the ery-
The major components of the red cell cytoskeleton throcyte membrane are crucial to normal RBC survival. In
include spectrin, ankyrin, actin, adducin, and other cytoskele- addition, they play a critical role in two important RBC char-
tal proteins.'' Spectrin is clearly the most abundant peripheral acteristics: deformability and permeability. Table 3-4 lists
protein of the RBC membrane cytoskeleton, comprising characteristics of some major components of the red cell
approximately 25% to 30% of the total membrane protein and membrane.
75% of the cytoskeleton.!”
Spectrin, a flexible rodlike molecule, is composed of a
Deformability
helix of two polypeptide chains, an alpha (a) chain (molecular
weight 240,000 d) and a beta (8) chain (molecular weight RBC membrane deformability or flexibility is critical not only
225,000 d).'° These chains intertwined side to side form het- to RBC survival as the cell travels through the microvascula-
erodimers, which link together with other aB chains to form ture, but also for its function of oxygen delivery.'° Decreased
(af), tetramers.'* Spectrin is an important factor in RBC mem- cellular deformability and red cell shape changes have been
brane integrity, because it binds with other peripheral proteins recognized as distinguishing features of a number of congen-
such as actin, ankyrin, adducin, and other cytoskeletal proteins to ital or hereditary hemolytic anemias leading to decreased
form a skeletal network of microfilaments on the inner surface of RBC survival in these disorders (see Chaps. 9 and 10).'°'*
the RBC membrane (see Fig. 3-2). These microfilaments It has already been mentioned that a loss of ATP (energy)
strengthen the membrane, protecting the cell from being broken levels leads to a decrease in the phosphorylation of spectrin
by circulatory shear forces, and also control the biconcave shape and, in turn, to a loss of membrane deformability. An accumu-
and deformability of the cell. In addition, the cytoskeletal net- lation or increase in deposition of membrane calcium also
work provides stability to the lipid bilayer interface.’ results, causing an increase in membrane rigidity and loss of
Spectrin complexes tie the RBC cytoskeleton network pliability.'? These cells are at a marked disadvantage when
together and stabilize the spectrin—actin junction.'* The they pass through the small (3- to 5-um diameter) sinusoidal
preservation of the spectrin complexes, and thus the integrity orifices of the spleen, one of the functions of which is
of the RBC membrane, requires phosphorylation of spectrin extravascular sequestration and removal of aged, damaged,

f the Red Cel

Protein Size Function

Spectrin* 0.1-um heterodimeric filamentous protein Principal structural element of RBC membrane,
consisting of a 240-kD o chain and a which plays a major role in the RBC cytoskeleton
225-kD B chain constitutes 25%-30% of membrane organization
the mass of membrane proteins
Ankyrin* 210-kD globular protein composed of Primary determinant of mechanical coupling of
1879 amino acids the lipid bilayer to the membrane skeleton
Adducin* Protein doublet of approximately 97 and 103 kD Promotes the association of spectrin with
F-actin in a manner similar to band 4.1
Band 4.1* Composed of 622 amino acids, constitutes Dual functions: promotes high-affinity
5% of the mass of membrane proteins association between spectrin and F-actin and
may link the skeleton to the membrane by virtue of
its associations with glycophorin and band 3
Major RBC transmembrane protein and Functions: catalyzes chloride—bicarbonate exchange;
major integral protein that has two distinct contains binding sites for ankyrin, band 4.1, band 4.2,
domains: the transmembrane and cytoplasmic; and several glycolytic enzymes; plays a role in
911-amino acid protein, comprising 15%-20% recognition and removal of normal senescent RBCs
of the total membrane protein

Band 4.2 Known to associate with the cytoplasmic Actual function unknown: however, a deficiency
___domain of band 3 of the protein is associated with several types of
inherited hemolytic anemias

*Denotes major components of the red cell cytoskeleton.

Source: Data from Mohandes, N, and Chasis, JA: Red blood cell deformity, membrane material properties and shape: Regulation by transmem-
brane, skeletal and cytoplasmic proteins and lipids. Semin Hematol 30:171, 1993; and Cohen, CM, and Gascard, P: Regulation and post-translated
modification of erythrocyte membrane and membrane-skeletal proteins. Semin Hematol 29:244, 1992.
68 Chapter 3. The Red Blood Cell: Structure and Function

or less deformable RBCs or fragments of their membranes

(Fig. 3-3). The loss of RBC membrane is exemplified by the
formation of spherocytes (Fig. 3-4), cells with a reduced
surface-to-volume ratio, and the so-called bite cells (Fig. 3-5),
in which the removal of a portion of membrane has left a per-
manent indentation in the remaining cell membrane. The
survival time of these forms is also shortened.
Shape change from the normal symmetric and resilient dis-
coid shape of the RBC can be stimulated by a variety of factors,
which include both mechanically induced and chemically
induced forces.'° Spectrin molecules exist in a folded conforma-
tion in the membrane of the nondeformed red cell. During
reversible membrane deformability, certain spectrin molecules
become uncoiled and extended, whereas others become more
compressed and folded, resulting in a rearrangement of
Figure 5-4 ™ Spherocytes.
the cytoskeletal network.*? This reversible RBC membrane
deformability results in a shape change while maintaining a con-
stant surface area. The limit to reversible RBC membrane maintains its volume and water homeostasis.** Normally, K*
deformability occurs when applied forces break the protein- is found primarily inside the red cell and Na* is found outside
to-protein associations, necessitating an increase in surface the RBC. The erythrocyte intracellular-to-extracellular ratio
area.*! RBC membrane deformability is characterized as irre- for sodium is 1:12, and for potassium it is 25:1.'° The passive
versible when red cells are exposed to forces great enough to influx of sodium and potassium is controlled by as many as
require an increase in surface area, leading to membrane frag- 300 cationic pumps that actively transport sodium out of the
mentation and instability of the cytoskeleton network.’! cell and potassium into the cell. Like other cationic pumps,
these sodium—potassium pumps are energy-dependent, requir-
Permeability ing ATP. The functional active transport of these particular
cations by these cationic pumps also requires the membrane
The RBC membrane is freely permeable to water and anions; enzyme sodium—potassium ATPase. Calcium (Ca**) is also
chloride (Cl) and bicarbonate (HCO, ) traverse the membrane actively pumped from the interior of the RBC and into
in less than a second. It is speculated that this massive the plasma through the energy-dependent calcium-ATPase
exchange of bicarbonate and chloride ions occurs through a cationic pump. Calmodulin, a cytoplasmic calcium-binding
large number of anion-exchange channels formed by the protein that contains four high-affinity calcium binding sites,
integral membrane proteins.” In contrast, the RBC membrane is speculated to control these calcium-ATPase pumps.'? When
is relatively impermeable to cations, with a_ half-time calcium—calmodulin complexes form, the calcium-ATPase
exchange of sodium (Na*) and potassium (K*) of more than pump is activated, preventing excessive intracellular calcium
30 hours.** It is primarily through the control of the sodium buildup, which is deleterious to the RBC, resulting in shape
and potassium intracellular concentrations that the RBC changes and loss of deformability.'°

Figure 5-3 @ Scanning electron micrograph (SEM) of red cells

(3 to 6) squeezing through fenestrated wall in transit from splenic
cords to sinus. Epithelial linings of sinus wall to which platelets (P)
adhere, along with “hairy” white cells (W), probably macrophages,
are shown. (From Weiss, L: A scanning electron microscopic study
of the spleen. Blood 43:665, 1974, with permission.) Figure 3-5 m Bite cells.
 Chapter 3. The Red Blood Cell: Structure and Function 69

The permeability properties of the RBC membrane, as GLYCOLIPIDS AND CHOLESTEROL

well as active cation transport, are crucial to the prevention of Most of the glycolipids are located in the outer half of the
colloid osmotic hemolysis and controlling the volume of the lipid bilayer and interact with glycoproteins to form many of
RBC.™ In addition, ATP-depleted cells allow the accumula- the RBC antigens.*° Cholesterol is approximately equally
tion of excess intracellular calcium and sodium followed by distributed, being located on both sides of the lipid bilayer
potassium and water loss. This results in a dehydrated, rigid inserted between the choline and amino phospholipids. Cho-
RBC which is subsequently sequestered by the spleen. The lesterol comprises 25% of the RBC membrane lipid and is
energy required for active transport and maintenance of mem- present in a 1:1 molar ratio with phospholipids. RBC mem-
brane electrochemical gradients is provided by ATP. Any brane cholesterol is in continual exchange with plasma cho-
abnormality that increases membrane permeability or alters lesterol and is, therefore, affected by changes in body lipid
cationic transport may lead to a decrease in RBC survival. In transport.
general, transport in RBCs is known to be diverse; it involves As mentioned, accumulation of cholesterol results in
not only proteins, ions, and fluids, but also carbohydrates, a more viscous membrane with subsequent morphological
gases, and other substrates.° changes in the RBCs such as target cells (Fig. 3-6), and may
cause RBC membrane damage. Acanthocytes, RBCs with
irregular, spiny projections called spicules (Fig. 3-7), have also
Red Blood Cell Membrane Lipids been associated with an excess accumulation of membrane
PHOSPHOLIPIDS cholesterol in association with liver disease and inherited lipid
The erythrocyte membrane lipid consists of a bilayer of phos- disorders such as abetalipoproteinemia and lecithin—cholesterol
pholipids interspersed with molecules of unesterified choles- acyltransferase deficiency (LCAT).*’** The former disorder is
terol that are present in nearly equimolar quantities. Free fatty caused by a decrease in apolipoprotein B (Apo B) in the blood
acids and glycolipids are present in small quantities. Two with subsequent decreases in plasma and membrane lipids.”
groups of phospholipids, choline phospholipids and amino The patient’s red cells appear as acanthocytes with increased
phospholipids, are known to possess a distinct asymmetry
within the bilayer matrix of the RBC. The asymmetry allows
selective movement of molecules into and out of the cell
membrane.
Choline phospholipids, consisting of phosphatidylcholine
and sphingomyelin, are located primarily on the outside half of
the lipid bilayer, readily accessible to the external environ-
ment.'> Because of their outward orientation in the lipid
bilayer, the choline phospholipids may represent controlling
points in the major pathways of lipid renewal, because there
is an exchange between plasma fatty acids and the RBC
membrane. Fatty acids are incorporated through an energy-
dependent process into membrane phospholipids. Therefore,
changes in body lipid transport and metabolism may cause
abnormalities in the plasma phospholipid concentration that
atts
may alter the RBC membrane composition, resulting in a
decreased RBC survival in circulation. In addition, choles- Figure 3-6 @ Target cells.
terol has a pronounced effect on membrane fluidity.
Membranes containing excess cholesterol are more viscous
and less fluidic. This leads to a decrease in membrane
deformability and RBC survival.
In contrast, amino phospholipids, consisting of phos-
phatidylethanolamine and phosphatidylserine, are located
almost exclusively on the inside half or cytoplasmic side of the
RBC membrane along with phosphatidylinositol. The specific
orientation of these phospholipids maintains a precise lipid
pattern that is critical to normal RBC survival in circulation.
Alteration of this arrangement, leading to the abnormal
appearance of these amino phospholipids on the outer surface
of the lipid bilayer, promotes activation of the clotting cascade
and may result in extravascular hemolysis. Stabilization of this
phospholipid asymmetry in the erythrocyte membrane is
maintained through the interaction with specific peripheral
membrane proteins.” Figure 3-7 @ Acanthocytes.
70 Chapter 3 The Red Blood Cell: Structure and Function

membrane rigidity. LCAT deficiency results in a reduction of Polypeptide

chains
plasma cholesterol esters and increased free cholesterol in the
plasma.** Target cell formation results from the subsequent
increase in membrane cholesterol. All of these RBCs have a B chains
(146 a.a.)
decreased survival rate because the excess lipid makes the
cell membrane less deformable.
The abnormalities that can lead to a change in RBC
morphology are summarized in Table 3-5.

Hemoglobin Structure and Function

Hemoglobin, a conjugated globular protein with a molecular
mass of approximately 64.4 kilodaltons (kD), constitutes 95%
of the RBC dry weight, or 33% of the RBC weight by volume.” o chains ©
Approximately 65% of the hemoglobin synthesis occurs during (141 aa)
Heme
the nucleated stages of RBC maturation, and 35% occurs during
(protoporphyrin + iron)
the reticulocyte stage.'° Normal hemoglobin consists of globin
(a tetramer of two pairs of globin polypeptide chains) and four Figure 3-8 @ Hemoglobin A molecule comprised of two alpha,
heme groups, each of which contains a protoporphyrin ring plus two beta, and four iron-containing heme groups. Beta chains have
ferrous iron (Fe**). See Figure 3-8. 146 amino acids and alpha chains have 141 amino acids.

Hemoglobin Synthesis body iron supply is bound to heme in the hemoglobin mole-
cule (see Chap. 6 for a discussion of iron kinetics).
Normal hemoglobin production is dependent on three processes
(Fig. 3-9): SYNTHESIS OF PROTOPORPHYRINS
1. Adequate iron delivery and supply Protoporphyrin synthesis begins in the mitochondria with the
2. Adequate synthesis of protoporphyrins (the precursor of formation of delta-aminolevulinic acid (SALA) from the amino
heme) and acid, glycine, and succinyl coenzyme A (CoA), which is the
3. Adequate globin synthesis major rate-limiting step in heme biosynthesis (Fig. 3—10).*! The
mitochondrial enzyme, 5ALA synthetase, which mediates this
IRON DELIVERY AND SUPPLY reaction, is influenced by erythropoietin and requires the pres-
Iron, in the ferric state (Fe**), is delivered to the membrane of ence of the cofactor pyridoxal phosphate (vitamin B,).*!
the RBC precursor by the protein carrier transferrin. Most of Porphyrinogens, not porphyrins, are the intermediates of
the iron that crosses the membrane and enters the cytoplasm heme synthesis. Porphyrinogens are unstable tetrapyrroles
of the cell is committed to hemoglobin synthesis and thereby that are readily and irreversibly oxidized to form porphyrins.
proceeds to the mitochondria (where ferric iron is reduced to In contrast, porphyrins are highly stable resonating molecules
ferrous [Fe**] iron for insertion into the protoporphyrin ring to that are normally found in small quantities in the urine as a
form heme). Excess iron in the cytoplasm aggregates as fer- result of normal RBC catabolism.*°
ritin, the amount of which depends on the ratio between the Excessive formation of porphyrins can occur if any one
level of plasma iron and the amount of iron required by the of the normal enzymatic steps in heme synthesis is blocked
erythrocyte for hemoglobin synthesis. Two-thirds of the total and can result in one of a number of metabolic disorders col-
lectively called the porphyrias.**-™ (See Chap. 6.)

GLOBIN SYNTHESIS
Globin chain synthesis occurs on RBC-specific cytoplas-
mic ribosomes, which are initiated from the inheritance of
Vicenaea various structural genes. Each gene results in the formation
of a specific polypeptide chain. Each somatic diploid cell,
Abnormality Be Morpholosy. including the RBC, contains four alpha (a), two zeta (oy
Cholesterol Re nntaton in the RBC” Target cells two beta (B), two delta (5), two epsilon (€), and four gamma
membrane (i.e., liver disease) (y) genes.'° The a and ¢ genes are located on chromosome
Abetalipoproteinemia with Acanthocytes 16, and the B, 5, €, and y genes on chromosome 11
cholesterol accumulation (Fig. 3-11). The resulting gene products formed have been
LCAT deficiency with Hemolysis with red
called a, C, B, 8, € and y globin chains. Throughout embry-
cholesterol accumulation cell fragmentation
Decreased phosphorylated Bite cells and onic and fetal development, activation of the globin genes
spectrin or altered spectrin spherocytes progresses from the ¢ to the a gene and from the € to the y,
5, and B genes.*>
 Chapter 3 The Red Blood Cell: Structure and Function 7\

Polyribosomes

Hemoglobin (oB)2
Globin

PORPHYRIN SYNTHESIS Ferritin

», Porphobilinogen->
URO > COPRO
Transferrin

Figure 3-9 = Hemoglobin synthesis in the reticulocyte. 8-ALA = delta-aminolevulinic acid; URO = uroporphyrinogen; COPRO = copropor-
phyrinogen; PROTO = protoporphyrin.

HEMOPROTEINS

Tricarboxylic
acid cycle
Succinyl-CoA
+

Glycine
BgPO, ALA-

synthase
i
HOOC-CH,—CH,-C=CH,NH>
6-Aminolevulinate 5 Proto IX

Porphobilinogen CYTOSOL
synthase
COOH IX |p
HOOC CH,
HC CH A HC Koon 5
Uro’gen Uro’gen Uro’gen
eee SE SET ETTTSESS Sa ea ae eee
synthase cosynthase Pp P decarboxylase Pp
ye H HC CH
HoN
P A P M
Uro’gen III Copro’gen III

Figure 3-10 m Synthesis of heme. The heme biosynthetic pathway, showing the distribution of enzymes between the mitochondria and the
cytoplasm. Condensation of glycine and succinyl coenzyme A yields SALA, which is irreversible; two molecules of ALA undergo condensation
by the enzyme ALA dehydrase to yield porphobilinogen (PBG). In the presence of uroporphyrinogen III cosynthase and uroporphyrinogen |
synthase, PBG yields uroporphyrinogen Ill. Uroporphyrinogen Ill undergoes four decarboxylation steps, catalyzed by the enzyme uropor-
phyrinogen decarboxylase, to yield coproporphyrinogen Ill. Coproporphyrinogen Ill is transported from the cytosol into the mitochondria,
where the enzyme coproporphyrinogen oxidase acts on the propionic acid side chains to yield protoporphyrinogen IX. Catalyzed by protopor-
phyrinogen IX oxidase, protoporphyrinogen IX is oxidized to protoporphyrin IX. Protoporphyrin IX combines with ferrous iron to yield heme
(catalyzed by heme synthase). (Intermediates between uroporphyrinogen and coproporphyrinogen, designated by X, remain unidentified.)
BPO, = pyridoxal phosphate. (From Tietz, MW: Textbook of Clinical Chemistry. WB Saunders, Philadelphia, 1986, with permission.)
72 Chapter 3. The Red Blood Cell: Structure and Function

GENE PRODUCTS (GLOBIN CHAINS) 100 ae

HEMOGLOBINS (Hb)
B
Genes Genes
80
S
Ce €2 2
3 60 4

Hb Gower 1 & | —— Fetal to adult

Hb switch
2B
©
|
On Ep o 40 | HEMOGLOBINS
Hb Gower 2 > / F = Op Yo
& ere | A = 02 Be
20 (embryonic) Ao = O2 82
Ce Gye
Co Aye 0 as
Hb Portland

Infant

Time (months)
Oo Gyp

10) Ay2 Figure 3-12 = Changes in globin chain synthesis during fetal devel-
Hb F opment, birth, and infancy. (From Hillman, RF, and Finch, CA: Red
Cell Manual, ed 7. FA Davis, Philadelphia, 1996, p 11, with
Oy So permission.)
Hb A,

On Bo
Hb A In the cytoplasm, each synthesized globin chain links with
heme (ferroprotoporphyrin IX) to form hemoglobin, which
Chromosome 16 Chromosome 11 primarily consists of two a chains, two 6 chains, and four
heme groups (see Fig. 3-8). The structure of the chains and the
Figure 5-1 1 @ Genetic control and formation of human
hemoglobins. resulting hemoglobin molecule is described in the following
four steps:

The € and ¢ chains normally appear only during embry- 1. The primary structure relates to the number and sequence of
onic development (Table 3-6). These two chains, plus the a amino acids constituting each globin chain. The a chains
and y chains, are constituents of embryonic hemoglobins: Hb have 141 amino acids; the non-a chains have 146 amino
Gower | (¢,€,), Hb Gower 2 (a,€,), and Hb Portland (C,7,).*° acids. The sequence of amino acids is different in each chain.
The € and ¢ chains are produced up to approximately 3 months 2. The secondary structure occurs with the twisting of the
after conception. The a chain is always present. Production amino acid chain around an axis in a helical conformation.
of y chains is active from the third month of fetal development 3. The tertiary structure consists of bending the twisted amino
until 1 year postnatally.*° In the fetus, the major hemoglobin is acid chain into a three-dimensional shape resembling an
ayy, (hemoglobin F). The y chains occur as a mixture of two “irregular pretzel.” The polar groups are oriented outward,
types of chains, differing only by one amino acid at position and the nonpolar groups are interior. The heme molecule is
136. G-gamma (“y) contains glycine, whereas A-gamma (“y) nestled in a nonpolar pocket and attached to a proximal his-
has alanine at that position.” The ratio of Sy to “y is approxi- tidine residue.
mately 3:1 at birth and 2:3 by | year of age.'’ By the age of 4. The quaternary structure is the assembling of the four
2 years, hemoglobin F comprises less than 2% of the total three-dimensional chains with their respected heme groups.
hemoglobin. Production of 8 chains rise gradually prenatally The result is a complete, functional hemoglobin molecule.
and reaches adult percentages between 3 and 6 months postna- (Review Fig. 3-8.)
tally.*° Figure 3-12 depicts the time sequence of globin chain
Table 3-6 shows the composition of hemoglobin found dur-
synthesis during fetal development, birth, and infancy.
ing normal human development. The precise order of amino
All normal adult hemoglobins are formed as tetramers
acids is critical to the structure and function of the hemoglo-
consisting of two a chains plus two (non-a) globin chains. Nor-
bin molecule.
mal adult RBCs contain the following types of hemoglobin:
The rate of globin synthesis is directly related to the rate
° 95% to 97% of the hemoglobin is HbA, which consists of of porphyrin synthesis, and vice versa: protoporphyrin synthe-
a8, chains sis is reduced when globin synthesis is impaired. There is, how-
° 2% to 3% of the hemoglobin is HbA,, which consists of a6, ever, no such relationship with iron uptake when either globin
chains or protoporphyrin synthesis is impaired; iron accumulates in
° 1% to 2% of the hemoglobin is HbF (fetal hemoglobin), the RBC cytoplasm as ferritin aggregates. The iron-laden,
which consists of ay, chains nucleated RBC is termed a sideroblast, and the anucleated
 Chapter 3. The Red Blood Cell: Structure and Function WES

of the deoxyhemoglobin molecule is known as the tense (T)

form, which has a lower affinity for oxygen. When hemoglobin
loads oxygen and becomes oxyhemoglobin, the established salt
bridges are broken and B chains are pulled together, expelling
2,3-DPG. This relaxed form of the hemoglobin molecule has a
higher affinity for oxygen. Each iron-containing heme group in
Globin Chains Hemoglobin Stage of Development each of the polypeptide chains of the hemoglobin molecule can
bind one oxygen molecule.*’
Q€, Gower 2
These changes in shape that occur as the hemoglobin
Co€> Gower | Embryo
OY2 Portland loads and unloads oxygen are referred to as the respiratory
movement.*° The dissociation and binding of oxygen by hemo-
Q“y> globin are not directly proportional to the oxygen tension
a,°Y> (PO) of its environment, but instead exhibit a sigmoid-curve
relationship known as the hemoglobin—oxygen dissociation
2B,
0485 curve depicted in Figure 3-14. The shape of this curve is very
important physiologically because it permits a considerable
amount of oxygen to be delivered to the tissues with a small
drop in oxygen tension. For example, in the environment of
form, a siderocyte, when stained with Prussian blue for visual- the lungs, where the PO,, measured in millimeters of mercury
ization of iron (Fig. 3—13).'° When protoporphyrin synthesis is (mm Hg), is nearly 100 mm Hg, the hemoglobin molecule is
impaired, the mitochondria become encrusted with iron, which almost 100% saturated with oxygen (see Fig. 3-14, point A).
is visible around the nucleus of the RBC precursor when As the RBCs travel to the tissues, where the PO, drops to an
stained with Prussian blue. Such an RBC is termed a ringed average 40 mm Hg (mean venous oxygen tension), the hemo-
sideroblast and is diagnostic for a pathogenesis linked to defi- globin saturation drops to approximately 75%, releasing
cient protoporphyrin synthesis*™ (see Fig. 3-13). approximately 25% of the oxygen to the tissues (see Fig. 3-14,
e point B).
This is the normal situation of oxygen delivery at basal
HEMOGLOBIN FUNCTION metabolic rate. In conditions such as hypoxia, a compensatory
The primary function of hemoglobin is delivery and release of “shift to the right” of the hemoglobin-oxygen dissociation
oxygen to the tissues and facilitation of carbon dioxide excre- curve (Fig. 3-15) occurs to alleviate a tissue oxygen deficit.
tion. Because of hemoglobin’s multichain structure, the mole- This rightward shift of the curve, mediated by increased levels
cule is capable of a considerable amount of allosteric movement of 2,3-DPG, results in a decrease in the affinity of hemoglobin
as it loads and unloads oxygen.*° One of the most important con- for the oxygen molecule and an increase in oxygen delivery to
trols of hemoglobin affinity for oxygen is the RBC organic the tissues.** The RBCs, therefore, have become more efficient
phosphate 2,3-diphosphoglycerate (2,3-DPG). The unloading of in terms of oxygen delivery.
oxygen by hemoglobin is accompanied by the widening of the Therefore, a patient who is suffering from an anemia,
space between 8 chains and the binding of 2,3-DPG on a mole- caused by a loss of RBCs, may be able to compensate by shift-
for-mole basis, with the formation of anionic salt bridges ing the oxygen dissociation curve to the right, making the
between the B chains* (Fig. 3-14). The resulting conformation RBCs, although fewer in number, more efficient. Some patients
may be able to tolerate anemia better than others because of this
compensatory mechanism. A shift to the right also may occur
in response to acidosis or a rise in body temperature.*° The shift
to the right of the hemoglobin—oxygen dissociation curve is
only one way in which patients may compensate for various
types of hypoxia; other ways include increases in total cardiac
output and in erythropoiesis.
A shift to the left of the hemoglobin—oxygen dissocia-
tion curve conversely results in an increase in hemoglobin—
oxygen affinity and a decrease in oxygen delivery to the
tissues (see Fig. 3-15). With such a dissociation curve,
RBCs are much less efficient in releasing oxygen to the
tissues. Among the conditions that can shift the oxygen dis-
sociation curve to the left are alkalosis; decrease in body
temperature; increased quantities of abnormal hemoglo-
bins, such as methemoglobin and carboxyhemoglobin;
increased quantities of hemoglobin F; or multiple transfu-
Figure 3-13 m Ringed sideroblast and siderocytes as detected by
Prussian blue staining of a bone marrow aspirate. (Prussian blue
sions of 2,3-DPG-depleted stored blood (attesting to the
stain, < 1000) importance of 2,3-DPG in oxygen release).*”
74. Chapter 3 The Red Blood Cell: Structure and Function

OXYHEMOGLOBIN

OXYHEMOGLOBIN —>

N oO
~—-- 25% Oo RELEASED
Mean venous
l Oo tension
OooO
|
Pso | Normal Ps9 = 26 to 30 mmHg
Saturation
(%) |
25 DEOXYHEMOGLOBIN

25 { 40 50 TS 100
POs (mmHg)
DEOXYHEMOGLOBIN
TISSUE VENOUS OXYGEN TENSION

Figure 3-14 ® Normal hemoglobin-oxygen dissociation curve. (From Hillman, RF, and Finch, CA: Red Cell Manual, ed 7. FA Davis,
Philadelphia, 1996, p 18, with permission.)

Hemoglobin—oxygen affinity also can be expressed by In addition to the reasons listed previously for shifts in
Ps) values, which designate the PO, at which hemoglobin the curve, inherited abnormalities of the hemoglobin mole-
is 50% saturated with oxygen under standard in vitro con- cule can result in either situation. These abnormalities are
ditions of temperature and pH.” The P;, of normal blood is described by the P;, measurements. Abnormalities in hemo-
26 to 30 mmHg." An increase in P;, represents a decrease globin structure or function can therefore have profound
in hemoglobin—oxygen affinity, or a shift to the right of the effects on the ability of RBCs to provide oxygen to the
oxygen dissociation curve. A decrease in Ps) represents an tissues.
increase in the hemoglobin—oxygen affinity, or a shift to
the left of the hemoglobin—oxygen dissociation curve.
Abnormal Hemoglobins of Clinical
Importance
The hemoglobins previously described, oxyhemoglobin and
ee << Normal reduced hemoglobin, are physiologic hemoglobins because
“Left-shifted” (2 they function in the transport and delivery of oxygen within
~“Right-shifted” the circulation. Abnormal hemoglobins of clinical significance
Abn Hb ‘—

i iat
that are unable to transport or deliver oxygen include the
70 TpH following:
60 Temp
P50 TTemp ° Carboxyhemoglobin
50 4-5---->f- TPs0 ¢ Methemoglobin
P50 ¢ Sulfhemoglobin

In carboxyhemoglobin, the oxygen molecules bound to heme

have been replaced with carbon monoxide (CO). This replace-
Oxyhemoglobin
saturation)
(% ment process is relatively slow and dependent on the concen-
tration of carbon monoxide in the blood. Once attached,
however, the binding of carbon monoxide to the heme of the
Normal P59 = 28 mm Hg
hemoglobin molecule is 200 times tighter than the binding of
30 40 50 60 70 80 90 100 oxygen to heme.” The concentration of carbon monoxide can
PO2 (mm Hg) be increased in a number of conditions, including chronic
heavy smoking.
Figure 3-15 @ Hemoglobin—-oxygen dissociation curve. Blue line is Methemoglobin is formed when the iron of the hemoglo-
the normal curve. The green line represents a “shift to the left” that bin molecule is oxidized to the ferric (Fe**) state.*° Normally,
can occur with an increase in abnormal hemoglobins, increase in
less than 1% of the total circulating hemoglobin is in the
PH (alkalosis), a decrease in 2,3-DPG, and a decrease in body tem-
perature. The red line represents a “shift to the right” that can methemoglobin form. Increased formation of methemoglobin
occur with a decrease in pH (acidosis), an increase in 2,3-DPG, and can occur as a result of an overload to oxidant stress, owing
an increase in body temperature. to the ingestion of strong oxidant drugs or to an enzyme
 Chapter 3 The Red Blood Cell: Structure and Function 75

deficiency (see the following section on RBC metabolic

pathways).*!4?
Sulfhemoglobin is formed when a certain situation or
condition, such as ingestion of a sulfur-containing drug or
chronic constipation, causes the sulfhemoglobin content of ne Maturation
the blood to build up.** Sulfhemoglobin is incapable of carry-
ing oxygen and represents an irreversible change of the hemo- Nucleated Adult
globin molecule that persists until the RBCs are removed Ae _ Reticulocyte_ RBC
from the circulation. Both carboxyhemoglobin and methemo-
Replication 0
globin, however, can be reverted to oxyhemoglobin through
DNA synthesis
the use of oxygen inhalation and the administration of strong RNA synthesis
reducing substances, respectively.*® The toxic levels for the Lipid synthesis
concentration of circulating abnormal hemoglobins are listed RNA present
in Table 3-7. Heme synthesis
Protein synthesis
At these toxic levels, the tissue oxygen deficit results in Mitochondria
cyanosis and anemia, and eventually death may occur. Krebs’ tricarboxylic tet
praetor
iat
tectacteact oa
ar
ap
Wars
SSS
acid cycle
Embden—Meyerhof +

Maintenance of Hemoglobin Function: pathway

Active Red Blood Cell Metabolic Pentose phosphate
pathway
ob

Pathways Maturation and/

or senescence
Active erythrocyte metabolic pathways are necessary for the
production of adequate ATP levels. Such generated energy is
crucial to RBC survival and function in that it is necessary for
maintaining: Of the ATP needed by RBCs, 90% is generated by the
* Hemoglobin function Embden—Meyerhof glycolytic pathway.** Here, the metabo-
¢ Membrane integrity and deformability lism of glucose results in the net generation of two mole-
* RBC volume cules of ATP. Although this ATP synthesis is inefficient
¢ Adequate amounts of reduced pyridine nucleotides when compared with cells that use the Krebs cycle (aerobic
¢ Protection of metabolic enzymes“ metabolism), it provides sufficient ATP for the require-
ments of the RBCs. Glycolysis also generates NADH from
RBCs generate energy almost exclusively through the anaero- NAD*, which is important in some of the other RBC meta-
bic breakdown of glucose, because the metabolism of the anu- bolic pathways.
cleated erythrocyte is more limited than that of other body Another 5% to 10% of glucose is metabolized by
cells. The adult RBC possesses little ability to metabolize fatty the hexose monophosphate shunt (also called the phosphoglu-
acids and amino acids. In addition, mature RBCs contain no conate pathway). This pathway produces the pyridine
mitochondrial apparatus for oxidative metabolism (Table 3-8). nucleotide NADPH from NADP*. NADPH, together with
The RBC metabolic pathways are mainly anaerobic, fortu- reduced glutathione, provides the main line of defense for the
nately, because the function of the RBC is to deliver oxygen RBC against oxidative injury.*° Oxidant drugs, as well as
and not to consume it. Four pathways of RBC metabolism will infections, can cause the accumulation of hydrogen peroxide
be considered: the anaerobic glycolytic pathway, and three and other oxidants, which can be toxic to cell proteins. The
ancillary Sk that serve to maintain the function of hemo- sequence of biochemical reactions shown in Figure 3-16
globin (Fig. 3-16). All of these processes are essential if the occurs within the normal RBC with adequate levels of appro-
RBC is to transport oxygen and maintain the physical charac- priate enzymes and substrate to prevent the accumulation of
teristics required for its survival in circulation. these agents.
When the hexose monophosphate pathway is function-
ally deficient, the amount of reduced glutathione becomes
insufficient to neutralize intracellular oxidants. This results in
: ee Levels for globin denaturation and precipitation as aggregates (Heinz
bodies) within the cell. If this process sufficiently damages
the membrane, cell destruction occurs. Inherited defects in the
pentose phosphate glutathione pathway, the most common
Abnormal Hemoglobin pene Level @%) — of which is glucose-6-phosphate dehydrogenase (G6PD) defi-
ciency, result in the formation of Heinz bodies with subse-
Carboxyhemoglobin
quent extravascular hemolysis. (Glutathione is not only
Methemoglobin
Sulfhemoglobin crucial to keeping hemoglobin in a functional state but it is
also important in maintaining RBC integrity by reducing
76 Chapter 3 The Red Blood Cell: Structure and Function

PHOSPHOGLUCONATE
PATHWAY
(oxidative)

EMBDEN-MEYERHOF
PATHWAY GSH GSSG
(non-oxidative)
Glucose
ATP
ADP [HK]
Glucose 6-P
NADP NADPH
6-P-Gluconate

[ce]}
G-6-PD
2

Fructose 6-P Pentose-P

[Prk]
Fructose 1,6-diP

METHEMOGLOBIN
REDUCTASE [All
Glyceraldehyde <> DHAP
PATHWAY LUEBERING-RAPAPORT
a Hemoglobin ‘
Methemoglobin [A] (yaon> —[earo]|1,3-diP-Glycerate
PATHWAY

HK Hexokinase

[rex]
Glucose-6-phosphate isomerase ADP 2,3-diP-Glycerate
PFK Phosphofructokinase ATP
A Aldolase 3-P-Glycerate

[row]}
TPI Triose phosphate isomerase
GAPD Glyceraldehyde-3-phosphate dehydrogenase
PGM Phosphoglycerate mutase
2-P-Glycerate
E Enolase
PK
LDH
Pyruvate kinase
Lactic dehydrogenase
DPGM __ Diphosphoglyceromutase
fel!
P-Enolpyruvate
DPGP _ Diphosphoglycerate phosphatase ADP
G-6-PD
6-PGD
Glucose-6-phosphate dehydrogenase
6-Phosphogiuconate dehydrogenase
ATP
[x] Pyruvate

foal
GR Glutathione reductase
GP Glutathione peroxidase NADH
DHAP _ Dihydroxyacetone-P NAD
PGK Phosphoglycerate kinase Lactate
R NADH-methemoglobin reductase

Figure 3-16 @ Red cell metabolism. (From Hillman, RF, and Finch, CA: Red Cell Manual, ed 7. FA Davis, Philadelphia, 1996, p 15, with
permission.)

sulfhydryl groups of hemoglobin, membrane protein, and 2,3-DPG, which is important because of its profound effect
enzymes subsequent to oxidation.) on the affinity of hemoglobin for oxygen.“ Stores of this
The methemoglobin reductase pathway is another impor- organic phosphate can serve as a reserve for additional ATP
tant component of RBC metabolism. Two methemoglobin generation.
reductase systems are important in maintaining heme iron in
the reduced (Fe**, ferrous) functional state.“° Both pathways Erythrocyte Senescence and Hemolysis
depend on the regeneration of reduced pyridine nucleotide and
are referred to as the NADH and NADPH methemoglobin The RBC, a 6- to 8-um biconcave disc (Fig. 3-17), travels
reductase pathways. In the absence of the enzyme methemo- 200 to 300 miles during its 120-day life span. During this
globin reductase and the reducing action of the pyridine time, circulating RBCs undergo the process of senescence or
nucleotide NADH, there is an accumulation of methemoglobin, aging. Various metabolic and physical changes associated
resulting from the conversion of the ferrous iron of heme to the with the aging of RBCs are listed in Table 3-9. Each day
ferric form (Fe**). Methemoglobin is a nonfunctional form of 1% of the old RBCs in circulation are taken out by a system
hemoglobin, having lost oxygen transport capabilities, as the of fixed macrophages in the body known as the reticuloen-
metheme portion cannot combine with oxygen. Normal effi- dothelial system (RES) or the mononuclear phagocytic system
ciency of the methemoglobin reductase pathway is exemplified (MPS). Modifications of the RBC membrane proteins that
by the fact that usually no more than 1% of RBC hemoglobin normally occur during RBC senescence play a role in enhanc-
exists as methemoglobin in the RBCs of healthy individuals." ing the recognition of aging red cells and their removal by
Another important pathway that is crucial to RBC func- phagocytic cells of the MPS. These RBCs are replaced by the
tion is the Leubering—Rapaport shunt. This pathway causes an daily release of 1% of the younger RBCs (reticulocytes) from
extraordinary accumulation of the RBC organic phosphate the bone marrow storage pool. As erythrocytes become older,
 Chapter 3. The Red Blood Cell: Structure and Function 77

NORMAL EXTRAVASCULAR HEMOLYSIS

RETICULOENDOTHELIAL SYSTEM
(spleen and bone marrow) NORMAL

Bilirubin + albumin |
Liver
General
circulation
Figure 3-17 @ Scanning electron micrograph (SEM) of a normal
red cell. Small
intestine

certain glycolytic enzymes decrease in activity, resulting ina Enterohepatic

decrease in the production of energy and loss of deformabil- circulation

ity. At a certain critical point, the RBCs are no longer able bilirubin
to traverse the microvasculature and are phagocytized by Conjugated = normal
the RES cells. Although RES célls are located in various 0-0.2 mg/100 mL
Unconjugated= normal , Large
organs and throughout the body, those of the spleen, 0.2—0.8 mg/100 mL a intestine
called littoral cells, are the most sensitive detectors of RBC
abnormalities.*’ Urine f j

Urobilinogen = normal (1*)

0.5-4.0 E.U./d Urobilinogen and urobilin = normal
Bilirubin = negative (2+) 75-400 E.U./d
Extravascular Hemolysis
Figure 3-18 ™ Normal extravascular hemolysis. (From Tietz, MW:
Ninety percent of the destruction of senescent RBCs occurs
Textbook of Clinical Chemistry, WB Saunders, Philadelphia, 1986,
by the process of extravascular hemolysis (Fig. 3-18). with permission.)
During this process, old or damaged RBCs are phagocytized
by the RES cells and digested by their lysosomes. The he-
moglobin molecules are disassembled and broken down into tetrapyrrole, biliverdin, is converted to bilirubin and carried
their various components. The iron recovered is salvaged by the plasma protein albumin to the liver.** In the liver,
and returned by the plasma protein carrier, transferrin to the bilirubin is conjugated to bilirubin glucuronide and excreted
erythroid precursors in the marrow for synthesis of the new along with bile into the intestines. Here it is converted
hemoglobin. Globin is broken down into amino acids and further, through bacterial action, into urobilinogen (sterco-
redirected to the amino acid pool of the body. Finally, the bilinogen) and excreted in the stool. A small amount of
protoporphyrin ring of heme is disassembled, and its @ car- urobilinogen is reabsorbed through enterohepatic circula-
bon exhaled in the form of carbon monoxide. The opened tion, filtered by the kidneys, and excreted in small amounts
in the urine. Both unconjugated (prehepatic) and conjugated
(posthepatic) bilirubin can be measured in the plasma as
indirect and direct bilirubin, respectively, and used to monitor
Table39 Changes Occurring © RON the amount of hemolysis."
! _ During Aging of RBCs ae
Decreases
Intravascular Hemolysis
ee

Several enzyme activities Only 5% to 10% of normal RBC destruction occurs through
ae toned 1G
Density Sialic acid intravascular hemolysis (Fig. 3—19).'° During this process,
Spheroidal shape Deformability RBC breakdown occurs within the lumen of the blood ves-
MCHC MCV sels. The RBC ruptures, releasing hemoglobin directly into
Internal viscosity Phospholipid the bloodstream. The hemoglobin molecule dissociates into
Agglutinability Cholesterol
af dimers and is picked up by the protein carrier, haptoglo-
Na* Kt
Methemoglobin Protoporphyrin bin.** The haptoglobin—hemoglobin complex prevents renal
Oxygen affinity excretion of the hemoglobin and carries the dimers to the
liver cell for further catabolism. The hepatocyte uptake
MCHC= mean cell earecastnconcentration;1 MCV = mean cell
volume.
and processing are identical at this point to the process
Source: Garratty, G: Basic mechanisms of in vivo cell destruction. previously described for extravascular hemolysis (see
In Beil, C (Ed): A Seminar in Immune-Mediated Cell Destruction. Fig. 3-18). Haptoglobin levels, therefore, fall in plasma as
American Association of Blood Banks, Bethesda, MD, 1981, with
permission.
haptoglobin is removed by the hemoglobin—haptoglobin
complex formation. It is estimated that as little as 1 to 2 mL
78 Chapter 3 The Red Blood Cell: Structure and Function

of the RBC intravascular hemolysis can totally deplete sequent degradation. As a result, the methemalbumin circu-
the amount of plasma haptoglobin.** Normally, 50 to lates until additional hemopexin is produced by the liver to
200 mg/dL of plasma haptoglobin is available and repre- serve as the protein carrier. Table 3-10 lists the various
sents the hemoglobin-dimer binding capacity.** As hapto- plasma proteins and the substances they carry. It is this circu-
globin is depleted, unbound hemoglobin dimers appear in lating methemalbumin that imparts a brown tinge to the
the plasma (hemoglobinemia) and are filtered through the plasma or blood. Intravascular hemolysis that occurs as a
kidneys and reabsorbed by the renal tubular cells. The renal result of RBC senescence is so minimal that it is limited to the
tubular uptake capacity is approximately 5 g per day of fil- involvement of only haptoglobin, which is rarely depleted.
tered hemoglobin.** Beyond this level, free hemoglobin Hemoglobinemia and hemoglobinuria, as well as the other
appears in the urine (hemoglobinuria). processes discussed, come into play only with excessive
Hemoglobinuria is always associated with hemoglobine- intravascular hemolysis, which can occur in patients having
mia. A normal plasma hemoglobin level is approximately 2 to various hemolytic anemias (see Chaps. 9 through 14).
5 mg/dL, which is released as a result of intravascular hemoly- This chapter has outlined and described three important
sis.** Depending on the amount of hemolysis and type of hemo- areas of RBC structure and metabolism: the red cell mem-
globin, the plasma may be pink, red, or brown. Likewise, in brane, hemoglobin structure and function, and red cell meta-
hemoglobinuria the urine also may be pink, red, brown, or bolic pathways. An understanding of these aspects of the
black. Two hemoglobin pigments, oxyhemoglobin and methe- RBC is important to appreciating the development and patho-
moglobin, are produced by auto-oxidation of the hemoglobin in genesis of the many forms of inherited and acquired RBC
the urinary tract when the urine is acidic.** Oxyhemoglobin is defects that result in hemolytic anemias.
bright red, and methemoglobin is dark brown. The color of the
urine, therefore, depends on the amount of hemolysis and the
concentration and relative proportions of these two pigments.
Oxyhemoglobin predominates in alkaline urine, and methemo-
globin predominates in acidic urine.**
Hemoglobin that is neither processed by the kidneys nor
bound to haptoglobin is oxidized to methemoglobin, which is
further disassembled as metheme groups are released and glo-
bin degraded. Free metheme is quickly bound by another
transport protein, hemopexin, and is carried to the liver cell to
be catabolized, as previously described. The heme-binding
capacity of hemopexin is approximately 50 to 100 mg/dL;
when this is exceeded, the metheme groups combine with
albumin to form methemalbumin.** Albumin cannot transfer
the metheme across the membrane of the hepatocyte for sub-

INTRAVASCULAR HEMOLYSIS

Globin

Hemoglobin <——— > Hemoglobin <—=> Methemoglobin

dimer tetramer
Heme
Haptoglobin Hemopexin

Methemalbumin

Hemosiderin Protein
<< Fecal urobilinogen Substance Carried
Hemoglobin
Methemoglobin Transferrin Iron
Haptoglobin Hemoglobin dimers
Figure 3-19 m@ Intravascular hemolysis. (From Hillman, RF, and Hemopexin Metheme
Finch, CA: Red Cell Manual, ed 7. FA Davis, Philadelphia, 1996, Albumin Bilirubin
p 23, with permission.)
 Chapter 3. The Red Blood Cell: Structure and Function 79

7. Which of the following is a complete list of abnormal he-

moglobins that are unable to transport ordeliver oxygen?
a. Carboxyhemoglobin and methemoglobin
b. Cell metabolism b. Methemoglobin and fetal hemoglobin
c. Intravascular hemolysis c. Carboxyhemoglobin, sulfhemoglobin, and fetal hemoglobin
d. Hemoglobin structure d. Carboxyhemoglobin, methemoglobin, and sulfhemoglobin
2. Which abnormal RBC is not caused by a structural . Which metabolic pathway generates 90% of the ATP
membrane defect? needed by RBCs?
a. Spherocyte a. Methemoglobin reductase pathway
b. Target cell b. Hexose monophosphate shunt
c. Siderocyte c. Embden—Meyerhof pathway
d. Acanthocyte d. Leubering—Rapaport shunt
3. Which list represents the complete set of processes nec- . What steps occur in the extravascular breakdown of
essary for normal hemoglobin production? senescent RBCs?
a. Iron delivery and supply, synthesis of protoporphyrins, a. RES cells phagocytize red cells; iron is coupled to trans-
and globin synthesis ferrin and returned to marrow; globin is returned to amino
b. [ron salvage, synthesis of conjugated bilirubin, and acid pool; biliverdin is converted to bilirubin; bilirubin is
haptoglobin synthesis coupled to albumin and transported to liver; bilirubin glu-
c. Iron accumulation, synthesis of hemopexin, and glo- curonide is converted to urobilinogen and excreted.
bin catabolism b. RBCs break down in lumen of vessel; the haptoglobin—
d. Iron catabolism, synthesis of uroporphyrinogen, and hemoglobin complex goes to the liver; unbound
ferritin synthesis hemoglobin dimers are excreted through the kidney
. What is the correct list for the number and type of globin as hemosiderin, hemoglobin, or methemoglobin; hapto-
chains in normal adult hemoglobin? globin is broken down to be excreted as urobilinogen
a. Four a, two B and two 6 chains c. RES cells phagocytize red cells; iron is coupled to
b. Two @ and two non-o chains transferrin and returned to marrow; globin is returned
c. Two a, four B, one 6, and one e€ chain to amino acid pool; the haptoglobin—hemoglobin com-
d. Two a, two B, two 6, and one e chain plex goes to liver; unbound hemoglobin dimers are
excreted through kidney as hemosiderin, hemoglobin,
. What is the composition of normal adult hemoglobin? or methemoglobin; haptoglobin is broken down to be
a. 92% to 95% HbA; 5% to 8% HbA,; 1% to 2% HbF excreted as urobilinogen.
b. 90% to 92% HbA; 2% to 3% HbA,; 2% to 5% HbF d. RBCs break down in lumen of vessel; haptoglobin
c. 80% to 85% HbA; 2% to 3% HbA;; 1% to 2% HbF picks up dissociated hemoglobin; the haptoglobin—
d. 95% to 97% HbA; 2% to 3% HbA,; 1% to 2% HbF hemoglobin complex goes to the liver; biliverdin is
. Which of the following cells is caused by iron accu- converted to bilirubin; bilirubin is coupled to albumin
mulation? and transported to liver; bilirubin glucuronide is
a. Acanthocyte converted to urobilinogen and excreted.
b. Ringed sideroblast 10. What steps occur in the intravascular breakdown of
c. Burr cell senescent RBCs? (Use answer choices for question 9.)
__d. Bite cell
See answers at the back of this book.
8O Chapter 3. The Red Blood Cell: Structure and Function

mw Iron is delivered to the membrane of the RBC precursor

é') SUMMARY by the protein carrier transferrin; two-thirds of total body
iron is bound to heme in the hemoglobin molecule.
= Glycophorin is the major integral membrane protein, rep-
resenting 20% of the total RBC membrane protein. = Globin chain synthesis occurs on RBC-specific cytoplas-
mic ribosomes, which are initiated from the gene inheri-
m Spectrin is the most abundant peripheral protein of the tance.
RBC membrane cytoskeleton, comprising 25% to 30% of
= Normal adult hemoglobin consists of 95% to 97% HbA,
the total membrane protein and 75% of the peripheral
2% to 3% HbA, and 1% to 2% HbF.
protein.
m The primary function of hemoglobin is delivery and
g The loss of ATP and subsequent decrease in the phospho-
release of oxygen to the tissues and facilitation of carbon
rylation of spectrin leads to a decrease in deformability
dioxide excretion.
and RBC survival.
w A shift to the left of the hemoglobin—oxygen dissociation
m=The RBC membrane is freely permeable to water and
curve results in a decrease in oxygen delivery to the
anions while relatively impermeable to cations (Na*, K*).
tissues.
m The erythrocyte intracellular-to-extracellular ratios for
w A shift to the right of the hemoglobin—oxygen dissociation
sodium and potassium are 1:12 and 25:1, respectively.
curve results in a decrease of oxygen affinity for hemoglo-
= RBC membrane lipids consist of a bilayer of phospho- bin and increased oxygen delivery to the tissues.
lipids interspersed with molecules of unesterified choles-
terol and glycolipids present in equimolar quantities. m Abnormal hemoglobins, which are unable to carry
oxygen, include carboxyhemoglobin, methemoglobin,
w Cholesterol comprises 25% of the RBC membrane lipid and sulfhemoglobin.
and is present in a 1:1 molar ratio with phospholipids.
m Ninety percent of RBC senescence occurs by extravascu-
gw Hemoglobin is a conjugated globular protein consisting of lar hemolysis, whereas 5% to 10% occurs through
globin (two pairs of polypeptide chains) and four heme intravascular hemolysis.
groups, each of which contains a protoporphyrin ring plus
ferrous iron. w Ninety percent of ATP needed for RBC survival is gener-
ated via the Embden—Meyerhof glycolytic pathway.
a Normal hemoglobin synthesis is dependent on adequate
iron delivery and supply, adequate synthesis of protopor- m The abnormal hemoglobins of clinical importance are car-
phyrins, and adequate globin synthesis. boxyhemoglobin, methemoglobin, and sulfhemoglobin.

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 Chapter

Anemia
Diagnosis and Clinical Considerations
Armand B. Glassman, MD

Definition of Anemia OBJECTIVES

Considerations by Age, Sex, At the end of this chapter, the learner should be able to:
and Other Factors
1. Describe clinical signs of anemia.
Causes of Anemia
2 . List causes of anemia.
Significance of Anemia and
Compensatory 3. State the laboratory criteria for the diagnosis of anemia.
Mechanisms
4 . Explain the most common method for the measurement of hemoglobin on automated
Red Blood Cell and
Hemoglobin Production INS LCUME RLS:
Clinical Diagnosis of Anemia . Explain how the hematocrit is calculated on automated hematology instruments.

Classification of Anemia . Calculate the significance of red blood cell indices as related to the diagnosis of anemia.
Hemoglobin and Hematocrit
. Describe the appearance of the peripheral blood smear in various anemias.
Red Blood Cell Indices
Red Blood Cell Indices and . Explain the diagnostic value of the reticulocyte count.
Other Tests
MN
=)
oO
So. List factors to be evaluated in the interpretation of a bone marrow aspirate smear.
OOO
Overview of the Treatment
of Anemias
Other Factors Involved in
Red Blood Cell Production
Tests in the Diagnosis of
Anemia
Hemoglobin
Hematocrit
Red Blood Cell Indices
Peripheral Blood Smear
Reticulocyte Count
Bone Marrow Smear and
Biopsy
Patient Studies of Anemia

Definition of Anemia There are varied types and causes of anemias. Anemia is
often caused by or associated with an underlying disease.
Anemia in its broadest sense is the inability of the circulat- Determining the specific cause of and characterizing an
ing blood pool to supply the tissue with adequate oxygen anemia in an individual is important to the physician so
for proper metabolic function.' Clinically the diagnosis of that he or she can base the appropriate therapy and assess
anemia is made by patient history, physical examination, the prognosis of the disease in that patient. Anemia is usu-
signs and symptoms, and hematologic laboratory findings. ally associated with decreased levels of hemoglobin or a

82
 Chapter 4 Anemia: Diagnosis and Clinical Considerations 83

decreased packed red blood cell (RBC) volume, also the geriatric age group, the difference between hemoglobin
known as the hematocrit (Hct). Under rare circumstances, levels of men and women narrows. Hemoglobin levels of
certain abnormal hemoglobins have very strong oxygen- geriatric men usually decrease slightly, and those of post-
binding capacities or oxygen is not released normally menopausal women approach those of men. The reference
to tissue, resulting in all the clinical signs and symptoms ranges used by each laboratory should be obtained, because
of anemia even though the hemoglobin or hematocrit value they best reflect the patient population served.
is normal or even raised. From a practical laboratory stand- Other factors, including geographic elevation, influ-
point, however, the usual diagnostic criterion for anemia ence individual “normal” hemoglobin levels. Persons
is a decreased hemoglobin (Hgb), hematocrit, or RBC living at elevations above 8000 feet may have persistently
count.* increased hemoglobin values secondary to decreased oxy-
Because most patients with anemia have lowered hemo- gen saturation in the ambient atmosphere. Lung diseases
globin levels, the anemia may be classified arbitrarily as may alter oxygen diffusion across the lung alveolar mem-
either moderate (7 to 10 g Hgb/dL) or severe (less than 7 g branes. A compensatory sequel to chronic lung disease
Hgb/dL).* Moderate anemias do not usually produce clinically may result in increased hemoglobin levels (secondary
evident signs or symptoms, especially if the onset is slow. polycythemia).
However, depending on the patient’s age or cardiovascular con- Various diseases and disorders are associated with lower
dition, even moderate amounts of anemia may be associated than usual hemoglobin levels; these include nutritional defi-
with exertional dyspnea (difficulty breathing), light-headedness, ciencies, external or internal blood loss, accelerated destruc-
vertigo, muscle weakness, headache, or general lethargy. tion of RBCs, ineffective or decreased production of RBCs,
Anemia of rapid onset, such as that resulting from gastrointesti- abnormal hemoglobin synthesis, bone marrow replacement
nal hemorrhage, may be associated with significant clinical by infection or tumor, and bone marrow suppression by
symptoms, such as hypotension, tachycardia, and dyspnea. toxins, chemicals, or radiation.*
These symptoms are usually associated with the precipitous
loss of intravascular volume as well as the oxygen-carrying
capacity of the RBCs. Another example of acute massive blood Causesof Anemia
loss is hemorrhage from extensive trauma.
Anemia has many causes (Table 4-2), which can be broadly
classified as nutritional deficiency (e.g., folate or vitamin
Considerations by Age, Sex, and Other B,, deficiencies); blood loss (hemorrhage); accelerated
Factors destruction of RBCs (immune and nonimmune hemolysis);
bone marrow replacement (e.g., by cancer); infection; toxi-
Newborn infants (younger than | week old) have a hemoglo- city; hematopoietic stem cell arrest or damage; and heredi-
bin value of 18 + 4 g/dL as a reference range. At approxi- tary or acquired defect of hemoglobin, RBC enzymes or
mately 6 months of age, the reference range is 12.5 + 1.5 other metabolic abnormalities. Categories may be simplified
g/dL. Childhood levels from the ages of | to 15 years have a to include conditions of increased RBC destruction, abnor-
reference range of approximately 13.0 = 2 g/dL. Adult mal or decreased RBC production, or some combination
hemoglobin reference ranges are approximately 16.0 + 2 thereof. There are many causes and a variety of classifica-
g/dL for men and 14.0 + 2 g/dL for women (Table 4—1). In tions of anemia.

cr
Table 4-1.Reference Range 'Values
for Hemoglobin nee
ee BUY Hemoglobin (g/dL)
- eea2aac r fA
Infants
Blood loss (hemorrhage, surgery, menses)
Newborns (< | week old) 14.0-22.0 Accelerated destruction of RBCs (immune and nonimmune
6 mo old 11.0-14.0 hemolytic)
Children (1-15 years old) 11.0-15.0 Nutritional deficiency (folate, vitamin B,;, iron)
Bone marrow replacement (cancer, infection)
Adults
Infection (viral, bacterial, microbacterial, mycobacterial,
14.0-18.0 parasitic)
WV Ona ae 10;9 Toxicity (hydrocarbons, medications, radiation)
12.0-16.0 Hematopoietic stem cell arrest or damage (aging, cytokines)
WHO 13.3a=) Hereditary or acquired defect (G6PD deficiency,
spherocytosis)
WHO = World Healthh Onnaion Unknown/idiopathic
84 Chapter 4 Anemia: Diagnosis and Clinical Considerations

Significance of Anemia and of new RBCs in response to proper nutrients, vitamins, and
other factors may be evaluated via the reticulocyte count.
Compensatory Mechanisms
Red Blood Cell and Hemoglobin Production
Clinical Diagnosis of Anemia
In a healthy ambulatory person, approximately 1% of the
senescent circulating RBCs are lost daily. Normally, the bone The clinical diagnosis of anemia is made by a combination of
marrow continues to produce RBCs. A laboratory measure of factors, including patient history, physical signs, and changes
this replacement is the reticulocyte count. In healthy people, in the hematologic profile. The signs and symptoms of anemia
reticulocytes, early circulating RBCs containing residual are generally nonspecific, such as fatigue and weakness, and
RNA, account for 0.5% to 2.0% of the circulating RBCs in the may include gastrointestinal symptoms such as nausea, con-
adult. Replacement of RBCs requires a bone marrow with stipation, or diarrhea. The patient may complain of dyspnea
adequate functioning stem cells, normal RBC maturation after a level of exertion that previously had not caused any
processes, and the ability to release mature RBCs from the problems.°°
bone marrow. Proper hemoglobin and RBC production require A patient example may be useful here. A man who had
a variety of nutritional factors, including iron, vitamin B,,, and been able to climb three flights of stairs without difficulty or
folic acid, and normal pathways of hemoglobin synthesis.* significant shortness of breath might report that now he must
Decreased production of hemoglobin and/or RBCs results in a stop after climbing one flight of stairs and is then very short of
hypoproliferative anemia. The role of hemoglobin synthesis in breath. Subsequent information indicates that the patient has
anemias is covered in greater detail in Chapter 6. passed very dark stools (melena) over the past week. Measure-
In severe anemias (less than 7 g Hgb/dL), symptoms of ment of his hemoglobin reveals a level of 8 g/dL. The initial
functional impairment of a number of organ systems may diagnostic impression from the clinical information is that the
be evident. With minimal exercise, the patient’s cardiac and patient’s anemia is secondary to gastrointestinal bleeding.
respiratory rates may increase dramatically. If the anemia is Physical signs of anemia are usually not specific for the
secondary to blood loss and decreased intravascular vol- underlying causative disease. Occasionally, however, the
ume, the patient’s blood pressure may drop significantly underlying diagnosis may be suspected from certain physical
when he or she is raised from the reclining to a sitting or findings. One example would be signs of malnutrition and
standing position. The heart rate will increase in order to neurologic changes with loss of proprioception (position
elevate the cardiac output to keep pace with peripheral sense) and vibration awareness in a patient with vitamin B,,
tissue oxygen demands in the face of a decreased oxygen- deficiency. Another example would be severe pallor, smooth
carrying capacity of the lowered hemoglobin level. Respi- tongue, and an esophageal web in a patient with severe iron-
ratory symptoms, including dyspnea on exertion, may also deficiency anemia.’ Light-skinned patients who are anemic
occur with anemia. may appear to have pale coloration of mucosal membranes,
An interesting compensatory mechanism in response nail beds, and skin. Occasionally, the temperature may be
to anemia is an increase in the 2,3-diphosphoglycerate slightly elevated, particularly in patients having certain types
(2,3-DPG) levels. This compound is a remarkable physiologic of hemolytic anemia. In the presence of anemia, heart mur-
regulator of normal hemoglobin oxygen-carrying capacity murs may be heard; these are sometimes secondary to the
and tissue oxygen delivery. In the presence of 2,3-DPG, cause of the anemia and sometimes related to the increased
hemoglobin can more readily release the oxygen it is carrying cardiac workload required to bring oxygen to the tissues.
to peripheral tissues. This enhanced release occurs regardless Patients with bacterial endocarditis have fever, heart mur-
of pH or blood arterial oxygen level. murs, and anemia. Bacterial endocarditis is a clinical example
A normal individual responds to anemia with elevated in which the damaged infected myocardial valve and heart
levels of erythropoietin (Epo) (see Chap. 1). The Epo level is murmur are related etiologically to the anemia. Prosthetic
sometimes used as an ancillary diagnostic aid in the differen- heart valves, arterial grafts, or disseminated intravascular
tial diagnosis of anemia. Epo is a hormone with molecular coagulation (DIC) can cause a form of mechanical hemolytic
mass of approximately 31,000 daltons. It has a plasma half- anemia known as microangiopathic hemolytic anemia.
life of between 6 and 9 hours and is produced by the peritubu- A broad category of anemias includes those termed
lar complex of the kidney. Epo levels vary as a result of aplastic. Aplastic anemia is defined as pancytopenia result-
altered oxygen tension in the tissues of the kidney. Increased ing from bone marrow production failure. Hypoprolifera-
Epo production occurs when there is a decreased hemoglobin tive anemia can be solely RBC associated. Additional infor-
level, a hemoglobin structural problem in which oxygen is not mation concerning aplastic anemia is found in Chapter 8.
released, or low ambient oxygen tension at high altitude. On In general, there are two types of aplastic anemia. The
the other hand, high oxygen levels in the kidney result in a first type includes congenital and so-called idiopathic
decrease in Epo production. Recombinant Epo is now avail- acquired forms. The second type is secondary or acquired.
able for treatment of certain types of anemias, particularly Causes of secondary aplastic anemia include exposure to
end-stage renal disease, anemia associated with human chemicals (benzene and some other fluorocarbons), therapeu-
immunodeficiency virus, and anemia associated with cancer tic agents (especially cancer chemotherapy agents), infection
and certain other chronic disorders.* Bone marrow production (some types of hepatitis and parvovirus infections), and
 Chapter 4 Anemia: Diagnosis and Clinical Considerations 85

ionizing radiation. This chapter deals primarily with the de- concentration (MCHC).'° The MCV is used as an estima-
crease in the RBC and oxyhemoglobin carrying capacity. It tion of the average size of the RBC. It may be calculated by
should be noted that aplastic anemia is a pancytopenia involv- dividing the hematocrit by the number of RBCs or mea-
ing red cells, white cells, and platelets. sured directly using automated cell counters. If the MCV is
within the reference range, the RBCs are considered nor-
mocytic; if less than normal, microcytic; and if greater than
Classification of Anemia normal, macrocytic. Both MCH and MCHC values are used
The individual types of anemias can be classified according to determine the content of hemoglobin in RBCs. Most
to several different criteria. A bone marrow dynamic classifica- automated hematology instruments also provide an RBC
tion is hypoproliferative or accelerated destruction (hemolytic), distribution width (RDW) value. The RDW is an index
or a combination of the two (sometimes called ineffective of size variation and has been used to quantitate the amount
hematopoiesis). Anemias are often classified clinically accord- of anisocytosis seen on a peripheral blood smear. The refer-
ing to their associated causes, such as blood loss, iron deficiency, ence range for RDW is 11.5% to 14.5% for both men
hemolysis, infection, metastatic bone marrow replacement, or and women. The MCV is not dependable when RBCs
nutritional deficiency. Anemias can also be categorized quanti- vary markedly in size. If MCHC is normal, the RBCs are
tatively by their hematocrit, hemoglobin level, blood cell referred to as normochromic; hypochromic RBCs have a
indices (e.g., normochromic, hypochromic [microcytic] or less than normal MCHC; and there are no truly hyper-
“hyperchromic” [macrocytic]), or reticulocyte count.” The chromic RBCs.
clinical laboratory scientist is frequently involved in the quan-
titative measurements that lead to these classifications and in Red Blood Cell Indices and Other Tests
subsequent evaluations.
The RBC indices are accurately calculated by the auto-
mated hematology instrumentation. These instruments pro-
Hemoglobin and Hematocrit vide precise numeric values for hemoglobin levels, the
numbers of RBCs, and the MCV (see Chap. 31). Although
Measurement of the hemoglobin level and packed cell volume
less precise, careful microscopic examination of a periph-
is the usual method ‘of determining whether a patient has
eral blood smear can tell the examiner whether the RBCs
anemia. As discussed earlier, reference ranges may vary by age
are normocytic, microcytic, macrocytic, normochromic, or
and sex (see Table 4-1) as well as state of hydration, patient
hypochromic. A proper specimen is required to obtain accu-
positioning, and local laboratory patient population determina-
rate answers.'!
tions. Hemoglobin assays (see Chap. 31) are based on the spec-
RBC index calculations and reference ranges are:
trophotometric absorbance readings of cyanmethemoglobin
MCV equals Hct (in %) multiplied by 10, divided by
compared with known amounts (a standard curve). Several
RBC count (in millions/uL); reference range: 90 + 10 fL.
companies manufacture automated instruments that include
these determinations as part of a hematologic profile (see Chap. Hct (%) X 10
MCV =
31). The hematocrit, or packed red blood cell volume (PCV), 1s RBC count (millions/uL)
determined by centrifugation of blood of either capillary or
MCH equals Hgb (in g/dL) multiplied by 10, divided by
venous origin or can be calculated on some automated instru-
RBC count (in millions/uL); reference range: 29 + 2 pg.
ments. The reference PCV for adult men varies by institution
(i.e., 47% + 5%). For adult women during the reproductive Nias Hgb (g/dL) x 10
years, the PCV reference range is 42% + 5%. On the basis of RBC count (millions/uL)
hemoglobin or PCV values and the duration of onset, anemias
MCHC equals Hgb (in g/dL) multiplied by 100, divided
may be classified as mild, moderate, or severe; and as either
by Hct (as a percentage); reference range: 34 + 2%.
acute or chronic. The approximate relationship of the hemoglo-
bin level to hematocrit is 1:3, a ratio that may vary with the MCHC —Hgb (g/dL) x 100
cause of the anemia and the effect of that cause on the RBC Het (%)
indices, particularly the mean corpuscular volume (MCV).
Use of the RBC indices in the differential diagnosis of
Microscopic examination of a properly prepared periph-
anemia can provide a general idea as to what is occurring
eral blood smear is a requirement for the clinical and labora-
clinically (Table 4-3). A normocytic normochromic anemia
tory evaluation of anemia. This technique is discussed later may be the result of bone marrow failure, hemolytic anemia,
under the tests in the diagnosis of anemia. Histologic exami- or some subset of either of these conditions.
nations of the bone marrow smear and aspirate are adjuncts to Making the differential diagnosis of bone marrow failure
elucidate the cause of the anemia further.
requires information about RBC production. This information
can be obtained from the reticulocyte count, which indicates
whether there is bone marrow capacity for increased RBC
Red Blood Cell Indices
production. Because RBC destruction may exceed produc-
The RBC indices are the mean corpuscular volume (MCV), tion, the reticulocyte count, in fact, measures effective RBC
mean cell hemoglobin (MCH), and mean cell hemoglobin production. Hemolytic anemia occurs when there is decreased
86 Chapter 4 Anemia: Diagnosis and Clinical Considerations

RBC survival, which in turn may be the result of extravascu-

Table 4-3 Classification of Anemias : = lar elimination, intravascular destruction, or a combination of
by RBC Indices Peat the two.
Macrocytic normochromic anemias usually occur
Size Hgb Content in association with folate or vitamin B,, deficiency. The
(MCV) (f£L) (MCHC) (%) Possible Causes
most commonly encountered anemias are the microcytic
Normocytic Normochromic Bone marrow failure, hypochromic anemias, usually related to iron-deficiency
(80-100) (32-36) hemolytic anemia, anemia. Beta-thalassemia, an inherited defect of hemoglo-
chronic renal disease, bin synthesis, is another cause of microcytosis. Less fre-
leukemia, metastatic
quently seen are the sideroblastic anemias, which are also
malignancy
associated with decreased MCV.
Macrocytic Normochromic Megaloblastic and The differential diagnosis of anemia is based on a
(>100) (32-36) nonmegaloblastic combination of laboratory findings (see Table 4-3) and
macrocytic anemias
clinical symptoms (Table 4—4). An abbreviated flowchart
(e.g., liver disease,
myelodysplasias) for the diagnosis of anemia using the RBC indices is pro-
vided in Figure 4—1.
Microcytic Hypochromic Iron deficiency,
(<80) (<32) sideroblastic anemia,
thalassemia, lead Overview of the Treatment of Anemias
poisoning, chronic
diseases, chronic Anemia is treated according to its cause(s). The cause
infection or
should be considered and determined before beginning
inflammation,
unstable either supportive therapy (such as a transfusion) or replace-
hemoglobins ment therapy. Table 4-3 and Figure 4—1 represent only
some of the possible causes of anemia. Indeed, more than

DECISION-MAKING FLOW CHART FOR ANEMIA

HEMOGLOBIN / HEMATOCRIT

‘Normal or high Low

Electrophoresis, : Check other RBC indices

Hb electrophoresis, causes of
eval. of symptoms
abnormal O2— €.g., cardiac,
binding to pulmonary
hemoglobin —

¢ History of acute
blood loss
¢ Autoimmune
hemolytic anemia
¢ Anemia of chronic
disease
¢ Dyserythropoiesis Normal or high
¢ Anemia of infection
¢ Aplastic anemia
Low iron Normal iron ¢ Congenital
¢ Pernicious ¢ Folate ¢ Myeloproliferative
dyserythropoiesis
anemia malnutrition disease
anemia (CDA)
¢ Severe * GI problem Liver disease
* lron deficiency ¢ Hemoglobin ¢ Bone marrow malnutrition ¢ Liver disease ¢CDA
¢ Anemia of chronic electrophoresis examination for ¢ Some Gl
disease for thalassemias sideroblastic problem
¢ Renal disease anemias

Figure 4-1 ® Decision-making flowchart (algorithm) for symptoms of anemia.

 Chapter 4 Anemia: Diagnosis and Clinical Considerations 87

Some factors act as growth-inhibiting agents. Among

these is transforming growth factor-beta (TGF-8), which
causes inhibition of growth of hematopoietic stem cells.
Myeloid progenitors are known to be inhibited by tumor
Pallor
necrosis factor (TNF) and interleukin-4 (IL-4).
Weakness
Fatigue
Lethargy or malaise
Exercise dyspnea Tests in the Diagnosis of Anemia
Palpitation
Pica (consumption of substance such as ice, starch, or clay, Hemoglobin
frequently found in iron deficiency anemia)
Syncope (particularly following exercise) Hemoglobin is the main component of the RBC. It is the
Dizziness
physiologic carrier of oxygen to tissues and acts as a buffer to
Headache
Tinnitus or vertigo handle carbon dioxide formed in metabolic activities.'* The
Irritability three methods for measuring hemoglobin are the cyanmethe-
Difficulty sleeping or concentrating moglobin method, the oxyhemoglobin method, and_ the
Gastrointestinal symptoms method in which iron content is measured. The cyanmethe-
moglobin method as modified in 1978 is recommended by the
International Committee for Standardization in Hematology
and is the only one discussed. In this technique, blood is
one cause of anemia can exist in a patient. Obtaining the diluted in a solution of potassium ferricyanide and potassium
proper diagnostic studies in the shortest and most cost- cyanide, which oxidizes the hemoglobin to form methemo-
effective manner is the responsibility of the attending globin. Subsequently, methemoglobin forms cyanmethemo-
physician and the laboratory professionals. More details globin in the presence of the potassium cyanide. Because the
concerning the appropriate treatment of anemias are pro- absorption maximum occurs at a wavelength of 540 nm, the
vided in Chapters 6 through 14. absorbance of the solution is read in a spectrophotometer at
The natural history of anemia depends on its cause. For 540 nm and compared with a standard cyanmethemoglobin
example, a patient with an iron-deficiency anemia associated solution. The advantages of this method are that most forms
with carcinoma of the colon may present with signs of an of hemoglobin are measured, the sample can be directly
iron-deficiency anemia associated with blood loss from the compared with a standard, the solutions are stable, and the
tumor. Later, with more extensive tumor involvement, the coefficient of variation for the method is less than 2% at phys-
anemia may have a bone marrow failure component because iologic ranges.
of bone marrow replacement by the tumor (a myelophthisic Errors in the measurement of hemoglobin can be pro-
anemia). Patients with pernicious anemia require a lifetime of duced by improperly drawing or handling the specimen, by
parenteral vitamin B,, supplementation. Patients with other poorly prepared or stored reagents, faulty equipment, and
forms of megaloblastic anemia may need only a balanced diet operator error.
and replacement of folic acid.”
Transfusions can obscure and confuse the findings of
diagnostic tests in patients with anemia. Transfusions can sup- Hematocrit
press erythropoiesis; alter vitamin B,,, folate, and iron levels;
The hematocrit, or packed RBC volume, is the ratio of the vol-
and thwart the interpretation of diagnostic tests seeking the
ume of RBCs to the volume of whole blood. Hematocrit is usu-
specific cause of the anemia. It is important that a diagnosis be
ally expressed as a percentage (e.g., 42%) but is expressed in
made, if at all possible, before transfusions are given.
Standard International (SI) units as a decimal fraction (e.g.,
0.42 L/L). The venous hematocrit agrees closely with the
Other Factors Involved in Red Blood Cell central blood hematocrit but is greater than the total body hema-
tocrit. Anticoagulants—usually ethylene diaminetetraacetic acid
Production
(EDTA), oxalate, or heparin—are used to prevent the blood
Bone marrow stem cells require various other factors to from clotting so that the hematocrit may be measured.
continue to synthesize hemoglobin and permit proper matura- Measurement of the hematocrit may be done by centrifu-
tion of erythrocytes and other cells. These include cytokines, gation or through calculations performed on many automated
for example, stem cell factor, erythropoietin, interleukin-3 hematology instruments. The calculated hematocrit is the
(IL-3), and other interleukins. The hormones of the androgen product of the MCV and the RBC count.
group and thyroxin are also necessary. Many vitamins, such as The adult reference range for hematocrit is 42% to 52%
vitamin B,>, folate, vitamin C, vitamin E, pyridoxine (B,), in men and 37% to 47% in women. Different reference ranges
thiamin, riboflavin, and pantothenic acid, play roles in bone are required for neonates, very young children, and male and
marrow productivity. Certain metals, such as iron, manganese, female patients, and may vary among institutions.
and cobalt, are part of the production process for RBCs and Problems in the measurement of hematocrit include incor-
other bone marrow cellular components. rect centrifuge calibration, choice of sample site, incorrect ratio
88 Chapter 4 Anemia: Diagnosis and Clinical Considerations

of anticoagulant to blood owing to improper amount of blood RNA, which may be associated with lead poisoning and some
drawn, and reading error—particularly for hematocrit values malignancies. Howell-Jolly bodies (see Chap. 5), which
determined after centrifugation. In our laboratory, the coeffi- are small, round, blue inclusions seen in RBCs, are the result
cient of variation for hematocrit, within the reference range, is of leftover fragments of DNA. Howell—Jolly bodies are often
approximately 2%. With centrifuge hematocrit techniques, the seen in hyposplenism or asplenism, pernicious anemia,
lower hematocrit values are associated with the higher coeffi- and some hemoglobinopathies—-particularly thalassemia.'*"'°
cients of variation. Pappenheimer bodies, which are iron-containing or siderotic
granules, appear as purplish-blue granules with Wright’s stain
and as coarse blue granules with Prussian blue iron stain. The
Red Blood Cell Indices clinical disorders associated with Pappenheimer bodies
include sideroblastic anemia, alcoholism, thalassemia, and
RBC indices were introduced earlier. The MCV is the average
some myelodysplastic syndromes. Nucleated RBCs with iron
volume, expressed in femtoliters (fL), of RBCs, and is mea-
granules are known as sideroblasts, and RBCs containing iron
sured directly or calculated from the hematocrit and RBC
granules but without a nucleus are referred to as siderocytes.
count. The MCH is the content of hemoglobin in the average
RBC. MCH is calculated from the hemoglobin concentration Ringed sideroblasts are those in which more than five granules
and the RBC count. MCHC, the average concentration of
are arranged in a ring around the nucleus of an orthochromatic
normoblast (Fig. 4-3). Ringed sideroblasts are indicative of
hemoglobin in a volume of packed RBCs, is calculated from
the hemoglobin concentration and the hematocrit.'° ineffective erythropoiesis.
RBC indices are readily available from the automated A threadlike blue ring contained entirely within an abnor-
hematology counting devices. In devices in which the MCV mal RBC, which may or may not have a “figure-of-8” and a
is derived from the voltage changes formed during the RBC round or oval configuration, is known as a Cabot ring (see
count and the hemoglobin is measured by spectrophotometric Chap. 5). This is a remnant of the nuclear membrane. This
determination of the cyanmethemoglobin, the values are cal- infrequent finding may be seen in several clinical disorders,
culated as follows: the hematocrit equals the MCV times the including pernicious anemia, lead poisoning, and other severe
anemias. Heinz bodies (Fig. 44) are small, rounded, angular
RBC count, the MCH equals the hemoglobin divided by the
RBC count, and the MCHC equals the hemoglobin divided by inclusions about | um in diameter that are aggregates of dena-
the hematocrit. The reference range for MCV is 80 to 100 fL; tured hemoglobin and are negative when stained with Prussian
for MCH it is 27 to 31 pg; and for MCHC it is 32% to 36%. blue or other iron stains. Heinz bodies can be demonstrated only
In various anemic states, the indices may be altered as by using supravital stains (e.g., crystal violet) and are not visible
follows: in microcytic anemia, from an MCV of less than with the usual Wright’s stain. The clinical disorders that have
80 fL down to a low of approximately 50 fL; from an MCH been associated with Heinz bodies include glucose-6-phosphate
of less than 25 pg to approximately 15 pg; and from an dehydrogenase (G6PD) deficiency after exposure to oxidizing
MCHC of less than 30% to 22%. In the macrocytic anemias, drugs, a variety of unstable hemoglobinopathies, and alpha-
MCV values are usually greater than 100 fL and may be as thalassemia; they can also be seen after splenectomy.
high as, and sometimes even higher, than 120 fL; the MCHC
may be normal or decreased. The MCHC may be increased
only in the presence of spherocytosis, if at all. Reticulocyte Count
The reticulocyte count is useful in determining the response
and potential of the bone marrow. Reticulocytes are
Peripheral Blood Smear
Much information concerning the cause of an anemia can be
determined from a peripheral blood smear. Coexistent neutrope-
nia, thrombocytopenia, and anemia may indicate bone marrow
failure or a lack of a nutritional substance to provide adequate
bone marrow production. The size and shape of the RBCs can
be noted. Alteration in size of the RBCs results in anisocytosis;
alterations in their shape result in poikilocytosis. The hemoglo-
bin (chromatic) content of the RBCs can be inspected visually
on the peripheral smear. In addition, cytologic details on the
peripheral smear may provide clues to the etiology of the ane-
mia, the bone marrow response, or both. The white cells may be
evaluated. For example, excess lobulations of the polymor-
phonuclear leukocytes are seen in the hypersegmented granulo-
cytes of macrocytic anemias (see Chap. 7).
Basophilic stippling in the RBCs may suggest the pres-
ence of increased bone marrow production and reticulocytosis
(Fig. 4-2). It may also indicate that there are remnants of Figure 4-2 @ Reticulocytosis (new methylene blue stain).
 Chapter 4 Anemia: Diagnosis and Clinical Considerations 89

Figure 4-5 @ Ringed sideroblast (center) and siderocytes (surround- Figure 4-4™ Heinz bodies. (From Bell, A: Hematology. Listen, Look
ing cells). (From Bell, A: Hematology, Listen, Look and Learn. and Learn, Health and Education Resources, Inc., Bethesda, MD,
Health and Education Resources, Inc., Bethesda, MD, with with permission.)
permission.)

non-nucleated RBCs that still contain RNA. Reticulocytes Bone Marrow Smear and Biopsy
may be visualized after incubation with a variety of so-called
supravital dyes, including crystal violet or brilliant cresyl blue Bone marrow aspiration and biopsy are important diagnostic
(see Chap. 31). RNA is precipitated as a dye-protein complex. tools in the determination of anemia. Bone marrow interpre-
Reticulocytes under normal circumstances lose their RNA a tation and evaluation are covered in Chapter 2. Factors to be
day or so after reaching the bloodstream from the marrow. evaluated in interpretation of a bone marrow aspirate smear
Reticulocyte activity can be expressed as an absolute count, a and biopsy include maturation of the red and white cell series,
production index, or a percentage. The reference values for presence of megakaryocytes, ratio of myeloid to erythroid
reticulocytes range from 0.5% to 2.0% as a percentage of all cells, abundance of iron stores, presence or absence of granu-
RBCs. Because anemia should be accompanied by increased lomas, tumor cells, and overall estimate of bone marrow
bone marrow activity, the reticulocytes should be expected cellularity.
to increase. One may correct the reticulocyte count for the Interpretation of the bone marrow requires a differential
following formula: count of the myeloid, lymphoid, and erythroid series; an iron
stain; and other appropriate techniques, such as immunohisto-
Patient Hct % X Reticulocyte %
Corrected reticulocyte % = Re(crenee ice means) chemical stains if a differential diagnosis of lymphoprolifera-
tive or myeloproliferative disorders is being considered.
Manual reticulocyte counting is associated with poor Other appropriate specific stains may be indicated if metasta-
reproducibility. In the reference range, the coefficient of tic tumor or infection is suspected or being evaluated.'7"'’ The
variation is said to be as great as 50%. Automated reticulocyte other tests that may be performed in the diagnosis of anemias
counting using fluorescent compounds and automated instru- and the chapters in which these tests are discussed are listed
ments results in better reproducibility.”'° in Table 4—S.
Interpretation of the reticulocyte count must take into Anemia has physiologic, functional, and quantitative
account the age and nutritional status of the patient. Nor- parameters that may be related to hemoglobin or hematocrit
mal adults have a reticulocyte count between 0.5% and levels. The differential diagnosis of anemia requires careful
2.0%, or from 24 to 100 X 10° reticulocytes per liter. The consideration of a wide variety of marrow, extramedullary,
newborn infant has a higher reticulocyte count, which falls and interrelating disease states. A large armamentarium of
to the adult range usually by the second or third week of tests is available to aid in the differential diagnosis of the mul-
life. Sources of error in the reticulocyte count include sam- tiple types of anemia. Successful studies of causes of anemia
pling error resulting from counting relatively few reticulo- require broad knowledge of clinical laboratory techniques and
cytes in a large number of erythrocytes. The 95% confi- medicine.
dence level when counting 100 RBCs, where the true
reticulocyte count is 1%, ranges from 0.4% to 1.6%; obvi-
Patient Studies of Anemia
ously, there is a very high coefficient of variation unless
large numbers of RBCs are counted. Usually 1000 cells are Individual patient studies of anemia are addressed in Chapters
counted for a manual reticulocyte count which is in con- 6 to 14.
trast to the automated method that counts 30,000 cells.
90. Chapter 4 Anemia: Diagnosis and Clinical Considerations

Test Diagnostic Use Chapter

Hemoglobin electrophoresis Hemoglobinopathies 3

Thalassemia syndromes
Antiglobulin testing Hemolytic anemias 13
Osmotic fragility test Hereditary spherocytosis* 6,9; liek
Severe iron deficiency
Sickle cell disease
6-Thalassemia
Sucrose hemolysis test (sugar-water test) Paroxysmal nocturnal hemoglobinuria*
Hypoplastic anemias
Myelodysplastic syndromes
Acidified serum test (Ham’s test) Paroxysmal nocturnal hemoglobinuria
Congenital dyserythropoietic anemia type II
Tests for red blood cell enzymes Hemolytic anemias
G6PD deficiency
PK deficiency
Serum iron and iron-binding capacity Tron-deficiency anemia
Folate and vitamin B,;, measurements ' Megaloblastic anemias

*Primary use.
G6PD = glucose-6-phosphate dehydrogenase; PK = pyruvate kinase.

Questions
_1. Which of the following laboratory results would not bea 5 Homi hematocrit measured on automated lemololosy
usual criterion for making a diagnosis of anemia? instruments?
a. Decreased hemoglobin level a. Centrifugation
b. Decreased hematocrit level b. Photometrically
c. Decreased platelet count c. Calculation (MCV X RBC count)
d. Decreased RBC count d. Calculation (MCH x Hgb)
i) . What condition is not a cause of anemia? . A patient has the following results: Hct 26%; Hgb 8
a. Dietary deficiency g/dL; and RBC count 3.5 X 10°/uL. Calculate the RBC
b. Moderate exercise indices—MCV, MCH, and MCHC—and determine the
c. Decreased RBC production classification of the anemia.
d. Increased RBC destruction or loss a. MCV 88 fL; MCH 30 pg; MCHC 33 g/dL; normocytic,
3. Which response represents the most complete and correct normochromic
listing of the most common clinical signs of anemia? b. MCV 101 fL; MCH 33 pg; MCHC 35 g/dL;
a. Fatigue, weakness, dyspnea, pallor macrocytic, normochromic
b. Urticaria, hypertension, inflammation, nausea c. MCV 74 fL; MCH 22 pg; MCHC 31 g/dL; microcytic,
c. Nausea, hypertension, temperature elevation, melena hypochromic
d. Rapid pulse, inflammation, temperature elevation, d. MCV 70 fL; MCH 22 pg; MCHC 38 g/dL; microcytic,
dehydration hyperchromic

4. What is the most commonly accepted method for . Which of the following would not be characteristically
measuring hemoglobin? found on a peripheral blood smear in a case of
a. Conversion of hemoglobin to oxyhemoglobin, anemia?
followed by spectrophotometric measurement a. Anisocytosis and/or poikilocytosis
b. Iron content measured by radioimmunoassay b. Basophilic stippling, Howell—Jolly bodies, and Pap-
technique penheimer bodies
c. Copper sulfate measured by specific gravity c. Cabot rings and Heinz bodies
d. Conversion of hemoglobin to cyanmethemoglobin, d. Dohle bodies and toxic granules
followed by spectrophotometric measurement
 Chapter 4 Anemia: Diagnosis and Clinica! Considerations 9]

8. What is the diagnostic value of the reticulocyte count in 9. Which of the following is a lesser factor to be considered
the evaluation of anemia? in the interpretation of a bone marrow aspirate smear?
a. Determines response and potential of the bone a. Maturation of red and white blood cell series
marrow b. M:E ratio
b. Determines compensation mechanisms for anemia c. Type and amount of hemoglobin
c. Determines the corrected RBC count after d. Estimate of bone marrow activity
calculation
d. Determines the potential sampling error for RBC See answers at the back of this book.
count

@ Erythropoietin, a hormone produced in the kidney, is

increased in states of anemia.
ll

@ Anemia is usually characterized by decreased RBC count, g The hematocrit (Hct), or packed red cell volume, is deter-
hemoglobin, and/or hematocrit levels. mined by centrifugation of blood of either capillary or
venous origin; it can be calculated by the mean corpuscu-
mw Moderate anemias (7 to 10 g Hgb/dL) that develop slowly
lar volume (MCV) multiplied by the RBC count.
may have few clinical symptoms.
g The ratio between the hemoglobin and hematocrit values
m Severe anemias or those that develop rapidly (e.g., fol-
Sees
lowing acute blood loss) may be associated with dyspnea,
general lethargy, and orthostatic hypotension. mw The hematocrit reference range of adult males is 47% +
5%: for women, it is 42% + 5%
m Hemoglobin levels vary with age and sex. Reference
ranges should be determined and reported for infants, g The MCV is used as an estimation of the average size of
children, and adult men and women. the RBC, and is calculated by the hematocrit multiplied
by 10, divided by the RBC count (reference range: 80 to
m There are many causes of anemia. They include nutri- 100 fL).
tional deficiencies, blood loss, increased destruction or
decreased production of RBCs, infections, toxicity, hered- g The mean corpuscular hemoglobin (MCH) is a measure of
ity, and acquired defects. hemoglobin content and is calculated by the hemoglobin
multiplied by 10, divided by the RBC count (reference
m= Chronic anemia is usually compensated for by increased range: 27 to 31 pg).
levels of 2,3-DPG, which enables enhanced release of
oxygen from hemoglobin. m The mean corpuscular hemoglobin concentration
(MCHC) is derived by hemoglobin multiplied by 100,
m Reference range values for red cell indices can be used to divided by the hematocrit (reference range: 32% to 36%).
aid in the classification and diagnosis of anemias.
w The red cell distribution width (RDW) is an index of size
m Treatment of anemia varies with the cause. Generally, the variation and has been used to quantitate the amount of
causes should be determined before a treatment is instituted. anisocytosis seen on the peripheral blood smear (refer-
gw The cyanmethemoglobin method is the reference method ence range: 11.5% to 14.5%).
for measuring hemoglobin. mAnemias may be classified as normocytic and _nor-
m= Morphological examinations of the peripheral blood mochromic, microcytic and hypochromic, or macrocytic
smear and bone marrow preparations are valuable aids in and normochromic.
the diagnosis of anemias. m The reticulocyte count is useful in determining the
m Clinical symptoms associated with anemia may include response and potential of the bone marrow. Staining tech-
vertigo, light-headedness, muscle weakness, headache, or niques include new methylene blue or brilliant cresyl
dyspnea. blue. Normal adult values are 0.5% to 2.0%. The count
can be determined by automated or manual methods.
m Adult hemoglobin reference ranges are approximately
14 to 18 g/dL for men and 12 to 16 g/dL for women. w The corrected reticulocyte count is determined by patient
packed corpuscular volume (PCV) multiplied by the retic-
m Persons living at elevations above 8000 feet may have
ulocyte count (%), divided by the reference PCV.
persistently increased hemoglobin values secondary to
decreased oxygen saturation in ambient atmosphere.
92 Chapter 4 Anemia: Diagnosis and Clinical Considerations

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Wyngaarden, JB, et al (Eds): Cecil Text- Wyngaarden, JB, et al (Eds): Cecil Text-
—_ . Beck, WS: Hematology, ed 3. MIT
book of Medicine, ed 19, vol 1. WB Saun- book of Medicine, ed 19, vol 1. WB
Press, Cambridge, MA, 1981, p 16.
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Allen, RH: Megaloblastic anemias. In pp 883-888.
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Wyngaarden, JB, et al (Eds): Cecil Text- . Forget, BG: Sickle cell anemia and asso-
(Eds): Clinical Laboratory Medicine.
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Mosby- Year Book, St. Louis, 1992,
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pp 898-937.
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. Hoffbrand, AV, et al: Erythropoiesis and
Yesterday, today, and tomorrow, Routine Philadelphia, 1992, pp 888-893.
general aspects of anaemia. In Hoffbrand,
Hematologic Testing. Clin Lab Med . Brunning, RD, and McKenna, RW: Atlas
AY, and Pettit, JE (Eds): Essential
EWS, IWS. of Tumor Pathology. Tumors of the Bone
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Oxford, 1993, pp 12-35.
specimen acceptability. A College of Pathology, Washington, DC, 1994,
. Woodson, RD, et al: Introduction to
American Pathologists Q-probes study of p 496,
hemopoiesis. In MacKinney, AA, Jr (Ed):
703 laboratories. Arch Pathol Lab Med . Young, NS: Aplastic anemia and related
Pathophysiology of Blood. John Wiley &
119:203, 1995. bone marrow failure syndromes. In
Sons, New York, 1984, pp 12-15.
2. Benz, EJ: Structure, function, and synthe- Wyngaarden, JB, et al (Eds): Cecil
Nn. Nathan, DG: Hematologic disease. In
sis of the human hemoglobins. In Wyn- Textbook of Medicine, ed 19, vol 1.
Wyngaarden, JB, et al (Eds): Cecil Text-
gaarden, JB, et al (Eds): Cecil Textbook WB Saunders, Philadelphia, 1992,
book of Medicine, ed 19, vol 1. WB
of Medicine, ed 19, vol 1. WB Saunders, pp 831-837.
Saunders, Philadelphia, 1992,
Philadelphia, 1992, pp 872-877. IIS) Ross, DW: Laboratory evaluation of the
pp 817-822.
. Benz, EJ: Classification and basic patho- patient with hematologic disease. In Bick,
ON. Lindenbaum, J: An approach to the ane-
physiology of the hemoglobinopathies. In RL, et al (Eds): Hematology. Clinical and
mias. In Wyngaarden, JB, et al (Eds):
Wyngaarden, JB, et al (Eds): Cecil Text- Laboratory Practice, vol 1. Mosby-Year
Cecil Textbook of Medicine, ed 19, vol
book of Medicine, ed 19, vol 1. WB Saun- Book, St. Louis, 1993, pp 7-16.
1. WB Saunders, Philadelphia, 1992,
ders, Philadelphia, 1992, pp 877-879.
pp 822-831.
. Benz, EJ: Hemoglobinopathies with
. Kushner, JP: Normochromic, normocytic
altered solubility or oxygen affinity. In
anemias. In Wyngaarden, JB, et al (Eds):
Wyngaarden, JB, et al (Eds): Cecil Text-
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book of Medicine, ed 19, vol 1.
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pp 837-839.
pp 879-883.
 Chapter |

Evaluation of Cell
Morphology and
Introduction to Platelet
and White Blood Cell
Morphology
Kathy W. Jones, MS, MT(ASCP), CLS(NCA)

Introduction OBJECTIVES
Examination of the Peripheral At the end of the chapter, the learner should be able to:
Blood Smear
A Define the terms flagged and reflex test as they pertain to automated hematology
The Normal Red Blood Cell
results.
Assessment of Red Cell
Abnormality . Describe the importance of maintaining competency in morphological identification.

Variations in Red Cell 3. List justifications for performing a manual morphology review.
Distribution . List the steps in the performance of a peripheral blood smear examination.
Normal Distribution
Abnormal Distribution . Identify normal red blood cell morphology on a peripheral smear.

Variations in Size Nn.

NON List the terms referring to abnormal red cell distribution, variation in red cell size,
Anisocytosis and variations in red cell color/hemoglobin content, being able to identify each
Normocytes abnormality on a peripheral smear and specify the particular clinical conditions
Macrocytes associated with these abnormalities.
Microcytes
. Define anisocytosis and poikilocytosis and list clinical conditions in which they may
Hemoglobin Content—Color be reported.
Variations
. Define the terms normochromic, hypochromic, microcytic, and macrocytic as they
Normochromia
relate to red cell indices.
Hypochromia
Hyperchromia . Correlate red cell indices with red cell morphology and the diagnosis of anemia.
Polychromasia
. Define the following terms: target cells, spherocytes, ovalocytes, elliptocytes, stoma-
Variations in Shape tocytes, and be able to identify these cells on a peripheral smear.
Poikilocytosis
Target Cells (Codocytes) . List diseases that may show fragmented red cells and describe their pathophysiology.
Spherocytes . Describe the most common red blood cell inclusions and their composition, relating
Stomatocytes each inclusion to clinical conditions in which they may be found.
Ovalocytes and Elliptocytes
Sickle Cells (Drepanocytes) . Correlate pathophysiology of clinical conditions associated with abnormal appear-
Fragmented Cells ance of red cells noted on the peripheral blood smear.
(Schistocytes, Helmet . Describe normal platelet morphology and specify some platelet abnormalities seen in
Cells, Keratocytes) pathologic conditions.
Burr Cells (Echinocytes)
. List conditions showing leukocyte cytoplasmic as well as nuclear changes.
94 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

Acanthocytes (Thorn Cells,

Spur Cells)
Teardrop Cells (Dacrocytes)
Red Cell Inclusions
Howell-Jolly Bodies
Basophilic Stippling
Pappenheimer Bodies and
Siderotic Granules
Heinz Bodies
Cabot Rings
Hemoglobin CC Crystals
Hemoglobin SC Crystals
Protozoan Inclusions
Examination of Platelet
Morphology
White Blood Cell Morphology
Case Study

From the educational perspective, it is essential that stu-

Introduction
dents be trained to recognize cellular abnormalities, given that
Hematology, as a laboratory discipline, has changed dramati- the majority of the blood smears they review will be abnormal.
cally over the last three decades, primarily as a direct result of With the development of sophisticated automated blood-cell
automation and enhanced technology. Multichanneled auto- analyzers, the proportion of CBC samples that require a blood
mated hematology analyzers provide a reliable and accurate smear has steadily diminished and in many clinical settings is
complete blood count (CBC). The analyzers have evolved from 10 to 15 percent or less, depending on facility patient popula-
basic direct current impedance enumeration of cells, to very tion.' Even with the wealth of information that may be derived
complex instruments involving multiple technologies in one from an analyzer, the blood smear remains a crucial diagnostic
instrument. These various technologies have significantly aid, and maintaining technologist competency in the identifica-
enhanced the ability of automation to reduce actual personnel tion of cellular abnormalities should be a priority in the labora-
time spent in performing and reporting CBC analysis. An exam- tory. There are morphologic abnormalities that are critical in
ple of such technology is flow cytometry, which actually pro- the differential diagnosis of anemia that can still be determined
duces qualitative as well as quantitative differential cell counts only from a blood smear. Because of financial constraints of
and analysis. With this technology, abnormalities are flagged, educational programs, students may have little or no training
which is an indication to the technologist the instrument has on sophisticated automation common in laboratories today.
identified results outside of the established parameters of the Regardless, students should have a strong background in man-
laboratory and some form of review must be completed before ual white blood cell (WBC) differential counting as well as red
reporting test results. The instrument, in most cases, will spec- cell and platelet morphology assessment.
ify the result with an indicator such as a symbol (1.e., an aster- This chapter guides the student in interpretation of red
isk) or a high (H) or low (L) designation depending on the cell, platelet, and white cell morphology by first defining
abnormality. The required review may include comparing cur- what is considered normal. Evaluation of abnormalities is
rent patient results with prior history or having set parameters discussed in terms of basic assessment techniques of the
that would automatically request further testing referred to as cellular morphology with particular emphasis on recogniz-
reflex testing. Reflex testing of an abnormal or flagged result ing a distinct morphology and relating it to the clinical con-
would include a slide review of abnormal morphology (usually dition. Physiologic mechanisms are explained to give the
scanning 8 to 10 oil immersion fields) and if necessary, a com- reader a better understanding of an abnormality and how it
plete white cell differential count. The proportion of required relates to different disease states. This description of blood
manual morphology reviews are determined by the parameters cell morphology maximizes the ability of each slide reviewer
set through laboratory policies which are based on financial to recognize and correlate the blood morphology to the
and regulatory standards as well as medical considerations. In clinical pathology and abnormal results. A careful and
comparison with the procedure for an automated CBC, the thorough examination via light microscopy in the optimal
examination of a peripheral blood smear is labor intensive, and area on a well made, well stained peripheral smear provides
therefore a relatively expensive investigation and must be used an experienced observer with valued information about
judiciously. morphology normal or abnormal.’
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 95

Examination of the Peripheral Blood 3. Find an optimal area for the detailed examination and
Smear enumeration of cells.
¢ The RBCs should not quite touch each other.
A blood smear examination may be performed for a variety ¢ There should not be areas containing large amounts of
of reasons. It may be requested by the physician in response broken cells or precipitated stain.
to perceived clinical features or to an abnormality discovered ¢ The RBCs should have a graduated central pallor.
in a previous CBC. The examination may also be initiated by
High-Power (40) Scan
the technologist as a result of an abnormality in the CBC or
in response to a flagged result reported from the hematology — . Determine the WBC estimate.
analyzer. A flagged result is an indication that a particular ° The WBC estimate is performed under high power (400
result has not met established laboratory criteria and must be magnification). WBCs are counted in ten fields and aver-
reviewed. All laboratories should have a documented proto- aged. The estimate is reported according to the values
col for the examination of a laboratory-initiated blood smear given in Table 5-1.
examination. This protocol may be derived from studies per- * The WBC estimate can also be performed using a factor
formed in the facility or may be based on consensus stan- which is based on the fact that each WBC seen in 400x
dards published by nationally recognized organizations. magnification (high power field) is equivalent to
The microscopic examination of a peripheral blood approximately 2000 cells per uL of blood. For example,
smear provides a wealth of information to the clinician. It is if the average number of WBCs counted per high power
used to detect or verify abnormalities and subsequently may field was 5, the WBC estimate would be 5 2000 or
provide the physician with information from which they may 10,000/uL.
be able to make a differential diagnosis. Various forms of 2. Correlate the WBC estimate with the WBC counts per mm?
anemia may actually be diagnosed from abnormal red cell from the automated instruments.
morphology reported on a blood smear examination. The 3. Evaluate the morphology of the WBCs and record
report of abnormal white cell morphology may in fact indi- any abnormalities, such as toxic granulation or Dodhle
cate what additional testing may be required. Abnormal bodies
platelet morphology may detect a platelet function deficiency
Oil Immersion (100) Examination
even when sufficient numbers of platelets have been reported
from the analyzer. = . Perform a 100 WBC differential count.
The examination of the blood smear should include eval- ° Counting should be performed by moving in a zig-zag
uation of the red cell, white cell, and platelet morphology. To manner on the smear (Fig. 5—1).
evaluate the smear thoroughly the technologist should review ¢ All WBCs are to be included until a total of 100 have been
at least 8 to 10 oil immersion fields (OIF). The red cell mor- counted.
phology evaluation should include examination for deviations 2. Evaluate the RBCs for anisocytosis, poikilocytosis,
in size, shape, distribution, concentration of hemoglobin, hypochromasia, polychromasia, and inclusions.
color, and the appearance of inclusions. The white cell mor- 3. Perform a platelet estimate and evaluate platelet morphology.
phology evaluation should consist of differentiation of the ¢ Count the number of platelets in 10 OIFs.
white blood cells and their overall appearance including ¢ Divide by 10.
nuclear abnormalities, cytoplasmic abnormalities, and the ¢ Multiply by 15,000/mm‘* if the slide was prepared by an
presence of abnormal inclusions that may denote a disease automatic slide spinner; multiply by 20,000/mm+ for all
process. Platelet counts should be verified, and in addition the other blood smear preparations.
smear should be reviewed for platelet shape and size abnor- 4. Correct any total WBC count per mm* that has greater than
malities and for clumping. 10 nucleated red blood cells (NRBCs) per 100 WBCs.
When abnormal morphology is identified on the smear, ¢ When performing the WBC differential, do not include
the technologist must determine if the abnormality is possibly NRBCs in your count, but report them as the number of
artifactual and not pathological. For instance, refractile arti- NRBCs per 100 WBCs.
facts may be the result of water contamination and should not ¢ Use the following formula to correct a WBC count:
be confused with red cell inclusions. Echinocytes or crenated
WBC/mm? X 100
cells may also be artifacts if practically every cell in the thin Corrected WBCs/mm? =
100 + No. of NRBCs/100 WBCs
portion of the film has a uniformly spiculed membrane.
The following describes the necessary steps in the exam- The examination of the peripheral blood smear is per-
ination of the peripheral blood smear. formed as part of the hematologic laboratory workup called
Low-Power (10) Scan the CBE.

1. Determine the overall staining quality of the blood smear.

2. Determine if there is a good distribution of the cells on the The Normal Red Blood Cell
smear.
* Scan the edges and center of the slide to be sure there are To identify abnormal morphology, one must be competent in
no clumps of RBCs, WBCs, or platelets. normal morphology identification, and more importantly,
* Scan the edges for abnormal cells. capable of differentiating them from abnormal cells, so we
96 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

4000-7000
7000-10,000
10,000-13,000
13,000-18,000

begin this section with a description of normal red blood cell

(RBC) morphology. The mature erythrocyte (RBC, normo-
cyte, discocyte) has a remarkable structure in that it lacks a
nucleus and organelles, and yet all components necessary for
survival and function are present. It is described as a bicon-
cave disc with a survival time of approximately 120 days. On
a Romanowsky (1.e., Wright’s, Giemsa) stained blood smear,
this mature red cell has a reddish-orange appearance. The Figure 5-2 = Normal red blood cells.
RBC has an average diameter of 7 to 8 um and an average
volume of 90 fL. The area of central pallor is approximately
2 to 3 um in diameter (Fig. 5—2), and the size variation of red peripheral blood smear review. If these criteria are achieved,
cells from a normal patient is approximately 5%. The primary
the reviewer must make a general assessment of whether the
function of the red cell is the transportation of O, to the tis-
morphological abnormality is due to shape change (poikilocy-
sues of the body and transportation of CO, back to the lungs
tosis) or size change (anisocytosis) or a change in color. Most
for expulsion. Fundamental to the red cell is the formation of
assessments of anisocytosis are performed in concert with the
hemoglobin, which is ultimately responsible for binding the
red cell indices and the red cell distribution width (RDW)
oxygen molecule for transport. The oxygen-carrying capacity
rating obtained from the hematology analyzer. In assessing the
of each erythrocyte is dependent on the production of ade-
smear, the reviewer takes into account the percentage of cells
quate amounts of functional hemoglobin. Also fundamental
that vary in size in at least 10 OIFs. For example, if the mean
to the red cells functionality is the maintenance of the cellu-
corpuscular volume (MCV) was 65 fL (80 to 100 for adults),
lar membrane. The red cell membrane is composed of equal
the reviewer would expect to see a large percentage of small
weighted portions of lipids and proteins and is responsible
cells. If the MCV were 105 fL, the reviewer would expect to
for sustaining a constant surface-to-volume ratio. Maintain-
see primarily larger cells. More information on red cell indices,
ing the integrity of this membrane is essential to the cells
including calculations, may be found in Chapter 31 of this text.
shape and deformability which allows the RBC to traverse
The majority of laboratories use either qualitative
through the microvasculature of the body. An alteration of
remarks (few or marked) or a numerical grading (/+ to
this membrane may result in the inability of the red cell to
4+) based on percentage of variation and to describe the
function efficiently and ultimately may lead to the cell’s
type of cell or cells that have caused the variation from
early demise.
the normal. With this method, a reviewer can present to the
clinician a series of objective ratings that can translate to a
visual impression of a patient’s peripheral smear. This
Assessment of Red Cell Abnormality assessment may be critical to the physician’s differential
A well-stained and well-made blood smear with an even distri-
diagnosis of certain forms of anemia. Reviewers are urged
to avoid the use of terms that are vague (e.g., the term pre-
bution of RBCs in the area to be examined is essential for any
sent) owing to the wide variations in the implication it may
have to clinicians. See Table 5—2 for an example of guide-
lines in grading anisocytosis and poikilocytosis. Please note
that the assessment of RBC morphologic abnormalities
remains a manual task that is inherently subjective, and it is
imperative that laboratories establish guidelines based on
their own patient and physician population. It is essential to
patient care that the laboratory and the clinician have simi-
lar interpretations of the results reported for all RBC mor-
phology. Figure 5-3 is a composite chart of normal and
Figure 5-1 ™ Blood smear.
abnormal red cell morphology.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 97

should be in the thin portion of the smear where the red cells
are slightly separated from one another or at most, barely
touching, with no overlap. The thin area should represent at
least one-third of the entire film.* The reviewer should avoid
the thicker portion of the slide where cells are overlapping
and the edges of smear where cells may be artifactually dis-
Percentage of Cells that Differ in Size or Shape from Normal torted in size, shape, and color. An exception is to be made
RBCs when scanning for platelet clumping.
Normal 5%
Slight S%-10%
Se 10%-25%
Abnormal Distribution
2+ 25%-S50%
AGGLUTINATION
338 50%-15%
4+ >715% Agglutination is an aggregation of red cells into random
clusters or masses. Agglutination is the result of an antigen—
Sample Situations antibody reaction within the body, and in cases of autoag-
2+ Microcytes Few schistocytes glutination the reaction is actually with the patient’s own
1+ Macrocytes Few burr cells cells and the patient’s serum or plasma. Such is the case with
1+ Target cells cold antibody syndromes, for example, cold hemagglutina-
3+ Anisocytosis 2+ Poikilocytosis tion disease and paroxysmal cold hemoglobinuria (PCH).
Agglutination occurs at room temperature during sample
preparation and appears as interspersed areas of clumping
Included in this chapter are flowcharts that correlate the throughout the peripheral smear (Fig. 5—4). The use of saline
abnormal morphology with a possible pathology. This scheme will not disperse these agglutinated areas; however, warm-
should enable the learner to more easily associate an abnor- ing the sample to 37°C helps to break up the agglutinins,
mal morphology with the clinical condition. allowing for the possibility of normal slide preparation for
morphology review. The MCHC and MCV from these speci-
mens are usually falsely elevated in response to the agglutinin
Variations in Red Cell Distribution formation. Other forms of autoagglutination may also occur
Normal Distribution spontaneously but are more likely to be seen in connection
with certain hemolytic anemias, atypical pneumonia, staphy-
The area of smear that is reviewed for morphologic abnormal- lococcal infections, and trypanosomiasis. Agglutination is not
ities is of the utmost importance. The area to be reviewed to be confused with rouleaux which is described next.

RED BLOOD CELL MORPHOLOGY

Hemoglobin Red cell
Size variation | distribution Shape variation distribution
Target cell , Acanthocyte Pappenheimer bodies | Agglutination

‘e
(siderotic granules)

Spherocyte Helmet cell

(fragmented cell)

1
1
1
Schistocyte Basophilic stippling Rouleaux
1 (fragmented cell) (coarse)
1

9 »-
1
1
1
1
‘
1
4
Oval macrocyte Stomatocyte 1 Tear drop Howell-Jolly
1
1
'
'
'
'
'
'
'
Hypochromic Tr

macrocyte Polychromasia Sickle cell '

'
Burr cell
i

aN
1
1
'
1
1
1
(Reticulocyte) 1

Figure 5-3 ® Normal and abnormal red blood cell morphology.

98 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

rouleaux. This is considered artifactual and should not be

reported until it is verified in the thin portion of the smear or
a new slide is prepared.

Variations in Size
Anisocytosis
Any significant variation is size is known as anisocytosis. This
size variation is frequently found in the leukemias and in most
forms of anemia. The severity of the variation should also
correspond to an increased RDW. Anisocytosis results from
abnormal cell development, and typically results from a defi-
ciency in the raw materials (i.e., iron, vitamin B,,, folic acid)
needed to manufacture them or by a congenital defect in the
Figure 5-4 m™ Note the agglutination on the smear from a patient cell’s structure. Cell size may deviate, from measuring smaller
with cold hemagglutinin disease.
than the normal 7 um, to being larger than normal. The terms
ROULEAUX used to describe these abnormalities are microcyte (<6 um) and
Rouleaux is a condition in which red cells appear as stacks of macrocyte (29 wm). These terms are used in conjunction with
coins on the peripheral smear. The stacks may be short or the terms microcytosis and macrocytosis and should also corre-
long, but regardless of the length, the red cells appear stacked late with the red cell indice results. Anisocytosis is graded in
on one another. These stacks are rather evenly dispersed most facilities as 1+ to 4+ (see Table 5—2). When reporting
throughout the smear. Rouleaux formation is the result of anisocytosis, it is important to describe the morphology picture
elevated globulins or fibrinogen in the plasma where the red in terms of microcytosis or macrocytosis, or in cases of a dimor-
cells have been more or less “bathed” in this abnormal plasma phic population there may be the appearance of both (Fig. 5-6).
which gives them a sticky consistency. This lowers the zeta (C)
potential, thus facilitating the stacking effect (Fig. 5-5). The
Normocytes
use of a saline dilution of the serum disperses rouleaux.
Rouleaux formation correlates well with a high erythrocyte The average size of the erythrocyte is indicated by the mea-
sedimentation rate. surement of the MCV, a result generated by the automated
Rouleaux is seen in patients with hyperproteinemias hematology analyzer. The MCV is considered an integral part
such as multiple myeloma and Waldenstrom’s macroglobu- of a CBC. Observation of red cell morphology on the blood
linemia. It may also be seen in chronic inflammatory disor- smear provides a quality control check on the electronic
ders, and some lymphomas. It is important to note that in MCV, as well as the other two red cell indices, mean corpus-
cases of severe rouleaux it may be impossible to evaluate cell cular hemoglobin (MCH) and mean corpuscular hemoglobin
size or shape. concentration (MCHC).’ A “normal” MCV would correspond
Peripheral smears reviewed in the thick portions of the to the MCV reference range (80 to 100 fL for adults). Subse-
smear and entire smears made too thick may appear to exhibit quent review of the blood smear should yield no significant

Pigure 5-6 m Note the different size (anisocytosis) and shape (poikilo-
Figure 5-5 @ Peripheral blood showing marked rouleaux formation. cytosis) of the red cells. Compare the largest (macrocytic) cell below
Note the “stacked coin” appearance of the red cells. the arrow in the center of the field with the smaller (microcytic) cells.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 99

size variation from the normal 7- to 8-um red cell. This sce- excess plasma cholesterol may be taken up by the red cell
nario is referred to as normocytic and the red cells are referred which subsequently leads to an increase in the surface area of
to as normocytes. This information would prove useful to the the cell. However, this last mechanism may not be reflective of
physician in the diagnosis of anemia. In the case of a normal a “true” macrocytosis (obstructive liver disease). Macrocytes
MCV and a high RDW (normal RDW is 11.5% to 14.5%), the should be evaluated for shape (oval versus round) as shown in
reviewer would expect to see a mixture of large and small Figure 5—6, color (red versus blue), pallor (if present), and the
cells.* This scenario is referred to as a dimorphic population presence or absence of inclusions. The conditions in which
and may be the result of a recent blood transfusion, or possi- macrocytes may be seen are listed in Figure 5—7.
bly the patient may be in the recovery stages of anemia.
Patient history plays an important role in this situation.
Microcytes
Macrocytes A microcyte is a small cell having a diameter of less than 7 um
and an MCV of less than 80 fL. Anemias associated with
Macrocytes are cells that are approximately 9 um or larger in microcytes are said to be microcytic. The hemoglobin content
diameter, having an MCV of greater than 100 fL.* Anemias of these cells may be normal to decreased. A consequence of
associated with these cells are referred to as macrocytic. These any defect that results in impaired hemoglobin synthesis may
cells may appear in the peripheral circulation by several mech- produce a microcytic, hypochromic (MCHC <32% and cells
anisms. One mechanism is impaired deoxyribonucleic acid with increased central pallor) blood picture. When erythroid
(DNA) synthesis, which results in megaloblastic erythro- cells are deprived of any of the essential elements in hemoglo-
poiesis leading to a decreased number of cellular divisions, bin synthesis (see Chap. 6), the result is an increase in cellular
and consequently a larger cell. This form of erythropoiesis divisions and consequently a smaller cell in the peripheral
produces a megaloblastic anemia and may be the result of B,, blood. This form of abnormal hemoglobin synthesis is seen in
or folate deficiency, chemotherapy, or any process producing a iron deficiency, deficiency of heme synthesis (sideroblastic
nuclear maturation defect. Macrocytes with an oval shape anemia), deficiency of globin synthesis (thalassemia), and
(macroovalocytes), neutrophilic hypersegmentation, as well as chronic disease states. In the case of iron deficiency, microcy-
MCV values exceeding 120 fL are typically seen in this type tosis will not be visually apparent until iron stores in the body
of anemia. have been completely exhausted and iron deficient erythro-
The most common cause of nonmegaloblastic macrocyto- poiesis takes place, as in iron deficiency anemia (IDA). Iron
sis is accelerated erythropoiesis which results from conditions deficiency is the most common cause of anemia, affecting
such as acute blood loss or alcoholism. The cells are released some 30% of the world population and accounting for up to
prematurely from the marrow, are non-nucleated, and appear 500 million cases worldwide, which also makes it the most
larger than a mature erythrocyte. On a Wright-stained smear the common microcytic/hypochromic anemia in the world.° It is
cells will appear as round polychromatophilic macrocytes and especially common in women of childbearing age. The causes
on a supravitally (i.e., new methylene blue) stained smear they of this deficiency may vary depending on the age and sex of the
appear as reticulocytes. Neutrophilic hypersegmentation is not patient and it is important to determine what instigated the
typically seen in this form of macrocytosis.° deficiency before treatment begins.
Macrocytosis may result from other conditions, such as Decreased or defective globin synthesis also presents
hypothyroidism and various bone marrow disorders, as well as as a microcytic/hypochromic anemia, but in most cases this
occur in neonatal blood, postsplenectomy, and in cases where results from a genetic abnormality producing a hereditary

MORPHOLOGY - MACROCYTE

- Post- Chemotherapy Hypothyroidism

splenectomy |

Figure 5-7 @ Correlation of macro-

cytes to pathologic processes.
100. Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

anemia known as thalassemia. This microcytic/hypochromic

anemia is rare and in the homozygous form it may result in a
severe anemia with a high rate of mortality. In the milder het-
erozygous form this anemic picture may be confused with
IDA. The appearance of target cells, family studies, as well
as additional hematological testing may be needed for a dif-
ferential diagnosis.
It is important to note that other disease processes such
as sideroblastic anemia and lead poisoning may produce
significant numbers of microcytic red cells, in most cases
without hypochromia. Clinical conditions in which micro-
cytes may be seen as the predominant cell morphology are
illustrated in Figure 5-8.

Hemoglobin Content—Color Variations Figure 5-9® Note the large central pallor in many of the red cells
depicting hypochromia.
Normochromia
The term normochromic indicates the red cell is essentially red cells with increased central pallor or hypochromia. A lower
normal in color. A normochromic erythrocyte has a well MCHC result typically correlates with a larger central pallor in
hemoglobinized cytoplasm with a small but distinct zone of the affected red cells. In general, this is very reliable; however,
central pallor. The area of pallor does not exceed 3 um when it does not take into account the situation in which a true
measured linearly. In a well stained peripheral smear, the nor- hypochromia is observed in the presence of a normal MCHC.
mal red cells will appear reddish-orange in color. The term In many cases, the MCHC will not be concordant with what is
normochromic is used to describe an anemia with a normal observed on the peripheral smear. The morphologist should not
MCHC and MCH. When used in conjunction with a normal be unduly influenced by the RBC indices in the evaluation of
MCV, the anemia would be described as a normochromic/ hypochromia. True hypochromia will appear as a delicate
normocytic anemia (see Fig. 5—2). shaded area of pallor as opposed to pseudohypochromia (the
water artifact), in which the area of pallor is distinctly outlined.

Hypochromia It is important to note that not all hypochromic cells are micro-
cytic. Target cells possess some degree of hypochromia, and
Any RBC having a central area of pallor of greater than 3 um is there are macrocytes and normocytes that can be distinctly
said to be hypochromic. There is a direct relationship between hypochromic.
the amount of hemoglobin deposited in the red cell and the The most common condition manifesting hypochromia
appearance of the red cell when properly stained. The term is IDA. In severe cases of IDA, red cells exhibit an inordi-
hypochromia literally means “low color” and indicates that the nately thin band of hemoglobin. Patients with iron defi-
cells have less than the normal amount of hemoglobin. Typically ciency may have many hypochromic cells, depending on the
any irregularity in hemoglobin synthesis will lead to some magnitude of the deficiency. In addition to large numbers of
degree of hypochromia (Fig. 5—9). hypochromic cells, there may be large numbers of micro-
Most clinicians choose to assess hypochromia based on cytes as well. Iron deficiency anemia is commonly referred
the mean corpuscular hemoglobin concentration (MCHC), to as a microcytic/hypochromic anemia. Hypochromia in the
which by definition measures hemoglobin content in a given alpha (a) and beta (8) trait thalassemia syndromes is much
volume of red cells (100 mL). When the MCHC is <32% the less pronounced. However red cells in the a—-thalassemias
anemic process is described as being hypochromic and the slide and $-—thalassemia homozygous states show significant
reviewer should scan the peripheral smear for the presence of amounts of pallor.’ Sideroblastic anemias show a prominent

WNKe)stedale)Mole) acu l [elstolen

ay =

Possible pathology

Iron Thalassemic Sideroblastic Anemia of Lead

deficiency conditions anemias chronic disease Figure 5-8 @ Correlation of microcytes to pathologic
processes.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 10]

however, the reticulum cannot be visualized without supravi-

tal staining.
It is not uncommon to find a few polychromatophilic
1+ Area of central pallor is one-half of cell diameter
2+ Area of pallor is two-thirds of cell diameter
cells in a normal peripheral blood smear, because regenera-
3+ Area of pallor is three-quarters tion of red cells is a dynamic process. The reticulocyte count
4+ Thin rim of hemoglobin should reflect the degree of polychromasia. In the blood
smear, polychromatophilic red cells appear in varying
shades of blue. Any clinical condition in which the marrow
is stimulated, particularly RBC regeneration, will produce a
dimorphic blood picture—macrocytic, normocytic, and polychromatophilic blood picture. This represents effective
microcytic cells together, only some of which show true erythropoiesis as well as an assessment of bone marrow
hypochromia. Some hypochromic cells may be seen in function. Examples of several conditions in which polychro-
patients with lead poisoning. Refer to Table 5-3 for a guide- masia is noted include acute and chronic hemorrhage,
line to grading hypochromia. hemolysis, and any regenerative red cell process. The degree
of polychromasia is an excellent indicator of therapeutic
effectiveness when a patient is given iron or vitamin therapy
Hyperchromia
as a treatment for anemia. Refer to Table 5—4 for a guideline
Red cells with a decreased surface-to-volume ratio and a to polychromasia grading.
decreased or absent central pallor may be described as hyper-
chomic. True hyperchromia exists when the MCHC is >36%
and may be seen in the peripheral smears of patients with
Variations in Shape
hemolytic anemias, including hemolysis caused by burns. Even Poikilocytosis
though true hyperchromia does exist it is not reported as such. It
is reported in terms of the cell abnormalities resulting from the Poikilocytosis is the term used to describe a variation in red
increased volume of hemoglobin and the decreased surface area. cell shape. Normal erythrocytes vary only slightly from the
The cell produced front these phenomena appears as a solid red- concise round shape of a biconcave disc, so even a slight
dish-orange disc with no central pallor and is referred to as a variation in significant numbers may prove to be important.
spherocyte, and is discussed later in this chapter. These poikilocytic cells may take on such peculiar shapes
as teardrops, pencils, and sickles. The differential diagnosis
of anemia cannot be determined from a reported poikilocy-
Polychromasia tosis. The term should be used in conjunction with more
descriptive terminology which would specify the particular
When RBCs are delivered to the peripheral circulation prema-
morphological abnormality observed. Examples of specific
turely, their appearance in the Wright-stained smear is distinc-
poikilocytes are sickle cells, which result from abnormal
tive. These red cells are described as polychromatophilic
hemoglobin, and spherocytes, which result from a red cell
(diffusely basophilic) and are gray-blue in color and usually
membrane abnormality as many of the poikilocytic cells do.
larger than normal red cells (Fig. S—10). The basophilic color
The differential diagnosis of some forms of anemia may be
of the red cell is the result of the residual RNA involved in
determined by identification of a specific morphological
hemoglobin synthesis. Polychromatophilic macrocytes, as
abnormality.
seen on a Wright’s stained smear, are actually reticulocytes;
The term poikilocytosis refers to the entire red cell mor-
phology in the scanned area of a peripheral smear and is
graded as 1+ to 4+ (see Table 5—2). Many labs consider the
term poikilocytosis as a “catch all” phrase for abnormal red
cells and in lieu of grading the smear for poikilocytosis opt
only to grade the specific types of morphologically abnormal
cells seen. In these cases the particular cells should be
reported in terms of few, moderate, and many.

Percentage of Red Cells that are Polychromatophilic

Slight 1%
Te 3%
2+ 5%
3+ 10%
4+ >11%
Figure 5-10 @ Note polychromasia in the cell with the arrow.
102 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

Target Cells (Codocytes) (HS). This is an inherited, autosomal dominant condition and
is due to a deficiency of, or a dysfunction in, the membrane
Target cells appear on the peripheral blood as a result of an proteins spectrin, ankyrin, band 3 and/or protein 4.2.° The
increase in RBC surface membrane. They are artificially membrane cytoskeleton is dependent on these particular pro-
induced on the smear and their true circulating form, as seen teins to maintain the shape, deformability, and elasticity of
with an electron microscope, is a bell-shaped cell. The name the red cell. The deficiency and/or dysfunction of any one
codocyte is from the Greek word kodon meaning bell. In air- these membrane components will destabilize the cytoskele-
dried smears, however, they appear as “targets,” with a large ton, resulting in abnormal red cell morphology and a shorter
portion of hemoglobin displayed at the rim of the cell and a lifespan for the affected red cells in circulation.* Spherocytes
portion of hemoglobin that is central, eccentric, or banded are typically seen in large numbers in peripheral smears from
(Fig. 5-11). As the name implies, the cell actually resembles these patients. Premature destruction of these abnormal
a target and is sometimes referred to as a “bull’s eye” cell as erythrocytes in the spleen may produce a mild to severe
well as a “Mexican hat’ cell. hemolytic anemia depending on the severity of the abnormal-
The mechanism of targeting is related to excess mem- ity. Erythrocytes from patients with hereditary spherocytosis
brane cholesterol and phospholipid and decreased cellular he- have a mean influx of sodium twice that of normal cells.
moglobin. This is well documented in patients with liver dis- Because these spherocytes have increased ability to metabo-
ease, in whom the cholesterol/phospholipid ratio is altered. lize glucose, they can handle the excessive intracellular
Mature red cells are unable to synthesize cholesterol and sodium while in the plasma, but when they reach the microen-
phospholipid independently. As cholesterol accumulates in vironment of the spleen, the active—passive transport system is
the plasma, as seen in liver dysfunction, the red cell is ex- unbalanced with increased sodium and decreased glucose
panded by increased membrane lipid, resulting in increased resulting in swelling and hemolysis of these cells (see Chap. 9).
surface area. Consequently, the osmotic fragility is also de- In more than one-half of these patients, the MCHC is greater
creased (see Chap. 31). Target cells are seen in many types of than 36%. Yet, for individuals not exhibiting an increased
anemia (Fig. 5—12); however, they are most prominent in the MCHC, a careful observation of their peripheral smear is the
hemoglobinopathies, thalassemias and liver disease. key to diagnosis.’ Figure 5-13 depicts a blood smear from a
patient with hereditary spherocytosis.
The acquired forms of spherocytosis share the mutual
Spherocytes
defect with HS in that there is a loss of membrane. In the nor-
Spherocytes have a reduced surface-to-volume ratio that results mal aging process of red cells they gradually lose their func-
in a cell with no central pallor. Because of their density tionality through loss of cellular lipids, proteins, etc.; thus,
(intense color) and smaller size, they are easily distinguished spherocytes are produced as a final stage before senescent red
in a peripheral smear. Their shape change is irreversible and cells are detained in the spleen and trapped by the reticuloen-
may also be seen as microspherocytes. They are considered dothelial system. This natural process does not typically
the most common form of the erythrocyte morphological dis- result in anemia. Another mechanism of producing sperocytes
orders stemming from an abnormality of the cell membrane. that may result in a mild to severe anemia is autoimmune
This abnormality may be hereditary or acquired and may be hemolytic anemia. The coating of the red cells with antibodies
produced by a variety of mechanisms affecting the red cell and the detrimental effect of complement activation
membrane. Perhaps the most detailed mechanism for sphering results in the membrane loss of cholesterol accompanied by a
is the congenital condition known as hereditary spherocytosis loss of surface area without hemoglobin loss producing sphero-
cytes. The reduced surface-to-volume ratio of all spherocytes
renders them abnormally susceptible to osmotic lysis; conse-
quently, they have an increased osmotic fragility. Hemolysis is
known to result from membrane abnormalities; therefore, other
hemolytic processes may also produce spherocytes. They may
also be seen as microspherocytes in the peripheral smears of
burn patients. Figure 5—14 lists the more common pathologic
conditions in which spherocytes are seen.

Stomatocytes
The word stomatocyte is derived from the Greek word stoma,
which means mouth. They have a central pallor which is said
to be slit-like or mouth-like on peripheral blood smears. These
red cells are of normal size, but are not biconcave, and in wet
preparations appear bowl-shaped (Fig. 5-15). The abnormal
eo morphology resulting in the stomatocyte is thought to be the
result of a membrane defect. Stomatocytosis is associated with
Figure 5-11 ™ Note the target cell at the arrow.
abnormalities in red cell cation permeability that lead to
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 103

MORPHOLOGY - TARGET CELLS

Figure 5-12 ™ Correlation of target cells to

pathologic processes.

changes in red cell volume, which may be either increased (hy- condition or at most a mild normochromic/normocytic
drocytosis) or decreased (xerocytosis), or is some cases, near anemia), hemolytic anemia, alcoholic cirrhosis, and acute
normal.’ Hydrocytosis and xerocytosis represent the alcoholism. Stomatocytosis is also present on peripheral
extremes of a spectrum of red cell permeability defects.? The blood smears of patients with Rh deficiency syndrome, also
exact physiologic mechanism of stomatocytic shape is poorly known as Rh null disease, in which erythrocytes from these
understood and the molecular basis of this disorder is rare individuals have either absent (Rh,,,) or markedly
unknown. Stomatocytosis may be acquired or congenital. As reduced (Rh,,.g)Rh antigen expression. This may result in a
with hereditary spherocytosis, stomatocytes are seen in sig- mild to moderate hemolytic anemia, and mutations in the Rh30
nificant numbers in the hereditary form known as hereditary and RhAG genes have been associated with this syndrome.”
stomatocytosis and in smaller numbers in the acquired form.
Many chemical agents can induce stomatocytosis in vitro
(phenothiazine and chlorpromazine); however, these changes Ovalocytes and Elliptocytes
are reversible.'° Stomatocytes are known to have an increased Many investigators consider the terms ovalocyte and ellipto-
permeability to sodium; consequently, their osmotic fragility cyte to be interchangeable; however, for the purposes of this
is increased. discussion, they are viewed as distinct and separate. This
Stomatocytes are more often artifactual than a true man- morphological abnormality is thought to be the result of a
ifestation of a particular pathophysiologic process. The arti- mechanical weakness or fragility of the membrane skeleton
factual stomatocyte has a distinct slit like area of central and may be acquired or congenital. The pathogenesis of the
pallor, whereas the area of pallor in the genuine stomatocyte formation of either of these cells is unknown. Ovalocytes may
appears shaded. Several of the associated disease states in be considered as more egg-shaped and have a greater ten-
which stomatocytes may be found are hereditary spherocyto- dency to vary in their hemoglobin content. They can appear
sis (the stomatospherocyte is best viewed in wet prepara- normochromic or hypochromic, normocytic or macrocytic.
tions); hereditary stomatocytosis (which is usually a benign Megaloblastic anemia is characterized by oval macrocytes
(macroovalocytes) that may be 9 um or more in diameter and
lack central pallor (Fig. 5—16).*

MORPHOLOGY - SPHEROCYTES

Figure 5-13 m Note the spherocyte at arrow in a blood smear from Figure 5-14 @ Correlation of spherocytes to pathologic processes.
a patient with hereditary spherocytosis.
104 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

Figure 5-15 ™ Stomatocytes in peripheral blood.

Figure 5-17 ™ Note the oval macrocyte (microovalocyte) at the

Elliptocytes, on the other hand, are pencil, rod, or cigar- arrow. Smear from a patient with pernicious anemia.
shaped and hemoglobin appears to be concentrated on both
ends of the cell. They are invariably not hypochromic,
exhibiting a normal central pallor. Hereditary elliptocytosis Sickle Cells (Drepanocytes)
(HE) is an inherited condition with anywhere from 25% to
90% of all cells demonstrating the elliptical appearance. The Depranocytes or sickle cells are typically crescent or sickle
erythrocytes in HE, in most cases, have a normal survival shaped with pointed projections at one or both ends of the
time; patients are typically asymptomatic and are diagnosed cell. These cells have been transformed by hemoglobin poly-
incidentally during testing for unrelated conditions.’ In merization into rigid, inflexible cells no longer resembling the
approximately 10% of cases where red cell survival time is normal biconcave disc (Fig. 5-19). Patients may be homozy-
shortened, patients’ symptoms may vary from a mild to severe gous or in some cases heterozygous for the presence of the
transfusion-dependent hemolytic anemia. Mutations in the abnormal hemoglobin, hemoglobin S. In the homozygous
red cell membrane protein a-spectrin account for a majority patient, physiologic conditions of low oxygen tension (in vivo
of cases of HE, with the remaining cases arising from muta- or in vitro) cause the abnormal hemoglobin to polymerize,
tions in B-spectrin or protein 4.1R (Fig. 5—17)." forming tubules that line up in bundles to deform the cell. The
Ovalocytes/elliptocyte may be seen in association with surface area of the transformed cell is much greater, and the
several disorders in addition to those already mentioned such normal elasticity of the cell is severely restricted. These cells
as microcytic/hypochromic anemia, myelodysplastic syn- have lost their ability to deform and in many cases are unable
dromes, and myelophthistic anemia.’ Refer to Figure 5—18 for to negotiate the microvasculature of the tissues, which leads
a description of pathologic processes associated with ovalo- to oxygen depravation in those areas (see Chap. 11).
cytes and elliptocytes (see Chap. 9). Most sickled cells possess the ability to revert to the disco-
cyte shape when oxygenated; however, approximately 10% are
incapable of reverting to their normal shape. These irreversibly
sickled cells (ISCs) are the result of repeated sickling episodes.
On the peripheral smear, they appear as crescent-shaped cells
with long projections. When reoxygenated, the ISCs may
undergo fragmentation. During a symptomatic period, the per-
centage of ISCs varies tremendously, and, consequently, it does
not correlate with symptomatology. Sickle cells are not usually
seen in the peripheral smears of individuals who are heterozy-
gous (Hgb AS), and are only rarely seen in conjunction with
other abnormal hemoglobins (i.e., Hgb Cyaiem Hgb Syrempnis)-
Classically, sickled cells are best seen in wet preparations. Many
of the cells observed on the Wright—Giemsa stain are the oat
cell-shaped form of the sickled cell (Fig. 5-20). In this form, the
projections are much less pronounced and the central area of the
cell is fairly broad. This shape is reversible.'* The more promi-
nent pathologic conditions in which sickle cells may be observed
Figure 5-16 ® Note the high percentage of elliptocytes in this are listed in Figure 5-21. In this figure, a morphological distinc-
blood smear from a patient with hereditary elliptocytosis. tion is made between ISCs and reversibly sickled cells.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 105

MORPHOLOGY - OVALOCYTE/ELLIPTOCYTE

Figure 5-18 @ Correlation of ovalo-

cytes and elliptocytes to pathologic
processes.

Fragmented Cells (Schistocytes, Helmet Schistocytes are the extreme form of red cell fragmen-
Cells, Keratocytes) tation (Fig. 5-22). Whole pieces of red cell membrane
appear to be missing, and bizarre red cells are apparent.
Schistocytes are split, cut, or cloven cells resulting from some Schistocytes may occur in patients with microangiopathic
form of trauma to the cell membrane. It is recognized that not hemolytic anemia, disseminated intravascular coagulation
all membrane alterations occur pathologically. However, (DIC), heart valve surgery, hemolytic uremic syndrome,
there are certain triggering events in disease that invariably thrombotic thrombocytopenic purpura,'* renal graft rejec-
lead to fragmentation such as alteration of normal fluid circu- tion, vasculitis, in severe burn cases, as well as march hemo-
lation. Examples of fluid alterations are the development of globinuria (a form of hemoglobinuria seen in soldiers and
fibrin strands, damaged endothelium, or a damaged heart long-distance runners).
valve prosthesis. The flow of blood in the circulation may Keratocytes are red cells that have been caught on fibrin
actually sweep the erythrocytes through the fibrin strands, strands in circulation, and rather than splitting, the cell hangs
splitting the red cell. The shapes of these cells vary based on over the fibrin fusing two sides of the cell together, creating a
the shear forces and presentation of the red cells as they are vacuole. Once the cell escapes from the fibrin strand it
cut by the fibrin. Intrinsic defects of the red cell make it less appears in the peripheral blood as a red cell with a vacuole in
deformable and, therefore, more likely to be fragmented as it one end resembling a blister and is called a blister cell. It also
traverses the microvasculature of the spleen. Examples such is said to resemble a women’s handbag and may be called a
as antibody-altered red cells and red cells containing inclu- pocket-book cell (see Fig. 5—22). Once the vacuole ruptures,
sions have significant alterations that increase their likelihood the resulting cell appears to have two horns. This “horned”
of being fragmented, consequently decreasing their survival cell also resembles a helmet and is sometimes reported as
time. such, but is actually a keratocyte (Greek for keras, horn).* The

Figure 5-19 m Irreversibly sickled cells. Figure 5-20@ Reversible, oat-shaped sickle cell.
106 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

MORPHOLOGY - SICKLE CELLS “XX

Possible pathology

‘Sickle cell HemoglobinC

anemia (SCA) Harlem

Figure 5-25 ™ Note the bite cell at the arrow.

A helmet cell and a bite cell are, therefore, one and the same.
The helmet cells may also be seen in patients with pulmonary
emboli, myeloid metaplasia and DIC. All fragmented red cells
le es are considered fragile and their survival time is diminished sig-
Zins

uywv nificantly to days, if not hours, owing to splenic sequestration.

Please note that all laboratories may not report frag-
mented red cells in the same manner (i.e., all fragmented red
Figure 5-21 ™ Correlation of sickle cells to pathologic processes. cells reported as schistocytes) owing to the similarities in
their origins. Regardless of the specificity of the terminology
used, it is imperative that the morphologists give a qualitative
primary difference in the two cells is not in their appearance,
estimate of the abnormality seen in all fields. Especially in
but in their formation.
significant numbers, the appearance of fragmented red cells
The helmet cell also has distinctive projections, usually
will provide physicians with important information on the
two, surrounding an empty area of the red cell membrane.
condition of their patients.
Helmet cells are seen in hematological conditions in which
Refer to Figure 5—24 for a flowchart correlation of the
large inclusion bodies are formed (Heinz bodies, Howell—Jolly
fragmented cells matched to the pathologic processes in
bodies). Fragmentation occurs by the pitting mechanism of
which they may be observed.
the spleen. This pitting mechanism removes the inclusion from
the cell, giving the appearance of having taken a “bite out of the
cell” and is sometimes referred to as a bite cell (Fig. 5—23). Burr Cells (Echinocytes)
Burr cells (echinocytes) are red cells with approximately 10 to
30 rounded spicules evenly placed over the surface of the red
cells (see Fig. 5-22). They are normochromic and normo-
cytic, for the most part. They may be observed as an artifact,
usually as a result of specimen contamination, in which case
they will appear in large numbers and will present with evenly
dispersed smooth projections and may be referred to as cre-
nated. The terms crenated cell and echinocyte may be used
interchangeably by some reviewers and therefore are not
reported. “True” burr cells occur in small numbers and
appear irregularly sized with unevenly spaced spicules. They
may be seen in uremia, heart disease, cancer of the stomach,
bleeding peptic ulcer, immediately following an injection of
heparin, and in a number of patients with untreated hypothy-
roidism. In general, they may occur in situations that cause a
Figure 5-22 @ Peripheral blood from a patient with renal disease. change in tonicity of the intravascular fluid (e.g., dehydration
Note the presence of fragmented cells: A. burr cells; B. acanthocyte; and azotemia). Burr cells may be considered pathologic and
C. blister/pocketbook cells; D. schistocyte. should be reported.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cel! Morphology 107

MORPHOLOGY
- FRAGMENTED CELLS a >
yy

Burr cells Heimet cells |

Figure 5-24# Correlation of fragmented cells to pathologic processes. HA = hemolytic anemia; DIC = disseminated intravascular coagula-
tion; HUS = hemolytic uremic syndrome; TTP = thrombotic thrombocytopenic purpura.

Acanthocytes (Thorn Cells, Spur Cells) lecithin-cholesterol acyltransferase, which has been well doc-
umented in patients with severe hepatic disease. This enzyme
An acanthocyte is defined as a cell of normal or slightly reduced is synthesized by the liver and is directly responsible for
size, possessing 3 to 12 spicules of uneven length distributed esterifying free cholesterol; when this enzyme is deficient,
along the periphery of the cell membrane. The uneven projec- cholesterol is increased in the plasma. Acanthocytes may also
tions of the acanthocyte are blunt rather than pointed, and the be seen in myeloproliferative disorders, microangiopathic
acanthocyte can easily be distinguished from the peripheral hemolytic anemia (MAHA), and autoimmune hemolytic ane-
smear background because it appears to be saturated with hemo- mias. The presence of acanthocytosis in peripheral blood
globin. It appears essentially as a spherocyte with thorns. The smears remains the hallmark of the clinical diagnosis of most
MCHC is, however, always in the normal range (Fig. 5—25). neuroacanthocytosis syndromes, such as chorea-acanthocytosis
Specific mechanisms relating to the formation of acan- (ChAc) and McLeod syndrome."
thocytes are unknown; however, some details about these The red cell responds to this excess cholesterol in one of
peculiar cells are of interest. Acanthocytes contain an excess two ways, depending on the balance of other lipids in the mem-
of cholesterol and have an increased cholesterol-to-phospho- brane. It will become a target cell or an acanthocyte. Once an
lipid ratio; consequently their surface area is increased. acanthocyte is formed, it is very liable to splenic sequestration
The lecithin content of acanthocytes is decreased. The only and fragmentation, and the fluidity of the membrane is directly
inherited condition in which acanthocytes are seen in high affected. The most prominent pathologies in which acantho-
numbers is the rare condition abetalipoproteinemia. Most cytes may be observed are listed in Figure 5—26.
cases of acanthocytosis are acquired, such as the deficiency of

Teardrop Cells (Dacrocytes)

Teardrop cells appear in the peripheral circulation as tear-
shaped or pear-shaped red cells (Fig. 5-27). The extent to
which a portion of the red cells form tails is variable, and
these cells may be normal, reduced, or increased in size. The
exact physiologic mechanism is unknown, yet teardrop for-
mation from inclusion-containing red cells is well docu-
mented. As cells containing large inclusions attempt to pass
through the microcirculation, the portion of the cells contain-
ing the inclusion cannot pass through and consequently gets
pinched, leaving a tailed end. For some reason, the red cell is
unable to maintain the discocyte shape once this has occurred.
Teardrop cells are seen most prominently in idiopathic
myelofibrosis with myeloid metaplasia. This type of morpho-
logical finding can also be seen in patients with the thalassemia
Figure 5-25 m™ Note the acanthocytes on this peripheral smear. syndromes, in drug-induced Heinz body formation,‘in iron
108 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

MORPHOLOGY - ACANTHOCYTES gh

Figure 5-26 ® Correlation of acanthocytes to pathologic processes.

deficiency, and in conditions in which inclusion bodies are with thalassemic syndromes, sickle cell anemia as well as
formed. They may also be seen in megaloblastic processes as other hemolytic anemias, and in megaloblastic anemias.
large tear-shaped cells (macroteardrops). Refer to Figure 5—3
for a composite of abnormal red cell morphology. Basophilic Stippling
Red cells that contain ribosomes can potentially form stippled
Red Gell Inclusions cells; however, it is thought that the actual stippling is the result
Howell-Jolly Bodies of the drying of cells in preparation for microscopic examina-
tion. Coarse, diffuse, or punctate basophilic stippling may occur
Howell—Jolly bodies (Fig. 5-28) are nuclear remnants con- and consist of ribonucleoprotein and mitochondrial remnants
taining DNA. They are | to 2 jm in size and may appear (Fig. 5-29). These aggregates of ribosomes result from an alter-
singly or doubly in an eccentric position on the periphery of ation in the biosynthesis of hemoglobin.
the cell membrane. They are thought to develop in periods of Diffuse basophilic stippling appears as a fine blue dusting,
accelerated or abnormal erythropoiesis. They may be seen in whereas coarse stippling is much more clearly outlined and eas-
Romanowsky, i.e., Wright’s, Giemsa, or supravitally stained ily distinguished. Punctate basophilic stippling is a coalescing of
peripheral smears. smaller forms and is very prominent and easily identifiable.
A fragment of the chromosome becomes detached and is Stippling may be found in any condition showing defective
left floating in the cytoplasm after the nucleus has been or accelerated heme synthesis, such as alcoholism, thalassemia
extruded. Under ordinary circumstances, the spleen effec- syndromes, megaloblastic anemias, and arsenic intoxication. It
tively pits these nondeformable bodies from the cell. How- is also considered a characteristic feature in the diagnosis of lead
ever, during periods of erythroid stress, the pitting mechanism poisoning. Basophilic stippling may be seen on a Romanowsky
cannot keep pace with inclusion formation. or supravitally stained peripheral smear. It is important for the
Howell-Jolly bodies may be seen after surgical splenec- reviewer not to confuse stippling with Pappenheimer bodies.
tomy, congenital absence of the spleen, or splenic atrophy The primary differentiation factors are that stippling appears
after multiple infarctions. They may also be seen in patients

Figure 5-27 @ Teardrop cells (peripheral blood). Figure 5-28 ™ Howell-Jolly body.
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 109

that Romanowsky stains visualize Pappenheimer bodies by

staining the protein matrix of the granule, whereas Prussian blue
stain is responsible for staining the iron portion of the granule.
Once the presence of siderotic granules has been con-
firmed by iron stains, the cells in which they are found are
termed siderocytes. Siderocytes containing a nucleus are
described as sideroblasts and are commonly seen in siderob-
lastic anemias. Sideroblasts exhibiting numerous siderotic
granules found within the mitochondria forming a ring around
at least one-third of the nucleus, are labeled as pathologic
ringed sideroblasts. Siderocytes are seen in any condition in
which there is iron overloading such as hemochromatosis or
hemosiderosis. They may also be seen in the hemoglo-
binopathies (e.g., sickle cell anemia and thalassemia) and in
patients following splenectomy.
Figure 5-29 ™ Note the cells with red cell inclusions: basophilic
stippling seen on a peripheral smear in a patient with lead poisoning.
Heinz Bodies
homogeneously over the cell, whereas Pappenheimers tend to Heinz bodies are formed as a result of denatured or precipi-
appear as clusters in the cells periphery. tated hemoglobin. They are large (0.3 to 2 um) inclusions that
are rigid and severely distort the cell membrane. They can be
formed for visualization in vitro by incubation with phenylhy-
Pappenheimer Bodies and Siderotic
drazine (a strong oxidizing agent). On initial exposure to
Granules phenylhydrazine, small crystalline bodies appear, coalesce,
Pappenheimer bodies (siderotic granules) are small, irregular and migrate to an area beneath the cell membrane. This pro-
magenta inclusions seen along the periphery of red cells. They cedure is used before staining with crystal violet or brilliant
usually appear in clusters, as if they have been gently placed on cresyl blue where the presence of Heinz bodies may be seen
the red cell membrane. Their presence on a Wright’s or a on the peripheral smear. Heinz bodies cannot be visualized
supravital stained peripheral smear is presumptive evidence for with Romanowsky stains (Fig. 5-31).
the presence of iron. However, the Prussian blue stain is the Heinz bodies may be seen in the a-thalassemic syndromes,
confirmatory test for determining the presence of these inclu- glucose-6-phosphate dehydrogenase (G6PD) deficiency under
sions. These bodies/granules in RBCs are nonheme iron, oxidant stress, and in any of the unstable hemoglobin syn-
resulting from an excess of available iron throughout the body. dromes (1.e., hemoglobin K6lIn, hemoglobin Zurich). They may
Even though Pappenheimers and siderotic granules are the also be seen in red cell injury resulting from chemical insult.
same inclusion, they are designated differently depending on
the stain used. The inclusions are termed Pappenheimer Cabot Rings
bodies when seen in a Wright-stained smear (Fig. 5—30) and
siderotic granules when seen in Prussian blue or other kinds of The exact physiologic mechanism in Cabot ring formation
iron stain. The explanation for the difference in terminology is has yet to be elucidated. This structure may represent a part of

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‘ ‘s
= 4

Figure 5-351 ® Heinz body prep; note the appearance of Heinz

Figure 5-30 m Pappenheimer bodies (Wright stain). body inclusions.
110 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

Table 5—5 summarizes abnormal red cell morphologies and

associated disease states and RBC inclusions.

Hemoglobin CC Crystals
Hemoglobin (Hb) C crystals may be found in hemoglobin CC
disease. HbC disease is a mild chronic hemolytic anemia in
which the patient is homozygous for the abnormal hemoglobin
C.'5 HbCC crystals are formed by the crystallization of the
abnormal hemoglobin into one end of the red cell membrane.
The crystal forms in a hexagonal shape with blunt ends, leaving
the remainder of the cell with the appearance of being empty.
These crystals tend to stain dark red and are said to resemble a
“bar of gold” and may be referred to as such (Fig. 5—33).
HbCC crystals may not always be demonstrated in HbC
Figure 5-32 ™ Note the appearance of a Cabot’s ring in the cell at
disease, but their appearance has been found to increase after
the arrow.
splenectomy. HbCC crystals are not seen in HbC trait (HbAC).

the mitotic spindle, remnants of microtubules, or a fragment

of the nuclear membrane. Cabot rings are found in heavily Hemoglobin SC Crystals
stippled cells and appear in a figure-of-eight conformation
similar to the beads of a necklace (Fig. 5-32). Cabot rings Hemoglobin SC (HbSC) crystals may be found on the periph-
may be found in megaloblastic anemias, dyserythropoiesis, eral smears of patients diagnosed with HbSC disease. SC dis-
homozygous thalassemia syndromes, and postsplenectomy. ease is a chronic hemolytic disorder punctuated by acute

Microcytes Sickle Cells

¢ Iron-deficiency anemia ¢ Sickle cell anemia

¢ Thalassemias ¢ Sickle thalassemia
¢ Lead poisoning
Acanthocytes
e Sideroblastic anemia
¢ Congenital abetalipoproteinemia
Macrocytes
e Vitamin E deficiency
¢ Megaloblastic anemias ¢ Alcohol intoxication
¢ High reticulocyte count ¢ Postsplenectomy
¢ Liver disease
Burr Cells
¢ Myelodysplatic syndromes
¢ Liver disease
Target Cells
¢ Renal disease
¢ Liver disease ¢ Severe burns
¢ Hemoglobinopathies ° Bleeding gastric ulcers
¢ Thalassemias
Helmet Cells
¢ Sideroblastic anemia

Spherocytes
° G6PD deficiency
¢ Pulmonary emboli
¢ Hemolytic anemias
Schistocytes
* Posttransfusion
¢ Hereditary spherocytosis * Disseminated intravascular coagulopathy (DIC)
Elliptocytes * Thrombotic thrombocytopenic purpura (TTP)
¢ Hemolytic uremic syndrome
* Hereditary elliptocytosis
¢ Iron-deficiency anemia Teardrop Cells
¢ Thalassemias ¢ Severe anemias
Stomatocyte ¢ Myeloproliferative disorders
¢ Pernicious anemia
* Acute alcoholism
¢ Malignancies
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 11]

in order to recognize their appearance as an abnormality need-

ing further review or testing.
All four species of the malaria parasite will invade
RBCs. The species include Plasmodium vivax, Plasmodium
malaria, Plasmodium falciparum, and Plasmodium ovale and
are transmitted by the Anopheles mosquito. The parasite may
appear in different forms (i.e., ring, troph) and although it is
important to the physician for a differential diagnosis and
treatment, all reviewers are not expected to be proficient in
identification of the specific form. The primary concern is the
recognition of the abnormality as a parasite and that it is not
confused with normal morphology such as platelets superim-
posed over red cells.
Babesia microti is also an organism that invades red
cells. It is transmitted by tick bites and may appear as ring
Figure 5-35 ™ Note the hexagonal shaped crystal inclusions in a forms resembling some forms of malaria. The distinguishing
peripheral smear from a patient with HbC disease. These HbC
feature of Babesia is that it also invades blood circulation and
crystals leave the remainder of the cellular cytoplasm to appear as
“empty.” on blood smears may appear in groups outside the erythro-
cyte. Patient symptoms and travel history are also useful in
differentiating the two organisms (Fig. 5—35).
painful crisis and diverse chronic organ damage, secondary to
the presence of both HbS and HbC.' The pathophysiology of
the disease is exacerbated by the presence of both hemoglo- Examination of Platelet Morphology
bins, as they tend to exhibit traits that are common to each
The normal platelet has several distinctive morphological
such as sickling from HbS and crystallization from HbC. The
characteristics. This structure measures approximately 2 to
result of this combination is the formation of crystals with fin- 4 um, with a discoid shape and even blue granules dis-
gerlike blunt-pointed projections protruding from the cell persed throughout a light-blue cytoplasm (Fig. 5-36).
membrane. The projections have been said to resemble the In pathologic states, platelets may appear as blue or gray
Washington Monument and consequently SC crystals may be agranular discs; they may be extremely large and may show
referred to as “Washington Monument” crystals (Fig. 5-34). tailing or streaming of the cytoplasm. In rare instances,
one may see megakaryocytic fragments in the peripheral
Protozoan Inclusions circulation.
A close and thorough examination of platelet morphology
Two organisms are briefly discussed in this section because of provides important information about the patient’s hemostatic
their tendency to invade the red cells and the fact that their capability. Gross variation in platelet morphology may be seen
appearance on a peripheral blood smear is confirmation of infec- in infiltrative disease of the bone marrow (e.g., idiopathic
tion by the organism. Although only an experienced reviewer myelofibrosis or metastatic infiltrates). Large platelets may be
would be expected to differentiate these organisms, it is impor- seen in any disorder associated with increased platelet turnover,
tant that all slide reviewers have knowledge of these organisms such as may occur with idiopathic thrombocytopenic purpura

Figure 5-34 m Note the “fingerlike projections” in this peripheral

smear from a patient with HbSC disease. These HbSC crystals are Figure 5-55 @ Comparison of babesiosis (/eft) and malarial forms
said to resemble the Washington Monument. (right).
1 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

infection, strongly suggests direct marrow architectural involve-

ment. This may indicate an infiltrative, neoplastic, or myelopro-
liferative process. Regardless of the reason for the appearance of
the immature cells, the reviewer should be able to identify the
cells accurately. Proper cellular identification at this point may
be critical to the patient’s diagnosis and subsequent prognosis.
Mature white blood cells may exhibit several morpho-
logical changes. In the performance of a slide review/WBC
differential the reviewer should take note of the appearance of
the nucleus of the white cells as well as the cytoplasm. Neu-
trophils tend to exhibit a wider variety of morphologic
changes than the other cell types with these changes originat-
ing in the cytoplasm in response to various pathologic
processes. The specific alteration may involve the appearance
or lack of cytoplasmic inclusions. Severe infections, inflam-
Figure 5-56 = Normal platelet at arrow. matory conditions, or other leukemoid reactions may be
accompanied by toxic granulation, toxic vacuolization, or the
presence of Dohle bodies (see Chap. 15). Toxic granulation
or bleeding disorders. In addition to the elevated platelet count, and Dohle bodies are generally considered nonspecific reac-
morphological changes may also occur postsplenectomy. tive changes, whereas vacuolization strongly indicates a
serious bacterial infection. This must also be noted with
its importance determined by the physician based on the
White Blood Cell Morphology patient’s condition.
Evaluation of white blood cells (WBCs, leukocytes) is per- Toxic granulation describes medium to large granules that
formed primarily in response to abnormalities identified by the are evenly scattered throughout the cytoplasm of segmented
automated blood cell counter. As with the evaluation of red cell polymorphonuclear neutrophil leukocytes. These granules are
morphology, the technologist must be proficient in identifica- seen in metabolically active neutrophilia and are composed of
tion of normal WBC morphology in order to adequately iden- peroxidases and acid hydrolases. Although nonspecific, they may
tify morphologic abnormalities. Figure 5—37 includes a normal occur in patients with severe bacterial infections, toxemia of
neutrophil as well as a normal lymphocyte. Slide reviewers pregnancy, or vasculitis, or in patients receiving chemotherapy.
must be able to recognize the appearance of normal cells, their Toxic vacuolization refers to the round, clear unstained
general size, their shape, and their overall appearance. They areas that are dispersed randomly throughout the cytoplasm
must also distinguish between normal granularity and the pres- of neutrophils in patients with overwhelming infections. Addi-
ence of abnormal inclusions. If immature cells are present, a tional cytoplasmic inclusions—Dohle bodies—are oval, blue,
skilled reviewer should be able to identify the cell line and the single or multiple inclusions originating in RNA, and are | to
stage of maturation. 3 um in diameter. Dohle bodies may be seen in peripheral blood
The presence of immature cells is a significant finding, smears of patients with severe infections, in patients with severe
with the greater the immaturity, typically, the more severe the burns, in pregnant women, and in patients receiving chemotoxic
diagnosis. The presence of more immature forms such as drugs. In these conditions, they represent toxic changes; how-
promyelocytes or myeloblasts, in the absences of severe ever, Dohle body-like inclusions are also characteristically
observed in certain congenital qualitative WBC disorders such
as the May—Hegglin anomaly and Chédiak—Higashi disorder
(see Chap. 15).
In addition to these morphological changes, severe bacter-
ial infections are also commonly associated with a moderate
leukocytosis and a shift to the left in granulocytes. Mild infec-
tions are characterized by a slight leukocytosis, with or without
the shift to the left. “Shift to the left” implies a release of younger
granulocytes—specifically bands and metamyelocytes—from
the bone marrow storage pool. These particular cell populations
may often be observed during an infection or inflammatory
process. The degree of leukocytosis or neutrophilia is useful in
discriminating among bacterial, viral, or fungal conditions.
Leukocytosis commonly refers to an increase in peripheral blood
leukocyte (WBC) concentration of greater than 10,000 cells/uL.
Because acute infection can rapidly mobilize the neutrophilic
nondividing marrow storage pool, the patient usually has a WBC
Figure 5-37 @ A, Normal neutrophil, 8, normal lymphocyte.
count below 50,000 cells/uL (average is 25,000 cells/uL). A shift
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

to the left is seen in the peripheral blood smear; however, it is un-

usual to see cells as immature as myelocytes in the peripheral
CBC Results Patient Results Reference Range
blood. Fungal infections may also be associated with neu-
trophilia and an increased WBC count, but a monocytosis is WBC 17.0 X 10%/L 6.0-17.5 X 107/L
more commonly observed. Viral infections usually are not asso- RBC 2.4 X 102/L 3.8-5.5 X 10°/L
ciated with neutrophilia but rather with lymphocytosis (see Hgb 7.5 g/dL 12-14.5 g/dL
Het 22.0% 30% 43%
Chap. 15).
MCV 92 fL 80-100 fL
Leukemoid reactions are characterized by a peripheral RDW 18% 11.5%-14.5%
neutrophilia that may resemble a chronic leukemia. The WBC
WBC Differential RBC Morphology
count is between 50,000 and 100,000 cells/uL, with immatu-
rity observed in one or more cell types. However, a high blast 40% Granulocytes Anisocytosis 3+
count is not part of the WBC differential picture, which can 58% Lymphocytes Poikilocytosis 3+
be helpful in eliminating leukemia as part of the differential 2% Monocytes Hypochromia 1+
Macrocytosis 1+
diagnosis. Acute infections, chronic infections such as tuber-
Microcytosis 1+
culosis and chronic osteomyelitis, as well as severe metabolic Schistocytes 2+
inflammatory and neoplastic processes have all been associ- Sickled cells le
ated with leukemoid reactions. Extremely elevated WBC Targets 1+
counts (greater than 100,000 cells/uL) are more suggestive of Polychromasia T+
a myeloproliferative process (see Chaps. 17 and 18), although Additional Tests Ordered
exceptions have been reported.
The reviewer should also take note of the appearance of Reticulocyte count 13%
Sickledex Positive
the nucleus, and in particular the number of segmentations. The
Hgb electrophoresis Hgb SS pattern
appearance of increased segmentation (normal is three to five
lobes) may indicate a megaloblastic process. This is referred to
as hypersegmentation and is reported when neutrophils contain DISCUSSION
greater than five lobes or when significant numbers of neu- Sickle cell disease is a hereditary disease that results in a
trophils all contain at least five lobes or more. The decreased chronic moderate to severe hemolytic anemia. The disease is
segmentation should also be noted, as it may be an indication characterized by the substitution of valine from the normal
of a benign hereditary condition known as Pelger—Huet anom- glutamic acid at the sixth position in the B-chain, resulting
aly or may actually be the result of a leukemic process. This in an abnormal hemoglobin that polymerizes when exposed
decreased segmentation consists of neutrophils with two lobes to low oxygen-tension conditions. This polymerization
or less and is described by the term hyposegmentation. results in the formation of sickle-shaped cells that are capa-
ble of temporarily or permanently blocking microcircula-
Physiologic leukocytosis is defined as an increased
tion, and the resulting stasis may lead to hypoxia and
WBC count without a shift to the left or any associated mor-
ischemic infarcts of various organs.
phological changes previously described for granulocytes. The disease is not evident at birth and does not manifest
This transient condition may be associated with such stimuli itself until the gamma chains of the newborn are replaced by
as exercise, intense emotional stress, anesthesia, or the B°-chains after 3 to 6 months of life.'!? Clinical manifesta-
administration of epinephrine or glucocorticoids. tions may be divided into acute and chronic episodes. Acute
problems result from a vaso-occlusive crisis termed “sickle
cell crisis.” This “crisis” typically includes an acute
hemolytic episode as well. Patients in sickle cell crisis pre-
sent with acute pain, fatigue, and possibly jaundice. All
three of these symptoms were present in our case study, as
was evidence of some form of anemic process.
Case Study Chronic manifestations of sickle cell disease usually
appear after mid-childhood.'® These include disturbances in
A 1-year-old African American child is brought to the growth and development, bone and joint disease, and organ
emergency department by her mother because the child had damage involving mostly all of the organ systems of the
no appetite and had not eaten in the last 2 days. Additional body at some point during the process of the disease.
information from the mother described a normally happy
child who had recently become restless and irritable. She QUESTIONS
was learning to walk, but now would not even attempt
standing. She has had a low-grade temp which is now ele- 1. Are the morphological findings on the blood film com-
vated to 102° F. On examination there is a definite yellow patible to the results from the analyzer?
tinge-to the sclerae and the spleen is palpable. Also noted 2. Did the other tests ordered confirm a probable diagnosis?
was a spindle-shaped deformity of two fingers on the right 3. Is the reticulocytes count useful?
continued
hand that were swollen and obviously painful to the touch.
The CBC results were as follows:
continued
L114 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

ANSWERS instrument, for example, the RDW is elevated and this is

reflected on the smear as 3+ anisocytosis. Many more
ike (Note: manual differential and morphology review per- abnormalities are detected by reviewing the peripheral
formed due to flagging of analyzer results, Fig. 5-38.) smear. These abnormalities are typically seen in patients
Morphology results correlate with the values from the in crisis as was this child.
. Yes. The sickledex is a screening test performed on
whole blood and is positive in both sickle cell disease
and sickle cell trait. The confirmatory test is the
hemoglobin electrophoresis, which will differentiate
abnormal hemoglobin traits from abnormal hemoglo-
bin disease. In this case the presence of the SS pattern
is diagnostic of Hgb S disease which will result in
sickle cell anemia.
. Yes. The reticulocytes count is a valid indicator of
bone marrow performance. In cases of sickle cell
crisis, in which the nonfunctional sickle cells are be-
ing destroyed, a properly functioning marrow should
accelerate the hematopoietic process. The 13% reticu-
locytes count is indicative of a proper response by the
bone marrow to the hemolytic process the patient was
experiencing.
Figure 5-58 ® Case study; note abnormal red cell morphology.
a

continued

Soe

Ru extort sesh

L. A prominent morphologic clue when suspecting lead “5, Morphological abnormalities found in
cases of
poisoning is the presence of which of the following on a severe burns, microangiopathic hemolytic anemias,
peripheral smear? and disseminated intravascular coagulation
a. Heinz bodies (DIC) are:
b. Target cells a. Schistocytes
c. Siderotic granules b. Crenated cells
d. Basophilic stippling c. Ovalocytes
i) . In which of the following disease states would you d. Stomatocytes
expect to find oval macrocytes on the peripheral smear? . Oat-shaped cells may be associated with:
a. Iron deficiency anemia a. Myelofibrosis
b. Lead poisoning b. Hereditary spherocytosis
c. Megaloblastic anemia c. Burns
d. Hereditary spherocytosis d. Sickle cell anemia
. All but one of the following are possible mechanisms for . How would a cell be classified that has a diameter of
the production of macrocytes: 9 um and an MCV of 104 fL?
a. Liver disease a. Macrocytic
b. Postsplenectomy status b. Microcytic
c. Pernicious anemia c. Normal
d. Thalassemia minor d. Either normal or slightly microcytic
. An abnormal erythrocyte seen in liver disease and hemo- . Abnormal platelet morphology may be observed most
globinopathies and thalassemias and is characterized by prominently in:
the “bull’s eye” area is known as a: a. Idiopathic myelofibrosis
a. Stomatocyte b. Anemia of chronic disorders
b. Target cell c. Hereditary spherocytosis
c. Schistocyte d. Septic shock
d. Hypochromic cell
 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology 115

. Which type of red cell inclusion is a DNA remnant? The following answer pool is used for items 15 to 29
a. Heinz body (Match)
b. Howell—Jolly body
aa. Anisocytosis f. Polychromasia k. Schistocytes
c. Pappenheimer body
d. Cabot ring b. Poikilocytosis | g. Microspherocytes —_1. Rouleaux
. Which of the following are considered microcytic/ c . Hypochromasia_h. Target cells m. Acanthocytes
hypochromic anemias? d. Microcytic i. Stomatocytes n. Dacrocytes
a. Autoimmune hemolytic anemia
oO . Depranocyte J. Blister cells o. Echinocytes
b. Pernicious anemia
c. Iron deficiency anemia lhaks RBCs with a large area of central pallor
d. Megaloblastic anemia 16. Variation in the size of the red blood cells
11. A hypersegmented neutrophil may be seen in which of ____17. Also known as codacytes
the following anemias?
eS: RBCs appearing stacked on each other
a. Iron deficiency
b. Megaloblastic pe NS: MCV of 65%
c. Autoimmune hemolytic anemia eee): RBCs with a mouthlike central pallor
d. Anemia of chronic disorders
21 . RBCs without an area of central pallor
12. Precipitates of denatured hemoglobin found primarily in
22. RBCs with evenly distributed spicules on the
patients with hemolytic anemia resulting from oxidant
membrane
stress describe:
a. Howell—Jolly bodies 23. RBC fragments
b. Heinz bodies . RBCs appearing bluish in color
c. Basophilic stippling
. Variation in the shape of the RBCs
d. Pappenheimer bodies
. Congenital abetalipoproteinemia
. Pappenheimer inclusions are formed from:
a. Excess a-chains 27. The formation of a vacuole in an RBC “trapped”
b. Excess B-chains by fibrin
c. Excess iron . Formed when an RBC with an inclusion squeezes
d. Oxidant stress out of a tight space
14. RBC inclusions resulting from an acceleration in hemo- . Seen in HbS disease
globin biosynthesis and consists of RNA:
a. Howell—Jolly bodies See answers at the back of this book.
b. Heinz bodies
c. Basophilic stippling
_d. Pappenheimer bodies

m Macrocytes are associated with high reticulocyte counts

and liver disease; oval macrocytes are associated with
megaloblastic processes.
w A variation in cell size is termed anisocytosis.
m Red cells that appear polychromatophilic on a Wright’s
w A variation in cell shape is termed poikilocytosis. stained smear would appear as reticulocytes on a suprav-
gw On an automated cell counter, a flag is a signal that a sig- ital stain.
nificant abnormality may be present in the sample. @ Spherocytes are associated with hereditary spherocytosis
m Microcytes are associated with an MCV of less than 80 fL and an MCHC that is greater than 36% in many cases.
and are seen in IDA, thalassemias, sideroblastic anemias, m Spherocytes may also be seen in autoimmune hemolytic
and anemia of chronic disease. anemia (AIHA) and posttransfusion.
m Macrocytes are associated with an MCV of more than w There are two types of sickle cells, irreversible sickle cells
100 fL and are seen in megaloblastic and nonmegaloblas- (ISCs) and oat-shaped, reversible sickle cells that are both
tic processes. commonly seen in HbS disease.
(continued)
l] 6 Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology

w Helmet cells are seen in patients with G6PD syndrome

"7 SUMMARYCH and occur as a result of Heinz body formation.

g Any regenerative red cell process will result in inclusions w Heinz bodies cannot be seen on a Wright-stained periph-
such as Howell—Jolly bodies, basophilic stippling, and eral smear.
Pappenheimer bodies. w Abnormal platelet morphology includes lack of granula-
= Howell—Jolly bodies, Pappenheimer bodies, and basophilic tion, giant platelets, and megakaryocytic fragments.
stippling may be seen in peripheral smears stained w Abnormal white cell morphology includes the presence or
with both Romanowsky type stain (i.e., Wright’s, absence of cytoplasmic granulation, or presence of cyto-
May-—Grumwald) and supravital stain (i.e., new methylene plasmic vacuolizaton, as well the appearance of the cellu-
blue, brilliant cresyl blue) lar nucleus.
w Siderotic granules and Pappenheimer bodies are basically a Hyposegmentation describes decreased neutrophil seg-
the same inclusion. The differentiating factor is that on an mentation (<2 lobes).
iron stain the inclusions are known as siderotic granules w Hypersegmentation describes increased neutrophil seg-
where on Wright’s stain they are known as Pappenheimer
mentation (25 lobes).
bodies.

m Howell—Jolly bodies are seen in patients postsplenectomy.

REFERENCES Is Orkin, SH, and Nathan, DG: The tha- Approach. Williams & Wilkins, Balti-
lassemias. In Nathan and Oski’s Hema- more, 1996, p 77.
| . Bain, B: Current concepts: Diagnosis
tology of Infancy and Childhood, ed 5. . Womack, EP: Treating thrombotic
from the blood smear. N Eng] J Med
WB Saunders, Philadelphia, 1998, p 818. thrombocytopenic purpura with plasma
353(5):498, 2005.
8. Bolton-Maggs, P, et al: Guidelines for exchange. Lab Med 30:276, 1999.
. Stiene-Martin, E, et al: Clinical Hematol-
the diagnosis and management of heredi- . Storch, A, et al: Testing for acanthocyto-
ogy Principles, Procedures, Correlations.
tary spherocytosis. Br JHaematol sis. J Neuro 1252(1):84, 2005.
ed. 2. Lippincott-Raven, Philadelphia,
126:455, 2004. . Lawrence, C, et al: The unique red cell
1998.
\O. Gallager, P: Red cell membrane disor- heterogeneity of SC disease: Crystal for-
WwW. Shojana, AM: Protein synthesis in mega-
ders. ASH Education Book, 2005, cited mation, dense reticulocytes, and unusual
loblastic disorders. In Gross, S, and
from, http://www.asheducationbook.org/ morphology. Blood 78(8):2104, 1991.
Roath, S (Eds): Hematology: A Problem
cgi/content/full/2005/1/13. pp 1-11. . Kaplan, L, et al: Clinical Chemistry The-
Oriented Approach. Williams & Wilkins,
Bicorct 100Gh a7 10. Chen, J, and Huestis, W: Role of mem- ory, Analysis, Correlation, ed 2. Mosby,
Bere One brane lipid distribution in chlorpromazine- St. Louis, 2003, p 686.
. Glassey, E (Ed): Color Atlas of Hematol- induced shape change of human erythro-
ogy. College of American Pathologists, cytes. Biochim Biophys Re eae
See Bibliography at the back of this book
Hematology and Clinical Microscopy 1323(2):299, 1997
Ae eae Nowafield IU; 11. Mohandas, N: A cell by any other name.
.
Ake
Davenport,
:
J: Macrocytic
;
anemia. Am 12
Blood 106(13):4017, 2005.
. Camilo, N, and Ravindranath, Y: Hemo-
Fam Physician 53(1):155(8), 1996.
globinopathies: Abnormal structure in
. Provan, D, and Weatherall, D: Red cells
hematology. In Gross, S, and Roath, S
II: Acquired anaemias and poly-
(Eds): Hematology: A Problem Oriented
cythaemia. Lancet 355:1260, 2000.
PART 2
ANEMIAS

Chapter

Iron Metabolism
and Hypochromic
Anemias
Kathleen Finnegan, MS, MT(ASCP)SH

Introduction OBJECTIVES
Normal Iron Metabolism At the end of this chapter, the learner should be able to:
Distribution and
Requirements . List the components of the hemoglobin molecule.
Daily Iron Requirements and . State the function of iron in relation to hemoglobin.
Absorption
lron Transport . Discuss the absorption and transport of iron into the protoporphyrin ring during
Ww
Oo

Iron Storage erythropoiesis.

Laboratory Evaluation of . Describe the physiologic factors that affect absorption and the amount of iron needed
Iron Status by the body.
Serum Iron
. Evaluate laboratory tests that are used to differentiate and identify categories of
Total lron-Binding Capacity
different types of anemia.
Transferrin Saturation
Ferritin . Discuss the pathophysiology of iron deficiency anemia, anemia of chronic disease,
Transferrin Receptor sideroblastic anemia, and the porphyrias.
Free Erythrocyte . List and compare the laboratory findings in iron deficiency anemia, anemia of
Protoporphyrin and Zinc
chronic disease, and sideroblastic anemia.
Protoporphyrin
. Define the porphyrias.
lron-Deficiency Anemia
Etiology . Compare and contrast the hypochromic anemias.
Pathophysiology
. Describe iron overload and hereditary hemochromatosis.
Clinical Features
Laboratory Findings . List the causes of iron overload.
Treatment
. List the characteristics of iron overload and hereditary hemochromatosis.
Anemia of Chronic Disease
Pathophysiology
Clinical Features
Laboratory Findings
Treatment
Sideroblastic Anemia
Pathophysiology
Laboratory Findings
Treatment
The Porphyrias
118 Chapter 6 Iron Metabolism and Hypochromic Anemias

Iron Overload
and Hemochromatosis
Hereditary
Hemochromatosis
Pathophysiology
Clinical Features
Laboratory Findings
Treatment
Secondary
Hemochromatosis
African Iron Overload
Case Study 1
Case Study 2
Case Study 3

hemosiderin.* Ferritin iron is easily mobilized by the body

Introduction
for utilization, and serum ferritin levels are used as an indi-
The microcytic hypochromic anemias are a group of red cell rect measure of the iron stores. Hemosiderin, another form of
disorders that involve a defect in hemoglobin synthesis due to storage iron, represents precipitated aggregates of ferritin
a deficiency of iron or an abnormal utilization of iron. These and is less readily available for utilization. Ten percent of tis-
anemias include iron-deficiency anemia (IDA), anemia of sue iron is unavailable or elemental iron and includes the iron
chronic disease (ACD), and sideroblastic anemia. The most in myoglobin and the cytochrome enzymes. A very small
common of these is IDA. Microcytic hypochromic anemias amount of iron is also present in plasma as transport iron.
also include disorders of globin chain synthesis, which com- Iron metabolism and maintenance of body stores is a tightly
prise the thalassemias. The globin chain defects are discussed regulated process. Daily iron intake, absorption, and losses
in Chapter 12. are usually very small. Body iron is repeatedly recycled, and
Erythropoiesis is a highly regulated process throughout the small amount of iron that is lost each day is replaced by
the entire life span of each individual. Hemoglobin synthesis the diet. This small amount of iron which is lost each day
is an integral part of erythropoiesis and requires three through cellular shedding and sweating is approximately
compounds: iron, globin, and protoporphyrin. Each hemoglo- | mg. In adult men, this | mg of iron represents the minimum
bin molecule consists of four heme groups and four globin daily requirement (MDR). The normal life span of the red
chains. Each heme group contains a protoporhyrin ring plus blood cell (RBC) is approximately 120 days. Each day | per-
an iron molecule. Adult hemoglobin A contains two cent of red cells are taken out of circulation. Ninety percent
alpha and two beta polypeptide chains. For a review of hemo- of these senescent red blood cells are removed by extravas-
globin formation and function, refer to Chapter 3. This cular hemolysis. Ten percent of these old red blood cells are
chapter reviews normal iron metabolism followed by a dis- removed by intravascular hemolysis. As a result, an average
cussion of IDA, ACD, sideroblastic anemia, and hereditary of 20 mg of iron is needed each day to replace the iron lost
hemochromatosis. by senescent red blood cells. The majority of this iron comes
from recycling, because nearly 100% of the iron from RBC
turnover is taken up by the mononuclear phagocytic system
Normal Iron Metabolism (RES cells) and reutilized. The daily iron turnover is illus-
trated in Figure 6-1.
Distribution and Requirements
Iron is an essential element for all living organisms. It is the
Daily Iron Requirements and Absorption
essential component of the heme complex and is required by
every cell in the body. It is important for cellular growth, Daily iron requirements are affected by a number of physio-
oxygen transport, and the proliferation of red blood cells. logic states, including menstruation, pregnancy, lactation, and
The average adult has a total body iron content of between growth. During menstruation, women can lose approximately
3500 and 4000 mg.' Two-thirds of the total body iron is 0.8 mg of iron per day, in addition to the | to 2 mg typically
found in the hemoglobin molecule and one third is found in lost daily through cellular shedding and sweating. The extent
the storage pools of the bone marrow, liver, and spleen. of menstrual bleeding is extremely variable, and some women
Nearly 90% of this stored iron is in the form of ferritin or may lose up to 1.5 to 2.5 mg of iron per day as a result of
 Chapter 6 Iron Metabolism and Hypochromic Anemias 119

DAILY FE*+ TURNOVER

CONTINUOUS PROCESS

Daily RBC turnover Daily RBC in bone marrow

of old cells production to replace
1% per day old cells 1%

Loss (cells from G.I.

“Absorption tract shed)
—
1—2 mg ONLY 1-2 mg DAILY

Figure 6-1 @ Daily iron turnover and body iron distribution.

menstruation. In pregnant and lactating women, the MDR iron ingested in the diet, only 5% to 10% is absorbed. The
increases to 3.0 mg per day. In the second and third trimesters, foods that increase and decrease iron absorption are listed in
the daily requirement of iron can increase to as high as 5 to Table 6-3.
6 mg. At delivery, there is a loss of 600 to 700 mL of blood. Regulation of iron absorption occurs within the intestinal
In total, during pregnancy approximately 1000 mg of iron is mucosa of the small bowel. The vast majority of iron is
utilized. This equals and at times exceeds the amount of stor- absorbed in the duodenum and the first portion of the jejunum.
age iron in an average woman of childbearing age. Lactation Iron molecules within the diet and iron complexes within the
and breast feeding also contribute to the loss of iron associ-
ated with pregnancy. Because milk is a poor source of iron,
many baby formulas are supplemented with iron. Infants who
are fed only mothers’ breast milk are at a significant risk of
developing IDA.
During periods of growth, such as infancy and adoles-
cence, iron requirements are substantially increased. During Individual MDR (mg)
the first year of life, absorption of 150 to 175 mg of iron is
required to maintain an appropriate hemoglobin concentra- Infant 1.0
Child 0.5
tion. The MDRs for iron for different individual groups are
Menstruating woman 2.0
listed in Table 6-1. Pregnant or lactating woman 3.0
Many foods are rich in iron, including meats, legumes, Adult man or nonmenstruating woman 1.0
green vegetables, cereals, and prunes (Table 6-2). Of the
120 Chapter 6 Iron Metabolism and Hypochromic Anemias

IRON (Fe) ABSORPTION AND TRANSPORT

Table 6-2 lron-Containing Foods
Ingestion

Foods High in [ron Foods Moderate in iron

Organ meats Muscle meats (beef, fish,fowl)

Wheat germ Prunes
Brewer’s yeast Cereals
Legumes Green vegetables
Whole grain breads Shrimp
Oatmeal Oysters

Table 6-3 Substances that Increase : Duodenum

and
and Decrease the BP orption Jejunum
of Iron

Increased TOS eased:

Ingestion ofee foods Phoechanee Bloodstream

Ascorbic acid (vitamin C) Phytates
Apoferritin
Foods that form insoluble
iron complexes Mucosal
Fecal cell
Loss

Figure 6-2 ™ Schematic of iron intake and absorption through the

body can be present in various iron states (Table 6-4). Ferric mucosal cells in the small intestine. Absorbed iron is converted to
iron (Fe**) is the most common dietary form of iron. The acidic ferritin for storage or transported bound to transferrin for distribu-
pH (<4) of gastric secretions in the stomach helps tion to body tissues.
reduce ferric iron (Fe**) to the ferrous form (Fe**) by ferrin
reductase.’ This strong acidic environment of the stomach, in transferrin compartment. Normally the majority of this iron is
conjunction with pancreatic enzymes, facilitates absorption. used for heme synthesis. Transferrin is available for binding
Optimal absorption occurs in the duodenum and jejunum. ferric iron (Fe**) and assists iron delivery to the erythroblasts
The ferrous form of iron is absorbed through the mucosal in the bone marrow by plasma circulation. The chelation of
cells of the duodenum and jejunum and oxidized back to fer- iron by transferrin binding maintains iron in a soluble form.
ric iron. The ferric iron enters the bloodstream and is trans- Two atoms of ferric iron can bind to one transferrin molecule.
ported by transferrin for distribution to body tissues, or for Transferrin levels are indirectly measured via the total iron-
storage in the bone marrow, liver, or spleen (Fig. 6—2). Trans-
binding capacity (TIBC). TIBC measures the amount of iron
ferrin is a beta, (8,) globulin protein that is responsible for
that the circulating transferrin could bind in a fully saturated
transporting iron through the bloodstream to the various state. Transferrin bound iron is moved into the nucleated red
organs of the body. The rate at which iron is transferred from blood cell (NRBC) and reticulocytes through the transferrin
the intestinal mucosal cell to the bloodstream is regulated by
receptor (TfR) which is located on the surface membrane of
the body’s iron levels and requirements. It has been postulated
the red cell. The iron transporter, divalent metal transport 1
that, as ferritin levels decrease (e.g., in iron deficiency), iron
(DMT1), is a protein that helps transfer iron across the RBC
absorption increases.
membrane. In the cytoplasm of the RBC, the iron is used for
Despite the large amount of iron in the Western diet, iron
hemoglobin synthesis. Any excess iron not needed for hemo-
deficiency continues to be a significant cause of morbidity in
globin synthesis is stored as ferritin. DMT1 is not specific for
North America and throughout the world. Many food prod-
iron alone. DMT! can transport other metals.2> DMT1 also
ucts, including flour and baby formulas, are now supple-
transfers iron from the intestinal lumen into the enterocytes
mented with iron to help alleviate the problem. Factors that
(mucosal cells). Once absorbed, iron may be transported from
affect the absorption of iron are listed in Table 6—S.
the enterocyte into the circulation by another iron transport
protein, ferroportin. Ferroportin is also important in the
Iron Transport recycling of iron by moving iron out of macrophages of vari-
ous organs, such as the bone marrow, into the bloodstream to
In plasma, the ferric iron circulates tightly bound to transfer- bind transferrin.
rin. The major pathway of internal iron exchange is a unidi- The hemochromatosis gene (HFE)° produces a protein
rectional flow from plasma transferrin to the erythron to the that binds to the transferrin receptor. This protein complex
macrophage and back to plasma transferrin.* The red blood has a role in determining receptor affinity for ferric transfer-
cell (RBC) uses about 80% of the iron passing through the rin and is an important modulator of this interaction. HFE
 Chapter 6 Iron Metabolism and Hypochromic Anemias 121

es e ‘Molecule/Compound BeState Location Function

Dietary Fe Fe or Fe Upper Gittract Roroclobi cerneloGie

enzyme synthesis
Hemoglobin Fe?* RBCs Bind oxygen
Transferrin Fe** Serum Transport Fe
Ferritin Fe*+ Serum and tissue sites Fe storage
Hemosiderin Fe** Bone marrow and Fe storage
other tissue sites

Iron Storage
Iron that is not used for erythropoiesis is stored in the
mononuclear phagocytic system (MPS) or reticuloendothelial
Amount and type of iron accessible from food (RE) cells of the bone marrow, liver, and spleen. Iron taken in
Functional state of gastrointestinal mucosa and pancreas
Current iron stores excess is stored in two forms, ferritin and hemosiderin. RE
Erythropoietic needs cells ingest old red cells and catabolize the hemoglobin to
recycle the iron. The major form of storage iron is ferritin.
Ferritin is water soluble and is easily mobilized by the body
for utilization.
protein interacts with the TfR to regulate iron uptake. Trans- The second storage form of iron is hemosiderin. Hemo-
ferrin receptor is capable of binding two molecules of trans- siderin is not water soluble. The iron in hemosiderin is
ferrin, each carrying ene or two molecules of iron. The iron released more slowly than that from ferritin and is less read-
content of the cell is regulated by control of the iron uptake ily available for utilization. Hemosiderin represents aggre-
and storage capacity. As cellular iron levels fall, the levels of gates of iron and can be visualized in tissue with the use of a
ferritin decrease and the transferrin receptors increase. When Prussian blue stain for iron. Hemosiderin appears as granules
cellular iron increases, ferritin increases, and TfRs fall. Proteins and aggregates. Ferritin is seen only via electron microscopy.
involved in iron metabolism are summarized in Table 6-6. Hemosiderin does become available in iron-deficient patients.

Protein ees Function

Transferrin Single:iad Faweommoten ithtwo iron Tron-pindine ianepe erorein in

binding sites plasma and extracellular fluid

Transferrin receptor Transmembrane glycoprotein diamer with Receptor mediated ferric transferrin
two transferrin binding sites

Ferritin Spherical protein binds up to 4500 iron Iron storage

atoms

HFE MHC class I glycoprotein complexes HFE binds with transferrin receptor
with B,-microglobulin reducing the affinity for transferrin

DMT1 Single chain glycoprotein Iron transport protein from GI lumen

into the duodenal enterocyte; from
erythroblast endosome to
cytoplasm

Hepcidin Liver synthesized peptide Involved with iron metabolism.

Increased in iron over-loading and
decreased iin iron eaten

“FE = eho gene protein;eMac = major Aistcectapateility Rpt. DMT1 = Hpac metal transporter 1;
GI= gastrointestinal.
122 Chapter 6 Iron Metabolism and Hypochromic Anemias

Table 6-7 Iron Status: Normal Val

Serum Iron TIBC Transferrin % Transferrin Ferritin
pe/dL pe/dL. ug/dL z Saturation hg/dL

Adult male 65— 170 250-450 200-400. 20-250

Adult female 50-170 250-450 200-400 10-120
Newborn 40-250 100-400 130-275 50-600
Adolescent os 1208 _ 250-450 = 200-400; 10-150_

* Please note that nt Oates vary by;institution, patient ei tea and testing oaoaayen

Laboratory Evaluation of Iron Status of iron depletion. It is increased in iron overload. Ferritin is an
acute phase reactant protein and is increased in inflammatory
The laboratory assessment of iron includes the measurement states, malignancy, infections as well as liver disease.’
of serum iron, total iron-binding capacity (TIBC), transferrin,
percent saturation of transferrin, serum transferrin receptors,
Transferrin Receptor
and ferritin. Zinc protoporphyrin (ZPP) is an indirect assess-
ment of iron availability. Reference ranges for iron status are Measurement of serum transferrin receptors (sTfRs) is
listed in Table 6—7. another tool for the assessment of iron status. The sTfRs are
»
inversely proportional to the amount of body iron. The con-
Serum Iron centration is increased in iron deficiency, but only when the
iron stores are depleted. The reason is that, with the lack of
Serum iron is a measure of transferrin-bound iron. Normal intracellular iron, the regulatory proteins directly increase the
serum iron concentration for males is 65 to 170 wg/dL. In synthesis of transferrin receptors.* TfRs do not increase with
women it is usually lower. The concentration of iron fluctu- anemia of chronic disease.
ates in individuals and is not always used for investigation of
iron metabolism without other laboratory tests. Early morning
Free Erythrocyte Protoporphyrin
specimens are preferred because of diurnal variation.
and Zinc Protoporphyrin
Total Iron-Binding Capacity Free erythrocyte protoporphyrin (FEP) is a heme precursor
that incorporates iron into the hemoglobin molecule. When
Total iron-binding capacity (TIBC) is the total amount of iron iron is not available to be incorporated into the protoporphyrin
that can be bound by transferrin in the plasma or serum. Each ring to form heme, excess protoporphyrins form. These excess
gram of transferrin will bind 1.4 mg of iron. The normal range rings complex with zinc to form zinc protoporphyrin (ZPP).
is 250 to 450 pg/dL. The binding capacity is normally one- ZPP is increased in iron-deficient erythropoiesis. The ZPP
third saturated. The measurement of TIBC is effectively an value correlates inversely with the ferritin level.
indirect measurement of transferrin concentration.

Transferrin Saturation
lron-Deficiency Anemia
Iron deficiency is described as a microcytic hypochromic
Percent saturation of transferrin is functionally measured as
anemia. The red blood cells are small in size and have an
the maximum amount of iron that is bound in plasma or
increase in central pallor, since they are lacking hemoglobin.
serum. Serum iron and TIBC are used to calculate the percent
The World Health Organization states that iron-deficiency
saturation. A transferrin saturation value below 16% is an in-
anemia (IDA) is the most common anemia* and is estimated
dicator of iron deficiency. Transferrin saturation is consis-
to affect approximately 2 billion people worldwide.’ IDA
tently increased in iron overload. The percent saturation is
results when there is a lack of adequate iron stores in the body
calculated as follows:
to meet physiological needs for the production of red blood
% saturation = S&UuMiron y iq9% cells. About 3% of children younger than 2 years of age, up to
DBE
3% of adolescent females, and fewer than 1% of adolescent
males have IDA.'° For high-risk groups see Table 6-8.
Ferritin
Etiology
Ferritin is directly proportional to the amount of iron stored.
Ferritin is a much better measurement than serum iron and IDA may occur because there is an increased demand for iron,
TIBC for the assessment of body iron stores. Normal range for or abnormal utilization for iron, poor diet, increased blood
ferritin is 20 to 300 g/dL. When low, ferritin is a good index loss, or malabsorption. IDA can develop slowly after the
 Chapter 6 Iron Metabolism and Hypochromic Anemias 123

i Pinan
6-9 Causes of Iron-Deficiency
Prenatal/Neonatal
Premature birth
Inadequate Absorption
Low birth weight
Inflammatory bowel disease
Anemia during pregnancy
Resection of the bowel
Low iron formula
Celiac disease
Lack of iron supplements after 6 months in breastfed infants
Antacid therapy
Infancy/Childhood High gastric pH
Restricted diets Loss or dysfunction of absorptive enterocytes
Lack of iron supplements Poor bioavailability
Growth spurts Excess bran
Chronic infection
Decreased Iron Intake
Chronic or acute blood loss
Meat-poor diet
Adolescent/Adult Cereal-rich diet
Fad diets Malabsorption
Menstruation Elderly
Excessive weight gain Increased [ron Utilization
Pregnancy _
Growth spurts
Lactation and breast feeding Pregnancy
Elderly
Improper diet Loss of Iron
Chronic bleeding Gastrointestinal blood loss
Gastrointestinal bleeding Ulcer
Gastritis
Social/Economic Epistaxis
Low socioeconomic groups Hemorrhoids
Recent immigration from developing countries Menorrhagia
Pulmonary blood loss
Malignancy or cancer
Trauma
Excess phlebotomy

normal stores of iron have been depleted, or may occur more

rapidly when there is an increased demand for iron. Causes of
IDA are summarized in Table 6-9.
MALABSORPTION
DIET AND INCREASED NEED Malabsorption is an uncommon cause of IDA. If iron absorp-
Nutritional deficiency occurs when insufficient iron taken in tion is impaired owing to the absence of gastric acids, which
through the diet does not meet the need for erythropoiesis. helps reduce dietary iron from ferric to the ferrous form, an
In most developed countries, inadequate dietary intake of IDA can develop. This can be seen with patients suffering
iron is not a major cause of anemia. If diet is inadequate in from malabsorption syndromes such as gastrectomy, gastric
iron, the body stores of iron will continue to deplete. Infants, bypass, celiac disease, atrophic gastritis, or any disease that
children, and adolescents are at a higher risk for developing will compromise iron absorption.
IDA because of higher iron requirements needed during
growth spurts. Iron absorption requirement on average is Pathophysiology
| mg per day to support normal growth and replace normal
loss. Toddiers have increased iron needs because cow’s milk IDA develops in the following stages over a period of time:
is low in iron. As mentioned previously, pregnancy also stage 1, iron depletion; stage 2, iron-deficient erythropoiesis;
makes demands on the mother’s body to provide iron for her and stage 3, the final stage, development of IDA. The stages
needs and iron for the developing fetus. of IDA are reviewed and summarized in Table 6-10.

BLOOD LOSS STAGE 1: [IRON DEPLETION

Excessive loss of iron from the body through blood loss can Generally this stage is asymptomatic. In stage I, the iron stores
also lead to IDA. IDA in adult males is usually caused by in the bone marrow are depleted. The hemosiderin content
chronic blood loss from the gastrointestinal tract (GI). Blood of the cells of the MPS or RES in a bone marrow aspirate
loss from the GI tract includes occult bleeding, peptic ulcers, is decreased or absent when stained with Prussian blue
tumors, malignancies, hemorrhoids, and hiatal hernia. Typi- (Fig. 6-3). As a result, serum ferritin (a measure of stored
cally blood loss from the GI tract occurs in elderly patients." iron) is decreased while mucosal absorption is increased. In
In women, IDA occurs via blood loss through menstruation. an attempt to compensate, the liver will synthesize more
Because the red blood cells are lost outside of the body, the transferrin, producing an increase in TIBC. An anemia may
iron cannot be recycled for reuse. not be evident (complete blood count [CBC] is normal), and
124 Chapter 6 Iron Metabolism and Hypochromic Anemias

oie ke

Table 6-10 Stages of lron-Deficiency Ane nia

Stage 2: Iron
Stage 1: Iron Deficient
Depletion Erythropoiesis
(IDA without (IDA with Stage 3: IDA
Laboratory Features Normal* Anemia) Mild Anemia) (Severe Anemia)

Serum iron level 65-170 ug/dL Normal to Decreased Decreased

decreased
Total iron binding 250-450 pg/dL Normal to Increased Increased
capacity increased
Serum ferritin level 12-300 pg/dL Decreased Decreased Decreased
Percent transferrin M: 20%-50%
saturation F: 15%-50% Normal <15% Decreased
TER 1.5-2.75 mg/L Normal Increased Increased
Zinc protoporphyrin 16-65 g/dL Normal to Increased Increased
(free erythrocyte increased
protoporphyrin)
Hemoglobin 13-15 g/dL Normal Normal to Very decreased
decreased
Hypochromia Not present Not present Slight Marked
Microcytes Not present Not present Slight Present
Stainable bone Present Absent or Absent , Absent
marrow iron decreased

TfR = transferrin receptor levels; ID = iron deficiency; M = male; F = female.

* Please note that normal values vary by institution, patient population, and testing methodology.

the RBC morphology is normal. This stage creates no overt (ZPP). The hemoglobin and hematocrit values are reduced and
effect on erythropoiesis. The red cell distribution width the red blood cells may appear microcytic. Ferritin levels
(RDW), however, is usually increased, which sometimes is are decreased, TIBC increases, and TfRs increase on the sur-
the first indication of an anemia developing. It is estimated face of the red blood cells. Other iron-dependent tissues may be
that nearly 50% of U.S. infants are in stage | of IDA at some- affected.
time during development.**
STAGE 3: IRON-DEFICIENCY ANEMIA
STAGE 2: IRON-DEFICIENT ERYTHROPOIESIS The final stage of anemia develops when the red blood cells
The second stage is iron-deficient erythropoiesis and is subclin- are severely deficient in iron. The advanced stage will show
ical. Plasma iron levels drop. When there is a decrease in iron a marked decrease in hemoglobin and hematocrit and hemo-
that is needed for heme synthesis, excess protoporphyrin accu- globin formation is delayed with the formation of red cells
mulates and complexes with zinc to form zinc protoporphyrin that are hypochromic and microcytic (Fig. 6-4). There is
ineffective erythropoiesis as a result of depleted storage iron
and diminished transport iron. The bone marrow shows
decreased hemoglobinization and ragged cytoplasm in red
blood cell precursors (Fig. 6-5). At this stage there is a
severe deficiency in total body iron. Most patients are not
diagnosed until this stage appears. Erythropoietin levels
increase, producing a slight reticulocytosis.

Clinical Features

IDA may occur over a period of months to years. The clinical

findings of IDA are varied. As the anemia develops in severity
and duration, the clinical symptoms increase. Typical symp-
toms of IDA include fatigue; irritability; headache; weakness,
especially with exercise; shortness of breath; tachycardia; pale
skin color or pallor. Other symptoms of a severe IDA are
Figure 6-3 ™ Bone marrow aspirate from a patient with iron-
koilonychias, the spooning of the nails (Fig. 6-6), pharyngeal
deficiency anemia stained with Prussian blue. Note the negative stain- webs, cheilitis, glossitis or sore tongue,!* blue sclera (whites of
ing indicated by a lack of any blue stain indicating an absence of iron. the eyes), and muscle dysfunction (Fig. 6-7).
 Chapter 6 Iron Metabolism and Hypochromic Anemias = 125

Figure 6-6 @ Koilonychias or spooning of the nails, characteristic

Figure 6-4 @ lron-deficiency anemia Characterized by microcytic, of iron-deficiency anemia.
hypochromic red blood cells.

PERIPHERAL BLOOD
In patients with severe IDA, a microcytic hypochromic anemia
Another manifestation that may be associated with IDA is seen on the peripheral blood smear (see Fig. 6-4). The mean
is pica.'* Pica is defined as the persistent eating or craving of corpuscular volume (MCV) can range from 55 to 74 fL; the
nonfood substances such as clay, dirt (geophagia), or ice mean corpuscular hemoglobin concentration (MCHC) can
(pagophagia). range from 22 to 31 g/dl; and the mean corpuscular hemoglobin
Infants with IDA are at risk for developmental, behavior, (MCH) can range from 14 to 26 pg. Microcytes, anisocytosis,
and motor difficulties. Symptoms include irritability, loss of and an increased RDW are the first morphological signs. The
memory, and difficulties in learning. They also have increased peripheral blood smear usually contains increased poikilocyto-
susceptibility to infection and poor growth.’ sis with the presence of a few codocytes (target cells), ellipto-
cytes or ovalocytes, and dacrocytes (teardrop cells) in addition
Laboratory Findings to microcytes. The reticulocyte count, which measures the abil-
ity for the bone marrow to produce new red blood cells, is
The features and indices associated with iron-deficiency ane- decreased in relation to the severity of the anemia as a result of
mia are summarized in Table 6-11. ineffective erythropoiesis. The white blood cell count is usually

Figure 6-5 ™ Bone marrow aspirate in iron-deficiency anemia

showing ineffective erythropoiesis, “ragged” erythroid precursors. Figure 6-7 ® Clinical manifestations of iron-deficiency anemia.
(From Bell, A: Hematology. In Listen, Look and Learn. Health and A. Cheilitis, before (top) and after (bottom) therapy. B. Glossitis
Education Resources, Inc., Bethesda, MD, with permission.) before (top) and after (bottom) therapy.
126 Chapter 6 Iron Metabolism and Hypochromic Anemias

Table 6-11 Indices and Features of

lron-Deficiency Anemia
Clinical
Clinical findings depend on the severity of the anemia.
Severe anemias may be associated with pallor, weakness,
and dyspnea.

Morphological
Usually hypochromic, microcytic RBCs
Mild to moderate anisopoikilocytosis
Decreased storage iron
Decreased sideroblasts
Absence of ringed sideroblasts

Laboratory
Decreased serum iron Figure 6-8 @ Peripheral blood of a patient with iron-deficiency
Decreased serum ferritin anemia after therapy. Note the two populations of red blood cells:
Decreased % transferrin saturation microcytic hypochromic and normal cells.
Increased total iron-binding capacity (TIBC)
Increased free erythrocyte protoporphyrin (FEP)
Increased serum soluble transferrin receptor ievels include ferrous gluconate and ferrous fumerate. In some cases
where intestinal absorption of iron is impaired, parenteral
administration of iron dextrans is used, which may also be
B
given for patients who cannot tolerate iron supplements. Par-
normal. The platelet count may be normal, increased, or enteral iron therapy may be given intravenously or intramuscu-
decreased. larly. Blood transfusions are used only if hemoglobin drops to
dangerously low levels. Each hospital will determine its own
IRON STUDIES critical level, but generally patients are transfused when their
Serum iron is decreased as a result of the depletion of iron hemoglobin level is less than 8 g/dL. After therapy, reticulo-
stores, TIBC is increased, percent transferrin is decreased, and cytes begin to rise within 3 to 5 days, and reach a maximum
TfRs are increased. Serum ferritin, the storage form of iron, is level within 8 to 10 days.'? Hemoglobin levels will increase in
decreased in all stages of IDA and is usually the first indication about 2 to 3 weeks and usually return to normal within 6 weeks.
of an IDA developing. Serum ferritin is a sensitive marker of The normal cell population will slowly predominate in about
iron storage and is an important laboratory test to help differen- 6 to 10 weeks. Microcytosis may take up to 4 months to
tiate IDA from other microcytic hypochromic anemias. The resolve. Restoration of the patient’s iron stores usually takes
sTfR is useful in detecting IDA. Patients with IDA have a mean about 6 months.'° A response to iron therapy is defined as an
sTfR level greater than two times the mean of the normal range. increase of | g of hemoglobin in | month (Fig. 6-8).

BONE MARROW
Usually a bone marrow assessment is not indicated for an Anemia of Chronic Disease
uncomplicated case of IDA. The bone marrow shows a mild
Anemia of chronic disease (ACD) or anemia of inflammation
to moderate erythroid hyperplasia with a decreased M:E ratio.
(AOI) is a common hematological disorder. ACD is the second
A common finding is the presence of poorly hemoglobinized
most prevalent anemia after IDA and the most commonly
noromoblasts with a scanty cytoplasm. Nuclear fragments,
found anemia in hospitalized patients.'°'* ACD is multifactor-
karyorrhexis, budding, and multinuclearity are also found.
ial in nature and is associated with chronic infections, autoim-
Iron content is decreased and nuclear cytoplasmic asynchrony
mune disease, chronic inflammation, and malignant neoplasms
is also evident during maturation. Cytoplasm maturation lags
(Table 6-12). ACD usually presents | to 3 months after the
behind nuclear maturation (see Fig. 6-5).
onset of a chronic disease. The anemia is characterized by
decreased serum iron levels, decreased TIBC, and decreased
Treatment saturation of transferrin. Serum ferritin levels, however, are
usually increased as a result of the iron being trapped in the
The first choice in the treatment for IDA is correction of the RES cells of the bone marrow. This iron can be visualized in
primary disease state. The next step is oral dietary supple- bone marrow aspirates stained with Prussian blue.
ments, which are necessary to replenish the body stores. Oral
supplements of ferrous sulfate are the standard treatment or Pathophysiology
treatment of choice.”'* The recommended dose of treatment is
3 tablets per day containing 60 mg of elemental iron'*'> The The pathogenesis of ACD is unclear. It has been suggested
major obstacle with oral supplements is the side effect of that chronic disease states block the transfer of storage iron to
nausea and stomach discomfort. Other oral supplements erythroid precursors within the bone marrow.
 Chapter 6 Iron Metabolism and Hypochromic Anemias 127

Clinical Features

Anemia may be present for several months after the develop-

ment of a chronic disorder. ACD presents as a mild to moder-
ate anemia. In 60% to 70% of the cases, the anemia presents
Infections
Tuberculosis
as a normocytic normochromic anemia and in 30% to 40% of
Chronic osteomyelitis the cases, present as a microcytic hypochromic anemia.'’ The
Fungal infections signs and symptoms of the disease are associated with the
underlying cause.
Neoplasms
Carcinomas
Malignant lymphoma
Multiple myeloma
Laboratory Findings
Autoimmune Disorders Anemia of chronic disease (ACD) is defined by an aggregate of
Systemic lupus erythematosus (SLE) clinical, morphological, and laboratory findings in Table 6-13.
Rheumatoid arthritis
Sarcoidosis PERIPHERAL BLOOD
On examination of the peripheral blood, the red blood cells
may be normocytic to microcytic in size and normochromic
to hypochromic in color (Fig. 6-9). Anisocytosis and poikilo-
The possible causes of ACD include a shortened red cell
cytosis are slight to absent. The reticulocyte count does not
survival, failure of the bone marrow to increase red blood cell
reflect the degree of the anemia. There is increased stainable
production, and impaired release of iron from the reticuloen-
bone marrow iron and decreased marrow sideroblasts. The
dothelial system.'®
WBC and platelet counts are usually normal, depending upon
ACD is the result of an activated immune system with
the underlying condition.
the production of cytokines. These cytokines and the RE cells
cause changes in iron homeostasis.'° Several cytokines are
IRON STUDIES
involved in the inflammatory response and are increased in
Patients with ACD have decreased serum iron and transferrin
diseases associated with ACD. Recent studies of the role of
saturation while the ferritin levels are normal to increased.
cytokines in ACD suggest that both impaired iron metabolism
There is an increase in free erythrocyte protoporphyrin (FEP)
and impaired erythropoiesis are involved in ACD. When there
or ZPP, and unlike IDA, the TIBC is decreased. The differen-
is an increase in microorganisms or malignant cells, or the
tial diagnosis between iron deficiency and anemia of chronic
presence of an autoimmune disease, T cells become activated.
disease is made via measurement of plasma TfR concentra-
The T cells induce immune effector mechanisms, thereby pro-
tion.* The TfR is normal in patients with ACD and elevated
ducing cytokines. These cytokine mechanisms lead to
with IDA. In addition, the TIBC is increased in IDA and
decreased iron concentration in the circulation and thus limit
decreased in ACD.'*>-?
the availability of iron for erythroid production.
It has also been suggested that erythropoietin (EPO) may
be the rate-limiting factor in the impaired marrow response.'*
Patients with ACD have lower levels of EPO, and EPO
response to the anemia is blunted.

BOTO%S OSC GPo B20” errPWe “50 Clinical

So: Se?C008
PP$9.0 °0.00% BPO
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, | Sock Anemia present for several months following development
@ a of a chronic disease
GS) ®
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; ® Oo” 00 9@ Vay
Morphological
Usually normocytic RBCs (normal MCV)
00 Ox ay
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May be hypochromic or normochromic
0%~98S.
»D80,O56 00 90,900
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Increased storage iron
; Decreased sideroblasts
e@c® @
eo “e050 ©,% 6
9&5 Rare to absent ringed sideroblasts

Laboratory
Decreased serum iron
Decreased total iron-binding capacity (TIBC)
Decreased % transferrin saturation
Normal to increased serum ferritin levels
Figure 6-9 ™ Normochromic, normocytic red blood cells in a Normal serum soluble transferrin receptor levels
patient with anemia of chronic disease. (Wright's stain, x 200)
128 Chapter 6 Iron Metabolism and Hypochromic Anemias

BONE MARROW
In the bone marrow there is an adequate number of erythroid
precursors, and the M:E ratio may be increased because of
decreased erythropoiesis. Sideroblasts are decreased but the
macrophages appear to have an increased amount of storage Inherited
Congenital sideroblastic anemia, sex-linked
iron. Hemosiderin is the long-term storage of iron and appears Autosomal recessive sideroblastic anemia
as very coarse aggregates of iron (Fig. 6-10). In contrast,
hemosiderin is absent in IDA. Acquired
Primary or idiopathic
Myelodysplasia (RARS)
Secondary
Treatment
Lead
When possible, treatment for ACD is to treat the underlying Alcohol
Drugs, including isoniazid and chloramphenicol
disease first. In cases in which treating the underlying disease
is not feasible, other options are necessary. Blood transfusions
are given for a rapid and effective intervention. Transfusions
are helpful when there is a severe or life-threatening anemia.
For patients with ACD associated with chronic infection Pathophysiology
or malignancy, supplements of iron should be avoided.* Ery- Sideroblastic anemias involve abnormalities of the enzymes
thropoietic agents for patients with ACD are currently regulating heme synthesis. The ringed sideroblast is a charac-
approved for use by patients with cancer who are undergoing teristic of this group of anemias. Several investigators have
chemotherapy. The therapeutic effect involves counteracting identified enzyme deficiencies, including deficiencies of delta
the antiproliferative effects of cytokines, along with the stimu- 5-aminolevulinic acid synthetase and uroporphyrinogen
lation of iron uptake and heme biosynthesis in the erythroid decarboxylase, in patients with sideroblastic anemia. No one
cells2 defect, however, can account for all inherited and acquired
disorders. The ringed sideroblasts are sideroblasts in which
iron is accumulated in the mitochondria that surround the
Sideroblastic Anemia
nucleus (Fig. 6-11). These siderotic granules are visible with
The sideroblastic anemias are a group of disorders character- Prussian blue stain.
ized by a hypochromic anemia, ineffective erythropoiesis, an The hereditary sideroblastic anemias are rare. The con-
increase in serum and tissue iron, and the presence of ringed genital sex-linked type is more common than the autosomal
sideroblasts in the bone marrow. It is a very diverse group of recessive variant. The anemias typically appear early, usually
anemias and can be inherited or acquired. The inherited sider- within the first few months or years of life. The molecular
oblastic anemias include sex-linked congenital sideroblastic defect involves the enzyme 5-aminolevulinic synthetase.*°
anemia and autosomal recessive sideroblastic anemia. The This enzyme initiates the heme synthetic pathway. Heme syn-
acquired sideroblastic anemias can be primary (also referred thesis is impaired as iron enters the erythroid precursor which
to as idiopathic) or secondary (Table 6-14). The secondary cannot be incorporated in the heme molecule because the pro-
sideroblastic anemias are typically the result of toxins or toporphyrin ring cannot be formed. Iron then accumulates in
drugs, such as chloramphenicol. the mitochondria.

Figure 6-10 @ Increased reticuloendothelial iron in a patient with Figure 6-11 @ Ringed sideroblast as detected by Prussian blue
anemia of chronic disease. (Prussian blue stain, x 200) staining of a bone marrow aspirate. (Prussian blue stain, x 1000)
 Chapter 6 Iron Metabolism and Hypochromic Anemias 129

The acquired sideroblastic anemias can be primary or

secondary. The primary or “idiopathic” sideroblastic ane-
mias are exemplified by refractory anemia with ringed
sideroblasts (RARS), one of the myelodysplastic syndromes
(see Chap. 19). The secondary sideroblastic anemias are the
result of ingestion of alcohol, lead, and various medications.
The medications that commonly cause sideroblastic anemia Clinical
Usually older than 50 years of age
include isoniazid, chloramphenicol, pyrazinamide, and
Weakness, pallor, fatigue associated with anemia
cycloserine.
Lead poisoning is a common form of sideroblastic Morphological
anemia. Lead poisoning impairs heme synthesis at several Dimorphic RBC population with prominent hypochromia
Anisopoikilocytosis of RBCs, which may be associated with
steps. The heme enzymes involved are 5-aminolevulinic acid
basophilic stippling
dehydrase and ferrocheletase. Coarse basophilic stippling of Hypercellular bone marrow with erythroid hyperplasia
the RBCs is also a common feature of lead poisoning More than 15% ringed sideroblasts
(Fig. 6-12).

Laboratory Findings
The principal clinical feature is a refractory or progressive
anemia with the characteristic ringed sideroblasts in the bone
marrow aspirates (Table 6-15).

PERIPHERAL BLOOD
The anemia is moderate to severe. The red blood cells are
dimorphic, ranging from microcytes to normocytes (Fig. 6-13).
The MCV, MCH, and MCHC are usually normal. The RDW is
increased, representing the dual population of red blood cells.
Other abnormalities of the red cells include anisocytosis, poik-
ilocytosis, target cells, Pappenheimer bodies (iron deposits in
the red cell), and basophilic stippling. The WBC and platelet : @e® ©

counts are usually normal.

Figure 6-13 & Dimorphic population of red blood cells with a
IRON STUDIES striking hypochromic component in a patient with sideroblastic
Iron studies show increases in serum iron, increased ferritin, anemia. Moderate anisopoikilocytosis is also present. (Wright's
stain, * 200)
normal to decreased TIBC, and an increase in percent satura-
tion levels. Serum soluble transferrin receptor levels are usu-
ally normal or decreased. BONE MARROW
The primary feature common to all sideroblastic anemias is the
presentation of the ringed sideroblast in the bone marrow. Inef-
fective erythropoiesis, erythroid hyperplasia, and increased
stainable bone marrow iron are also characteristic of siderob-
lastic anemia.
In the myelodysplastic syndrome of refractory anemia with
ringed sideroblasts, the bone marrow is typically hypercellular
with erythroid hyperplasia and dyserythropoiesis (see Chap. 19).
Iron stains reveal more than 15% ringed sideroblasts. In the
other sideroblastic anemias the marrow has changes of erythroid
hyperplasia and often megaloblastoid characteristics.

Treatment

It is important to know whether the anemia is hereditary or

acquired. In the case of secondary sideroblastic anemia
caused by drugs, rapid improvement can be seen with the dis-
continuation of the offending medication. Administration of
Figure 6-12 = Prominent basophilic stippling is found with
pyridoxine has also been proven to be effective because the
defective heme synthesis. offending drug decreases the level of pyridoxine.
130. Chapter 6 Iron Metabolism and Hypochromic Anemias

ifferential Diag!
d Bone Marrow

Indices Iron-Deficiency Anemia Anemia of Chronic Disease Sideroblastic Anemia

RBC count Decreased Decreased Decreased

Hemoglobin (Hgb) Decreased Decreased Decreased
concentration
Hematocrit (Hct) Decreased Decreased or normal Variable
MCV Decreased Normal or decreased Variable
MCH Decreased Usually normal or Variable
may be decreased
MCHC Decreased Usually normal or Variable
may be decreased
Red cell distribution Increased Usually normal or Normal or increased
width (RDW) may be increased

Peripheral Blood and

Bone Marrow Features
Anisocytosis Yes No Wes
Poikilocytosis, including Yes No Yes*
target cells
Basophilic stippling No No Yes
Stainable bone marrow iron Decreased or absent Increased Increased
Marrow sideroblasts/ringed Decreased Decreased Increased
sideroblasts

*In hereditary forms.

MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin
concentration.

The differential diagnosis comparing RBC indices and resulting from excess iron. In this disorder of iron storage,
peripheral blood and bone marrow features of IDA, ACD, and there is an inappropriate increase in intestinal iron absorption
sideroblastic anemia is presented in Table 6-16. that leads to excess iron in the tissues. Iron overload may be
primary or inherited or secondary to the chronic anemias and
their treatment. The excess iron is stored in your liver, heart,
The Porphyrias
The porphyrias are a group of rare inherited disorders that
involve a block in porphyrin synthesis that is due to a
defect in the enzymes in the pathway of heme synthesis
(Fig. 6-14). This causes porphyrin heme precursors to
accumulate in tissues and large amounts are excreted in
urine and feces. The porphyrias are classified as acute or
nonacute according to their clinical presentation, and as
erythropoietic or hepatic, depending on the site of abnormal
metabolism.
The porphyrias are associated with neurovisceral
attacks’! which include photosensitivity, motor dysfunction,
sensory loss, mental disturbances, and sometimes abdominal
pain. The classification and characteristics of the porphyrias
are summarized in Table 6-17. The most common is acute
intermittent porphyria characterized by colicky abdominal
pain, vomiting, diarrhea, constipation, and central nervous
system involvement.

Iron Overload and Hemochromatosis

Figure 6-14 m Erythropoietic porphyria. Note the precipitated
Iron overload is defined as the accumulation of excess iron in
porphyrins in the cytoplasm. (From Bell, A: Hematology. In Listen,
reticuloendothelial cells in varying tissues. Hemochromatosis Look and Learn. Health and Education Resources. Inc., Bethesda,
describes a clinical disorder that results in tissue damage MD, with permission.)
 Chapter 6 Iron Metabolism and Hypochromic Anemias 131

“Table 6-17 Classification and Characteristics of the Porphyrias

Erythroid/
Disease Dehcient oe Course Inheritance Hepatic Symptoms

Acute intermittent Barpropi norer Acute Autosomal H Neurological

porphyria deaminase dominant
Hereditary Coproporphyrinogen Acute Autosomal H Neurological
coproporphyria III oxidase dominant Photosensitive
Variegate Porphobilinogen Acute Autosomal H Neurological
porphyria oxidase dominant Photosensitive
Cutaneous Uroporphyrinogen Chronic Variable H Photosensitive
hepatic decarboxylase
porphyria
ALA dehydratase
deficiency ALA dehydratase Acute Autosomal Neurological
porphyria recessive
Congenital
erythropoietic Uroporphyrinogen Chronic Autosomal Photosensitive
porphyria III cosynthetase recessive
Erythropoietic
protoporphyria Heme synthetase Chronic Autosomal Photosensitive
dominant

ALA = aounclevalinne acid; E =Le ceH= Jiatane

and pancreas** and damages these organs. The different types The third stage is iron overload with early symptoms that
of hemochromatosis are summarized in Table 6-18. include lethargy and arthralgia. The last stage is iron overload
with organ damage and cirrhosis. The liver organ is com-
monly implicated and hepatomegaly is present in 95% of
Hereditary Hemochromatosis Cases ge
Hereditary hemochromatosis (HH) is a recessive genetic disor- The involvement of hepcidin in hemochromatosis has
der and is one of the most frequent genetic diseases in Caucasian also been studied. Hepcidin is a peptide synthesized in the
populations, affecting approximately | in 300 people.'*?* Preva- liver and is involved with iron homeostasis. The rate of iron
lence is especially high in the Irish, Scottish, and Welsh popula- influx into the plasma depends on the activity of hepcidin.*°78
tions. In 1996, a mutant gene linked to the HLA-A locus on the When plasma iron levels are increased, the synthesis of hep-
cidin increases, which diminishes the release of iron from the
short arm of chromosome 6, called the HFE gene, was first
described in patients with hereditary hemochromatosis.’*** This
mutation is a single-base change that results in the substitution
of tyrosine for cysteine at position 282 of the HFE protein
(C282Y). The C282Y mutation alters the conformation of the
Table 6-18 Classification Of
HFE protein and interferes with its function of regulation of iron _Hemochromatosis sean
absorption.
Hereditary Hemochromatosis
Classical hemochromatosis type |
Juvenile type 2
Pathophysiology Transferrin receptor type 3
African overload type 4
The disease is caused by a gradual accumulation of iron in
the tissues which leads to chronic liver disease, arthritis, Secondary Hemochromatosis
diabetes, pituitary damage, congestive cardiac failure, Hereditary disorders
and cardiac arrhythmias (Fig. 6-15). Patients with HH Thalassemia
Sickle cell anemia
absorb two to three times as much dietary iron as normal
Sideroblastic anemia
individuals.2> The end result is intestinal absorption of Enzyme-deficiency anemia
iron that generally exceeds iron loss by approximately Hereditary spherocytosis
3 mg per day.7 25
Acquired Disorders
Four stages of the disorder have been described.” The Anemia not due to blood loss in which multiple
first stage is a genetic predisposition with no abnormality transfusions are required
other than increased serum transferrin saturation. The second Dyserythropoietic anemia
stage involves 2 to 5 g of iron overload without symptoms.
132. Chapter 6 Iron Metabolism and Hypochromic Anemias

hepatomegaly develops, which leads to cirrhosis and fibrosis

of the liver. Iron deposit in the heart tissue causes cardiomy-
opathy. Other complications are diabetes mellitus, hypopitu-
itarism, hypogonadism, and hypoparathyroidism.

Laboratory Findings
Although iron metabolism is abnormal, erythropoiesis is
normal and hematologic abnormalities are usually not seen.
Other laboratory abnormalities include increased liver function
enzyme tests, particularly alanine and aspartate aminotransam-
inases (ALT, AST).

IRON STUDIES
In patients with HH, serum iron, serum ferritin and serum
Figure 6-15 @ Liver biopsy from a patient with idiopathic
hemochromatosis and cirrhosis. Note the excess deposits of iron. transferrin levels are increased. TIBC is usually within nor-
(Ferric ferricyanide stain) mal limits. Increased percent transferrin saturation is an
important hallmark of the disease. The laboratory criteria for
diagnosing HH include transferrin saturation greater than
mucosal cells and macrophages. When plasma iron drops, the 50% for females and transferrin saturation greater than 60%
synthesis of hepcidin is decreased, allowing these cells to for males.* Diagnosis can be confirmed by direct analysis of
release more iron. Hepcidin expression is increased in iron the HFE gene.
overload and decreased in iron deficiency.™*
Other polymorphisms have been found, including
mutation H63D. H63D is a histidine to aspartic acid substi-
Treatment
tution at position 63. C282Y and H63D account for the The goal of treatment of iron storage disease is to remove the
majority of the cases of HH. The roles of hepcidin and HFE excess amount of iron from the body. Patients who have
protein are related. symptoms of HH require therapeutic phlebotomy. Removal of
500 mL of blood once or twice a week is performed until
Clinical Features there is a marked decrease in serum transferrin percent satu-
ration and serum ferritin. The goal is to have the serum fer-
Signs and symptoms of HH usually occur in midlife. The ritin less than 50 g/L and transferrin saturation below 30%.'*
most common complaint is joint pain. Chronic arthritis of the Once this is achieved, phlebotomy frequency is reduced to
second and third metacarpophalangeal joints is highly sugges- two to four times per year. In the event that therapeutic phle-
tive of the disease. Early symptoms are usually nonspecific. botomy is not appropriate, desferrioxamine, a chelating agent,
They may include fatigue, arthralgia, bronze discoloration of is used to reduce iron stores. For patients with hemoglobins
the skin, and erectile dysfunction. As the disease progresses, greater than 10 g/dL, phlebotomy is the choice of treatment;

en Mle

Transferrin Transferrin Stainable Bone

Disorder Ferritin Saturation Receptor Marrow Iron
StESO ONESRLEH

Iron deficiency D I D I Absent

anemia
Anemia of I ; N/D N/D N/D N/I
chronic
disease
Sideroblastic N/D N/D ii
anemia Ringed
sideroblast
Hereditary N/D N/D
hemochromatosis

D = decrease; I = increase; N = normal; V = variable; TIBC = total iron-binding capacity; ZPP = zinc protoporphyrin.
 Chapter 6 Iron Metabolism and Hypochromic Anemias 133

for patients with hemoglobins less than 10 g/dL, desferriox- African Iron Overload
amine is the treatment of choice.
A distinct iron-loading disorder is prevalent in Africa,
affecting up to 10% of some rural populations.**?? These
Secondary Hemochromatosis individuals have a predisposition to iron overload as a
result of excessive dietary iron intake. African iron over-
Secondary hemochromatosis can be acquired or secondary to
load is not due to mutations of the HFE gene and is not
other inherited hemolytic anemias. The common characteristics
linked to the HLA locus, but is thought to be related to
of secondary hemochromatosis are anemia, ineffective erythro-
cooking in iron pots.
poiesis, and iron overload. Secondary hemochromatosis patients
A summary and comparison of iron studies in the
are transfused repeatedly. This leads to increased iron storage
microcytic hypochromic anemias and hemochromatosis are
because there is no mechanism for iron excretion. Iron overload
provided in Table 6-19.
from transfusion therapy is treated with chelation therapy.

c. 2 mg/day
Case Study I d. | mg/day
5. Prussian blue staining of the bone marrow in this patient
A mother brought her 17-month-old female child to the would reveal:
pediatrician for a general check-up. The child was recently a. Increased storage and increased sideroblastic iron
adopted from Central America. On physical examination, b. Increased storage and decreased sideroblastic iron
the child appeared pale and listless. The laboratory tests c. Decreased storage and decreased sideroblastic iron
included WBC count = 16.5 X 10°/L; RBC count = 2.97 x d. Increased ringed sideroblasts
10'°/L: HGB = 4.2 g/dL; Het = 15.8%; MCV = 53.3 fL;
MCH = 14.1 pg; MCHC = 26.5 g/dl; RDW = 23.2%; PLT ANSWERS
count = 496 x 10°/L. Examination of the peripheral blood
smear revealed microcytic, hypochromic red blood cells
with marked anisocytosis and poikilocytosis.

QUESTIONS
AR
WN Oy
5es
So
a
©

1. What is the most likely explanation of this patient’s

anemia?
a. Hereditary hemochromatosis
b. Lead poisoning
c. Iron-deficiency anemia
d. Sideroblastic anemia
2. What additional test may useful in confirming the Case Study 2.
diagnosis?
a. Hemoglobin electrophoresis A 78-year-old man who was diagnosed 3 months earlier
b. Vitamin B,, and folate levels with non-Hodgkin’s lymphoma has undergone an extensive
c. Measurement of serum iron, ferritin, and TIBC staging workup. The patient has not yet received chemother-
d. Chromosome analysis apy, and staging studies suggest localized disease. The
3. Iron-deficiency anemia 1s associated with: results of laboratory tests included WBC count = 10.3 X
a. Decreased hemoglobin, increased MCV, increased 10°/L; Hgb = 12.0 g/dL; Het = 33.1%; MCV = 81 fL; PLT
serum ferritin, and decreased serum iron count = 221 xX 10°/L. Examination of the peripheral blood
b. Decreased hemoglobin, decreased hematocrit, revealed normocytic, hypochromic red blood cells without
decreased serum ferritin, and increased stainable bone significant anisocytosis and poikilocytosis. A bone marrow
marrow iron biopsy was performed to rule out involvement by malignant
c. Decreased hemoglobin, decreased hematocrit, lymphoma. Examination of the bone marrow specimen
increased serum iron, increased serum ferritin, and revealed no evidence of malignant lymphoma, and iron
marked anisocytosis and poikilocytosis stains were performed.
d. Decreased hemoglobin, decreased serum iron,
decreased ferritin, increased TIBC, and decreased QUESTIONS
transferrin saturation
4. Assuming the patient has normal dietary iron absorption, 1. What is the best explanation for this patient’s anemia?
the minimum dietary requirement of iron for this patient a. Iron-deficiency anemia
would be: b. Sideroblastic anemia
a. 3 mg/day c. Anemia of chronic disease
b. 0.5 mg/day d. Hereditary hemochromatosis
continued continued
134. Chapter 6 Iron Metabolism and Hypochromic Anemias

QUESTIONS
Case Study ete ra
il, What is the most likely cause of this patient’s disorder?
2. If serum iron studies were performed on this patient, a. Iron deficiency anemia
they would reveal: b. Anemia of chronic disease
a. Decreased serum iron, decreased serum ferritin, and
c. Acquired sideroblastic anemia
increased TIBC
d. Hereditary hemochromatosis
b. Decreased serum iron, normal to increased serum fer-
. The serum iron studies in this patient would reveal:
ritin, and decreased TIBC a. Increased serum iron, increased serum ferritin, and
c. Increased serum iron, increased serum ferritin, and increased percent transferrin saturation
decreased or normal TIBC b. Decreased serum iron, normal serum ferritin, and
d. Increased serum iron, increased serum ferritin, and decreased percent transferrin saturation
increased TIBC c. Decreased serum iron, decreased serum ferritin, and
3. Prussian blue staining of a bone marrow aspirate speci- decreased percent transferrin saturation
men from this patient would reveal: d. Increased serum iron, decreased serum ferritin, and
a. Decreased stainable bone marrow iron and decreased increased percent transferrin saturation
ringed sideroblasts . What additional test would be most useful in confirming
b. Decreased stainable bone marrow iron and increased the diagnosis?
ringed sideroblasts a. Hemoglobin electrophoresis
c. Increased stainable bone marrow iron and decreased b. Vitamin B,, and folate levels
marrow sideroblasts c. Reticulocyte count
d. Increased stainable bone marrow iron and increased d. Measurement of transferrin saturation
ringed sideroblasts . What is the appropriate therapy or treatment for this
disorder?
a. Blood transfusion
ANSWERS
b. Chelation therapy
c. Phlebotomy
d. Iron supplements
OO
WN

ANSWERS

Case Study 3 gs
CS)tek
(Sr
ety
ey

A 45-year-old diabetic man visited his physician because he

noticed bronze discoloration on his arms and legs. It was
starting to spread to his abdomen. On physical exam his liver
was slightly enlarged. Laboratory results of the CBC are as
follows: WBC count = 6.8 X 10°/L; RBC count = 4.97 x
10°/L; HGB = 14.2 g/dL; Het = 44.8%; MCV = 89.3 fL;
MCH = 26.1 pg; MCHC = 34.5 g/dl; RDW = 23.2% PLT
count = 296 < 10°/L. Liver enzymes were elevated.
continued
 Chapter 6 Iron Metabolism and Hypochromic Anemias ee)

Questions
: 1. The average adult has a total body iron content of: c _ 6 In the older patients, iron deficiency most often develops
a. 25 mg from:
b. 100 mg a. Impaired absorption of iron
c. 250 mg b. Poor diet
(4.8500 mg c. Chronic gastrointestinal bleeding
d. The aging process
2. Two-thirds of the total body iron is present as:
a. Hemosiderin . Microcytosis of red blood cells is reflected by:
b. Ferritin a. Increased MCV
c. Transferrin b. Decreased MCV
@ Hemoglobin iron c. Increased hemoglobin concentration
d. Decreased hemoglobin concentration
3. One milligram of blood contains:
a. 2 mg of iron iil . Anemia of chronic disease is commonly associated with
b. | mg of iron each of the following except:
.) mg of iron : a. Infections
d. 10 mg of iron b. Malignant neoplasms
c. Autoimmune disorders
4. On average, what percentage of the ingested iron is ab- d. A congenital defect
sorbed each day?
25% to 35% . Each of the following are causes of secondary siderob-
5% to 10% lastic anemias except:
c. 80% to 90% a. Alcohol
d. 100% b. Lead
c. Medications
5. Dietary iron is absorbed predominantly in the: d. Radiation therapy
a. Stomach
uodenum and first part of the jejunum 13. A common feature of sideroblastic anemia is which of
c. Transverse colon the following?
d. Sigmoid colon: a. Ringed sideroblasts
b. Decreased iron
6. The protein responsible for transporting
iron in the c. Decreased ferritin
bloodstream is: d. Macrocytes
a. Hemoglobin
b. Hemosiderin . Which of the following is the most sensitive assay in
ransferrin screening for hereditary hemochromatosis?
d. Ferritin a. Serum iron
_b. Serum ferritin
7. The hypochromic anemias represent a related group of c. Transferrin saturation
disorders with: d. Total iron-binding capacity
a. A quantitative defect in hemoglobin synthesis
b. A qualitative defect in globin protein chains ile The most often cause of secondary hemochromatosis is:
a. Drugs
c. Excess hemoglobin synthesis
d. Vitamin B,, and folate deficiency
b. Diet
c. Repeated blood transfusions
8. The most common cause of hypochromic anemia 1s: d. Malabsorption
a. Sideroblastic anemia
b. Megaloblastic anemia See answers at the back of this book.
c. Iron deficiency anemia
d. Lead poisoning
 6 Chapter 6 |ron Metabolism and Hypochromic Anemias

w Iron deficiency anemia presents as a hypochromic and

RR vivi\vae microcytic anemia with an increased RDW.
m [ron deficiency occurs when there is inadequate iron
m The hemoglobin molecule consists of iron, globin protein
intake, excess iron loss, or increased need for iron.
chains, and protoporphyrin.
w Iron studies for patients with IDA include a decreased
= The total iron concentration of an adult is 40 to 50 mg/kg
serum iron, serum ferritin and percent transferrin satura-
of body weight.
tion, and increased TIBC and increased transferrin
w The majority of the total body iron is present as hemoglo- receptor.
bin iron.
# Anemia of chronic disease is the most common anemia of
a Ninety percent of tissue iron is present as storage iron in hospitalized patients.
the form of ferritin or hemosiderin.
gw Iron studies of patients with anemia of chronic disease
w Approximately 5% to 10% of the ingested iron is absorbed include: decreased iron, normal to decreased iron binding,
per day. decreased percent transferrin saturation, increased fer-
= The minimum daily requirement for iron for an adult man ritin, and a normal TfR level.
and nonmenstruating woman is | mg. mw Bone marrow evaluation of anemia of chronic disease
g Increased iron consumption states include growth spurts, reveals increased storage iron and decreased sideroblastic
menstruation, pregnancy, lactation, and iron deficiency. iron.
g Ferrous iron (Fe**) is easily absorbed into the intestinal gw Sideroblastic anemias are disorders of heme synthesis and
mucosa cells. may be inherited or-acquired.
w Ferritin is the primary storage compound for iron. w Sideroblastic anemia presents with the “ringed siderob-
w Transferrin is a globulin protein responsible for transport- last’ due to iron being blocked from incorporation to form
ing iron within the bloodstream. heme.

@ Ferrous iron combines with protoporphyrin in the mito- w Hereditary hemochromatosis is an inherited iron overload
chondria of the red blood cell to form heme. disorder.

m@ Hemoglobin is formed from two a and two B globin pro- w Increased transferrin saturation is an important hallmark
tein chains, four heme groups, and four oxygen mole- of HH.
cules. mSecondary hemochromatosis can be hereditary or
mw The transferrin receptor is a tool for the assessment of acquired and is most commonly caused by repeated trans-
iron. The TfR level is inversely proportional to the fusions.
amount of iron in the body. mwThe most common gene mutation found in hereditary
g@ Serum ferritin levels are an indirect measure of iron stores hemochromatosis is C282Y.
in the body.

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1996. Accessed 5/17/06.
Sy. Brittenham, GM: Red blood cell function
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metabolism. ACP Medicine Online 2002.
19(7):60, 2002. 13(5):353, 2000.
Accessed 5/17/06.
. Centers for Disease Control (CDC)
co . Beutler, E, et al: Iron deficiency and
tO. Andrews, NC: Disorders of iron metabo-
MMWR Weekly Iron deficiency-United overload. Am Soc Hem (1):40, 2003.
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States 1999-2000. . Loil, JG: Anemia. In Fox, JA (Ed); Pri-
1S) . Riedl, HD, et al: Characterization and
. Carley, A: Anemia: When is it iron mary Health Care of Infants, Children and
partial purification of ferrireductase from
deficiency? Pediatr Nurs 29(2):127, Adolescents, ed. 2. Mosby, St. Louis,
human duodenal microvillus membranes.
2003. 2002, pp. 471480.
Biochem J 309:745, 1995.
. Tender, J, and Cheng, TL: Iron defi- . Weiss, G, and Goodnough, LT: Anemia
. Brittenham, GM, et al: Clinical conse-
ciency anemia. In Burg, FD, et al (Eds): of chronic disease. N Engl J Med
quences of new insights in the patho-
Gellis and Kagan’s Current 352(10):1011, 2005.
physiology of disorders or iron and heme
Pediatric Therapy. W.B. Saunders, . Wians, FH, et al: Discriminating between
metabolism. Am Soc Hematol 1:39,
Philadelphia, 2002, pp. 633-637. iron deficiency anemia and anemia of
2000.
Wil. Joosten, E, et al: Upper and lower gas- chronic disease using traditional indices
Nn . Gunshin, H, et al: Cloning and character-
trointestinal evaluation of elderly inpa- of iron status vs transferring receptor con-
ization of a mammalian proton-coupled
tients who are iron deficient. Am J Med centration. Am J Clin Pathol 115(1):112,
metal-ion transporter gene. Nat Genet
107(1):24, 1999. 2001.
16:383, 1997.
. Brittenham, GM: Red blood cell function 18. Means, RT, and Krantz, SB: Progress in
. Feder, JN, et al: A novel NHC class I-like
and disorders of iron metabolism. understanding the pathogenesis of the
gene is mutated in patients with hereditary
 Chapter 6 Iron Metabolism and Hypochromic Anemias 137

anemia of chronic disease. Blood 22 . O'Neil, J, and Powell, L: Clinical 27. Fleming, RE, and Sly, WS: Hepcidin: A
80(7):1639, 1992. aspects of hemochromatosis. Semin putative iron regulatory hormone rele-
19. Means, RT: Recent developments in the Liver Dis 25(4):381, 2005. vant to hereditary hemochromatosis and
anemia of chronic disease. Curr Hematol 3. Hash, RB: Hereditary hemochromatosis. the anemia of chronic disease. PNAS
Rep 2(2):116, 2003. J Am Board Fam Pract 14(4):266, 2001. 98(15):8160, 2001.
20. Schrier, SL: Anemia production defects: . Meeker, J, and Miller, S: Hereditary . Barton, JC, et al: Management of
Production defects with marrow ery- hemochromatosis. MLO 37(10):10, 2005. hemochromatosis. Ann Intern Med
throid hyperplasia and ineffective ery- . Pietrangelo, A: Hereditary hemochro- 129:932, 1998.
thropoiesis. ACP Medline Online 2002. matosis-a new look at an old disease. 29. Gordeuk, V, et al: Iron overload in
Accessed on 5/17/06. N Engl J Med 350(23):2383, 2004. Africa: Interaction between a gene and
BM. Sassa, S, and Kappas, A: The porphyrias: . Kohli, M, et al: Long term survival dietary iron content. N Engl J Med
Clinical presentation of the porphyries. in patients with hereditary hemochro- 82605m L992:
ACP Medline Online 2002. Accessed matosis. Gastroenterology 110:1107,
5/17/06. 1996.
 Chapter /

| Megaloblastic
Anemias
Mitra Taghizadeh, MS, MT(ASCP), CLS(NCA)

Introduction OBJECTIVES
Biochemical Aspects At the end of this chapter, the learner should be able to:
Clinical Manifestations of
| . Define megaloblastic anemia.
Megaloblastic Anemia
Z . Compare and contrast the morphological characteristics of megaloblasts and
Hematologic Features
normoblasts.
Ineffective Hematopoiesis
Bone Marrow Morphology . Characterize the peripheral blood morphology of megaloblastic anemia.
Peripheral Blood
. Identify the bone marrow morphology of megaloblastic anemia.
Morphology
Etiology of Megaloblastic . Describe pernicious anemia, including the pathophysiology and clinical and
Anemia laboratory findings.
Vitamin B,, Deficiency . Describe the Schilling test as a diagnostic tool for pernicious anemia.
Folic Acid Deficiency
. List the causes of vitamin B,, and folate deficiencies.
Laboratory Diagnosis of
Megaloblastic Anemia . Evaluate laboratory tests used for the diagnosis of megaloblastic anemia.
Laboratory Tests for the
. Compare and contrast the treatment for vitamin B,, and folate deficiencies.
Diagnosis of Vitamin B,,
and Folic Acid Deficiencies . Using the peripheral blood findings, differentiate the anemia of liver disease from a
Other Laboratory Tests megaloblastic anemia caused by vitamin B,, or folate deficiency.
Treatment 1] . List other causes of macrocytic nonmegalobiastic anemias.
Vitamin B,, Deficiency
Folic Acid Deficiency De, Evaluate laboratory tests used for the diagnosis of macrocytic anemias.

Response to Therapy
Vitamin-Independent
Megaloblastic Changes
Inherited
Acquired
Drug and Toxin Induced
Macrocytic
Nonmegaloblastic Anemias
Causes of Macrocytic
Nonmegaloblastic
Anemias
Case Study

138
 Chapter 7 Megaloblastic Anemias 139

Introduction Hematologic Features

Megaloblastic anemia is a subgroup of macrocytic anemia char- Ineffective Hematopoiesis
acterized by defective nuclear maturation caused by impaired
deoxyribonucleic acid (DNA) synthesis. This defect is mani- Megaloblastic anemia is associated with ineffective erythro-
fested by the presence of megaloblasts (large and abnormal red poiesis and hemolysis. The mean corpuscular volume (MCV)
cell precursors) in the bone marrow and macro-ovalocytes in the is greater than 100 femtoliters (fL). Patients with megaloblas-
peripheral blood. The granulocyte precursors tend to be larger tic anemia may have MCV values as high as 160 fL. This
than normal, with giant metamyelocytes as a striking feature. An elevated MCV reflects the megaloblastic picture of the bone
abnormal nuclear pattern in megakaryocytes may be seen in marrow. Increased erythrocyte precursors in the bone marrow
severe anemia. The megaloblastic changes are not limited to the and decreased red cell release into the peripheral blood is
hematopoietic cells; changes are also present in other nucleated indicative of ineffective erythropoiesis which is supported by
actively proliferating cells, such as skin, vaginal, uterine, cervi- decreased reticulocytes.
cal, and buccal cells. Megaloblastic erythrocyte progenitors have a much shorter
life span than normal erythrocyte progenitors. They are more
fragile and, therefore, die prematurely in the marrow. Evidence
Biochemical Aspects of intramedullary hemolysis includes decreased haptoglobin,
increased levels of serum bilirubin, serum lactate dehydroge-
The defective nuclear maturation and the megaloblastic mor- nase (LD, in particular, LD-1 and LD-2 isomers), and increased
phology are caused by a decrease in thymidine triphosphate serum iron.'® Cell death occurs primarily at the later stages of
(TTP) synthesis from uridine monophosphate (UMP). This the megaloblast maturation (1.e., basophilic and polychro-
deficiency interferes with nuclear maturation, DNA replica- matophilic stages), causing a decrease in production and release
tion, and cell division. When TTP is not present in adequate of mature erythrocytes. A decreased level of erythrocytes in the
amounts, deoxyuridine triphosphate incorporates into the circulation stimulates erythropoietin release, which in turn stim-
DNA instead'> (Fig. 7-1). This misincorporation causes frag- ulates production of red cell progenitors.*
mentation of the nucleus and, ultimately, destruction of Ineffective granulopoiesis is defined by increased bone
immature cells. . marrow white cell precursors and failure to release mature
The primary causes of lack of thymidine and conse- forms into the peripheral blood. The giant metamyelocytes do
quently defective DNA synthesis are vitamin B,, and folic not mature to circulating neutrophils but, rather, die prema-
acid deficiencies. These vitamins, in the form of cofactors, turely in the bone marrow. The elevated serum muramidase is
play important roles in some key reactions involved in DNA a result of this increased turnover of white cells.
synthesis. In addition, drugs that interfere with the metabo- Ineffective thrombopoiesis is manifested by the presence
lism of these vitamins also cause DNA impairment. of increased abnormal megakaryocytes in the bone marrow
and thrombocytopenia in the peripheral blood.
Clinical Manifestations of Megaloblastic
Anemia Bone Marrow Morphology
Certain clinical manifestations are common to all patients Patients with megaloblastic anemia have a hypercellular bone
with megaloblastic anemias regardless of the cause. The marrow. The myeloid-to-erythroid (M:E) ratio is decreased
degree of anemia may be mild to severe, with the symptoms (Fig. 7-2) and may be as low as 1:1 to 1:3. The degree of
of weakness, fatigue, shortness of breath, and lightheaded- increased cellularity (megaloblastic picture) depends on the
ness. Congestive heart failure may or may not be present, severity of the anemia. Megaloblasts are large cells with
depending on the degree of anemia. In severe anemia, the increased RNA per DNA unit. Their nuclear chromatin appears
patient may have a lemon-yellow skin tint because of mild loose and less mature than the nuclear chromatin of the normal
jaundice and pallor. Increased bilirubin is reported in about red cells at the same stage of maturation (Fig. 7-3). The cyto-
30% of patients as a result of intramedullary hemolysis plasm maturation is, however, normal. This phenomenon is
caused by ineffective erythropoiesis.” referred to as nuclear to cytoplasm asynchrony. The mature

Thymidylate
— synthetase

Figure 7-1 ™ Thymidine synthesis pathway from uridine nucleotide. Uracil is incorporated into DNA in the absence of thymine. UDP =
uridine diphosphate; (UDP = deoxyuridine diphosphate; dUTP = deoxyuridine triphosphate; (UMP = deoxyuridine monophosphate;
dTMP = deoxythymidine monophosphate; dTDP = deoxythymidine diphosphate; dTTP = deoxythymidine triphosphate: CH, THF =
methylene tetrahydrofolate.
140 Chapter 7 Megaloblastic Anemias

concentration (MCHC) is normal. Not all patients with macro-

cytosis have megaloblastic anemia (i.e., alcoholism and liver
disease), and not all megaloblastic anemias are macrocytic. A
normal MCV may be present in patients with megaloblastic
anemia and coexisting iron deficiency or thalassemia.
The hemoglobin value may be normal to low. In a severe
anemia the hemoglobin may drop to below 7 to 8 g/dL. The
erythrocyte count is generally decreased. The leukocyte count
may be normal at the early stage of anemia but may decline
to as low as 1 to 3 X 10°/L. Although platelets are the least
affected cell line, platelet counts below 100 x 10°/L have
been reported in patients with severe anemia.°
The peripheral smear is pancytopenic, with the presence
of macrocytes and macro-ovalocytes (Fig. 7-4). The degree of
anisocytosis and poikilocytosis varies with the severity of ane-
Figure 7-2 @ A. Mitotic figures in megaloblastic marrow. B. Large mia. Other poikilocytes, such as schistocytes, teardrop-shaped
megaloblastic band neutrophil. C. Megaloblastic pronormoblast cells, spherocytes, and target cells, may also be seen on the
with open, sievelike chromatin. The myeloid-to-erythroid (M:E) ratio
peripheral blood smear. Increased anisocytosis causes an ele-
is decreased because of the increase in megaloblastic erythroid pre-
cursors labeled A and C. vated red cell distribution width (RDW) as determined via an
automated cell counter. Dimorphic red cell morphology may be
present in patients who have iron-deficiency anemia, tha-
megaloblastic red cells entering the circulation usually have a lassemia, anemia of chronic disease, or hyperthyroidism in
shorter life span than normal, mature red cells. addition to the megaloblastic anemia. A trimorphic blood smear
Megaloblastic changes are manifested in white cell precur- may be seen in transfused patients. Red cell inclusions such as
sors by the presence of large bands (see Fig. 7—2B) and giant Howell—Jolly bodies and basophilic stippling are frequently
metamyelocytes in the bone marrow. The nucleus of the giant present (Fig. 7-5). Cabot rings and megaloblastic nucleated red
bands may show abnormal staining characteristics. These white cells may be seen on the peripheral blood smear (Fig. 7-6). The
cell abnormalities are not seen in the megaloblastic bone mar- absolute reticulocyte count is decreased with a reticulocyte pro-
row present in patients with myelodysplastic syndrome or duction index (RPI) of less than 2, indicating ineffective
erythroleukemia.’ Megakaryocytes are also affected in severe erythropoiesis. With treatment, the number of reticulocytes
megaloblastic anemia. They may have abnormal nuclear or increases along with increased numbers of nucleated red cells.
cytoplasm morphology, such as increased nuclear lobulation In patients with untreated megaloblastic anemia, the
and hypogranulation.* macrocytes have a shortened survival time (27 to 75 days)
compared with the survival time of normal red cells (110 to
120 days). The survival of normal red cells is shortened when
Peripheral Blood Morphology
Megaloblastic anemia is a macrocytic, normochromic ane-
mia. Depending on the degree of anemia, the MCV may range
from 100 to 160 fL. The mean corpuscular hemoglobin
(MCH) is elevated but the mean corpuscular hemoglobin

Figure 7-4 m™ Extreme degree of anisocytosis (+4) and poikilocytosis

(+4) with oval macrocytosis (arrow) in a patient with severe pernicious
Figure 7-3 ® Bone marrow. A. Polychromatophilic megaloblasts. anemia. (From Bell, A: Hematology. In: Listen, Look and Learn. Health
B. Orthochromic megaloblast with multiple Howell-Jolly bodies. and Education Resources, Inc., Bethesda, MD, with permission.)
 Chapter 7 Megaloblastic Anemias 141

et
Figure 7-5 @ Howell-folly body in an RBC in pernicious anemia Figure 7-7 ™ Neutrophil hypersegmentation in pernicious anemia.
(arrow). (From Bell, A: Hematology. In: Listen, Look and Learn.
Health and Education Resources, Inc., Bethesda, MD, with
permission.)
The diagnosis of megaloblastic anemia is usually based on
the morphological characteristics of the peripheral blood and
the results of other biochemical tests. Bone marrow examina-
transfused to a patient with severe and untreated megaloblas- tion is generally not required. Bone marrow aspirates are per-
tic anemia, which is indicative of extracorpuscular hemolysis.° formed for diagnosis of other disorders with a megaloblastic
Multilobed neutrophils, termed hypersegmented neu- picture, such as myelodysplastic syndrome or erythroleukemia.
trophils, are seen in the peripheral smear in 98% of cases? The clinical features of megaloblastic anemia are summarized
(Fig. 7-7). Hypersegmented neutrophils refer to one or more in Table 7-1.
neutrophils with six lobes, or five or more neutrophils with
five or more lobes.’ They are larger than normal mature neu- Etiology of Megaloblastic Anemia
trophils. The number of hypersegmented neutrophils counted
per 100 white cells as determined by the differential should be The major causes of the megaloblastic anemias are vitamin
reported. Although hypersegmented neutrophils appear early B,, deficiency, folic acid deficiency, or a combination of both.
in the peripheral blood, they are the last morphological fea- Megaloblastic anemia can also be present in non-vitamin-
ture to disappear. Their absence does not rule out the diagno- deficient diseases, such as myelodysplastic syndromes and
sis of megaloblastic anemia. acute leukemias. Drug-induced megaloblastic anemia is
Other notable abnormal laboratory tests are elevated lac- caused by drugs that interfere with the metabolism of either
tate dehydrogenase (LD), indirect bilirubin and urobilinogen, vitamin B,, or folic acid. Examples of these drugs are
decreased haptoglobin, increased serum iron and ferritin, and discussed later in the chapter.
increased erythropoietin.*°'°

Bone Marrow
Hypercellular
Low ME ratio
Megaloblasts
Giant bands and metamyelocytes

Peripheral Blood
Pancytopenia
Macro-ovalocytes
Hypersegmented neutrophils

Biochemical Changes
Decreased haptoglobin
Elevated LD
Elevated indirect and direct bilirubin
Increased serum iron and ferritin
Increased erythropoietin
Figure 7-6 ™ Cabot ring in pernicious anemia (arrow).
142 Chapter 7 Megaloblastic Anemias

Vitamin B,, Deficiency TRANSPORT AND METABOLISM

Two important proteins are involved in the transport of vita-
SOURCES AND REQUIREMENTS min B,, from the duodenum to the ileum and from ileum to
Vitamin B,, is produced by microorganisms and fungi. It is tissues: the intrinsic factor (IF) and transcobalamin II.’°
present in foods of animal origin such as liver, fish, poultry, Dietary cobalamin is released from the food by gastric acids
meat, eggs, and dairy products. Liver is a major source of and intestinal enzymes. On release, it binds to a carrier pro-
vitamin B,,. Vegetables do not contribute B,, to the diet. tein called R protein (fast-moving protein electrophoreti-
Vitamin B,, is commercially available as a supplement for cally). On entering the duodenum, B,, releases from the R
treatment of deficiencies. protein by the action of pancreatic enzymes. The released
In the United States, the daily diet for adults contains an vitamin B,, then binds to IF, a glycoprotein with molecular
average of 5 to 30 ug of vitamin B,,, of which | to 5 ug is mass of approximately 44,000 daltons.' The gene for IF is
absorbed. The recommended dietary intake of vitamin B,, for located on chromosome 11.’ IF is secreted by parietal cells of
adults is 5 ug/day.' This requirement increases in pregnancy, the stomach. The parietal cells also secrete hydrochloric acid
infancy, during growth and increased metabolic stages, and in and other gastric juices. IF binds to vitamin B,, and forms
the elderly. Vitamin B,, is lost through the urine and feces. B,.-IF complex, which allows vitamin B,, to be absorbed
The rate of loss is about 0.1% per day. Body storage of vita- through the receptors present on the brush borders of the
min B,, is about | to 5 mg, of which approximately | to 2 mg ileum (the distal half of the small intestine) (Fig. 7-9). IF is
is stored in the liver.' Because the daily requirement of vita- not absorbed by the ileum and, therefore, cannot be reutilized.
min B,, is low and the storage rate is high, it takes several It is degraded on release from vitamin B,,.''' The released
years for a person to develop vitamin B,, deficiency as a vitamin B,, then enters the portal vein.
result of malabsorption. When vitamin B,, leaves the ileum and enters the portal
vein, it attaches to three proteins— transcobalamin I (TC 1),
STRUCTURE transcobalamin II (TC II), and transcobalamin HI (TC II).
Vitamin B,, (cobalamin) is a large, water-soluble molecule. It About 70% to 90% of vitamin B,, is bound to TC I, TC III, and
consists of a corrin nucleus composed of four pyrrol rings (A to other R proteins, whereas only about 10% to 25% of vitamin
D) with a cobalt atom at the center (Fig. 7-8), similar to the B,, is bound to TC II.'’ TC II is the main transport protein of
porphyrin structure (see Chap. 6). The corrin ring is attached to cobalamin to the tissues. It is a polypeptide with a molecular
the nucleotide 5,6-dimethylbenzimidazole. The cobalt atom mass of 43,000 daltons.’ TC II is synthesized by ileal cells,
can be attached to several different molecules, such as adeno- endothelial cells, liver, spleen, heart, and macrophages, and is
syl (5-deoxyadenoside), methyl, cyanide, and hydroxy, to form secreted into the plasma.'’° TC II transports vitamin B,, to the
the biologically active forms of cobalamins (see Fig. 7-8). liver for storage and to the bone marrow and other tissues for
DNA synthesis (see Fig. 7-9). Deficiency of TC II causes
megaloblastic anemia. It is suggested that the functions of TC
I and TC III are to bind to vitamin B,,, preventing its losses in
R:
Adenosyl thesunines>
Methyl
Cyanide
Hydroxy

Ce y |&
Cobamide

Byo ——>Byo + IF<—IF

5,6-Dimethyl-
benzimidazole

Bio
+TC ll

Figure 7-8 @ Structure of vitamin B,, (cobalamin). When R is

adenosyl, the compound is adenosylcobalamin (AdoCb); when R is
methyl, it is methylcobalamin (MeCb); when R is cyanide, it is
cyanocobalamin (CNCb); and when R is hydroxy, it is hydroxo- Figure 7-9 ® Transportation path of vitamin B,, from the diet to
cobalamin (OHCb). the tissues. IF = intrinsic factor; TC Il = transcobalamin II.
 Chapter 7 Megaloblastic Anemias 143

Deoxyuridine Thymidine
‘Table 7-2Causes ofVitamin Be |
| Deficiency
Dietary Deficiency
Strict vegans
synthetase
Malabsorption
NADPH +H Pernicious anemia
Gastrectomy (total or partial)
5, 10-CHo THF DHF < DHF reductase Blind loop syndrome
Fish tapeworm (Diphyllobothrium latum)
NADP + Diseases of ileum
Wale Chronic pancreatic disease

Methionine Other Disorders

Hemodialysis
Methyl Co Human immunodeficiency virus (HIV) infection
Tissue Acquired immunodeficiency syndrome (AIDS)
Homocysteine Drugs
CH3 THF Alcohol
Nitrous oxide (NO)
Para-aminosalicylic acid (PAS)

CH3 THF

Figure 7-10 The role of vitamin B,, and folate in DNA synthesis. DIETARY VITAMIN B,, DEFICIENCY Nutritional vitamin B,,
CH,THF = methylene tetrahydrofolate; THF = tetrahydrofolate; deficiency is uncommon in western countries and is limited to
DHF = dihydrofolate; CH;THF = methyl tetrahydrofolate; (UMP = strict vegetarians. In this group, the decrease in vitamin B,, is
deoxyuridine monophosphate; dTMP = deoxythymidine
accompanied by an increased plasma folate level.
monophosphate.
It is worth noting that children born to a vegan mother (one
who consumes no animal food) or to a mother who has an
untreated vitamin B,, deficiency are vitamin B,, deficient, espe-
Vitamin B,, plays an important role in two key reactions
cially if they are breast-fed.’ Untreated infants are severely
in the body. First, it is necessary in the synthesis of methionine
megaloblastic, with retarded growth and psychomotor develop-
from homocysteine. In this biochemical reaction, both vitamin
ment. Neurologic complications, such as irritability, anorexia,
B,, and folic acid are involved. Vitamin B,, acts as a coenzyme,
and failure to thrive, have been reported in severely vitamin B,-
methylcobalamin (MeCb), for the enzyme methyltransferase
deficient infants.”!°
(Fig. 7-10). Second, vitamin B,, is important in conversion of
The major cause of vitamin B,, deficiency is malabsorp-
methylmalonyl CoA, a Krebs cycle intermediate, to succinyl
tion. The most common form of intestinal malabsorption is
CoA. In this reaction vitamin B,, also acts as a coenzyme,
pernicious anemia. Other causes of vitamin B,, deficiency are
adenosylcobalamin (AdoCb), for the enzyme methylmalonyl
summarized in Table 7—2 and discussed later.
CoA mutase. Adenosylcobalamin acts as a hydrogen (H) car-
rier, taking the H from methylmalonyl CoA to make succinyl
PERNICIOUS ANEMIA
CoA (Fig. 7-11).
DEFINITION Pernicious anemia is the most common cause
of vitamin B,, deficiency. It is a chronic disease caused by the
CAUSES OF VITAMIN B,, DEFICIENCY
deficiency of IF. The cause for a decrease in IF is gastric parietal
Vitamin B,, deficiency progresses through four stages: stage I,
cell atrophy, which is associated with a concomitant decrease in
negative vitamin B,, balance; stage II, vitamin B,, depletion;
other gastric juices. Lack of IF leads to defective vitamin B,5
stage III, vitamin B,,-deficient erythropoiesis; and stage IV,
absorption and, consequently, megaloblastic anemia.
vitamin B,,-deficient anemia. Stages | and II are referred to as
the depletion stages. Stages III and IV are referred to as the
deficient stages.'*

COR
Sa CON CO oe Con

Ado Co
CH3 — CH2>—CO — S—CoA——*»CH3—CO—COOH ~—® CH2—CH2— COOH
Propionyl CoA Methylmalonyl CoA Succinyl CoA

Figure 7-11 m Conversion of methylmalony! CoA to succinyl CoA. AdoCo = adenosylcobalamin.

144. Chapter 7 Megaloblastic Anemias

Pernicious anemia is more common in people of the cobalamin-binding site of IF and is found more often. Type
Scandinavian, English, and Irish descent, with a prevalence in Il, or binding antibody, is detected as a complex with IF.°"°
women (female-to-male ratio of 4:1).° Pernicious anemia 1s Thyroid antibodies have often been found in the serum of
more common after age 50. In blacks, the disease may start patients with pernicious anemia or their relatives.°
earlier.'° Pernicious anemia occurs rarely in children, and, if Lymphocytotoxic antibodies have also been detected in
it occurs, it may be in the congenital form. Congenital perni- one-third of patients with pernicious anemia. A decrease in
cious anemia is characterized by the total absence of IF and suppressor T cells and an increase in the CD4:CD8 ratio sup-
normal secretion of other gastric juices. There are no antibod- port the presence of cell-mediated immunity in patients with
ies present against IF or the parietal cells. Juvenile pernicious pernicious anemia.°
anemia is similar to adult pernicious anemia, with the age of An association between pernicious anemia and other
onset in the second decade.°® autoimmune diseases, such as thyroid disease, diabetes melli-
tus, and rheumatoid arthritis, has also been noted.®° The posi-
PATHOPHYSIOLOGY The main cause of pernicious anemia is tive response to steroids in some patients with pernicious
atrophic gastritis characterized by atrophy of the gastric mucosa anemia supports the autoimmune mechanism.°
with decrease of gastric secretions and IF. The cause of gastric The Helicobacter pylori microorganism has been identi-
atrophy is probably autoimmune, with poorly defined genetic fied as a major cause of gastritis and peptic ulcers. This
predisposition.® IF is essential for absorption of vitamin B,,. In microorganism induces autoantibodies against the gastric pro-
the absence of IF, only a small amount of vitamin B,, is ton pump H+, K+-ATPase in 20% to 30% of infected patients.
absorbed, causing a gradual deficiency in vitamin B)>. These antibodies are associated with gastritis, increased
atrophy, and apoptosis in the corpus mucosa. Patients who
Genetic Factors develop these antibodies present clinical features similar to
The congenital form of pernicious anemia is inherited as an those of patients with autoimmune gastritis.'*
autosomal-recessive trait and is primarily seen in children
before age two.*!” The genetic contribution to the adult form of CLINICAL MANIFESTATIONS OF VITAMIN B,,
pernicious anemia is supported by: DEFICIENCY
The onset of pernicious anemia is generally insidious. Patients
|. The concordant presence of pernicious anemia in identical
with pernicious anemia and other vitamin B,, deficiencies have
twins
all the signs and symptoms of megaloblastic anemia mentioned
2. The increased risk in relatives of patients with pernicious
earlier. Fever is usually present in severe anemia. Loss of
anemia
appetite is a common complaint. Glossitis (sore tongue) 1s
3. The presence of achlorhydria with or without malabsorp-
reported in 50% of patients.° Although the initial presentations
tion in relatives of patients with pernicious anemia
may vary among patients with vitamin B,, deficiency, the clas-
4. The findings that relatives of patients with pernicious ane-
sic symptoms include weakness, glossitis, and paresthesias.°
mia may produce antibody to gastric parietal cells (24%, as
The bone marrow morphology of patients with vitamin B,5
compared with 3.8% in patients with a negative family
deficiency is megaloblastic, and the peripheral smear contains
history).'°!?
macro-ovalocytes. In addition to hematologic abnormalities,
The exact cause of the genetic predisposition of pernicious vitamin B,, deficiency is associated with gastrointestinal, throm-
anemia is not yet clear. A weak association has been made botic, psychiatric, and neurologic complications.
between pernicious anemia and the human leukocyte antigen
(HLA), but it is not conclusive. An association between HLA- NEUROLOGIC MANIFESTATIONS Neurologic problems are
B7 and pernicious anemia has been reported in whites. The more common in pernicious anemia than in other types of
association of HLA-D antigens Dw2, Dw5, and DR2 with vitamin B,, deficiencies. The degree of neurologic involvement
pernicious anemia is more significant than that of other HLA is not directly related to the degree of anemia. Neurologic man-
antigens.°'* IF antibodies are associated with HLA-Dw2.° ifestations in the absence of hematologic abnormalities have
been reported in many cases.'°
Immunologic Factors The neurologic abnormalities may be mild, moderate, or
The serum of patients with pernicious anemia contains autoan- severe and may involve degeneration of peripheral nerves,
tibodies to parietal cells, to IF, and to thyroid tissue. The pari- posterior columns of the spinal cord, and posterior and lateral
etal cell antibodies are found in the serum of approximately columns of the spinal cord (Table 7-3). Because multiple neu-
80% to 90% of all patients with pernicious anemia. The anti- ropathies are involved, the terms subacute combined degener-
bodies are also present in the gastric juices of 75% of patients ation (SCD) and combined system disease may be used.°”°
with pernicious anemia. This antibody is specific for the pari- In the earlier stage of pernicious anemia, the peripheral
etal cells only, and does not show any cross-reactivity.° nerves are affected, causing paresthesia and areflexia.2°?! The
Antibodies to IF are demonstrated in the serum of patient often experiences symmetric tingling or “pins-and-
approximately 50% to 70% of patients with pernicious ane- needle” sensations in the toes and later in all four limbs. Less
mia. These antibodies are more specific for diagnosis of ane- often, the patient may complain of numbness. At the later
mia than antiparietal antibodies. Two types of IF antibodies stage, the posterior spinal columns may be involved. At this
have been reported. Type I, or blocking antibody, attaches to stage, the patient may complain of clumsiness and have an
 Chapter 7 Megaloblastic Anemias 145

DISEASES OF ILEUM Vitamin B,, deficiency can also be

seen in diseases of the ileum, for example, after ileal resection
or bypass, and in regional enteritis.®

CHRONIC PANCREATIC DISEASE In pancreatic disease,

¢ Degeneration of peripheral nerves
vitamin B,, deficiency develops as a result of a decrease in
¢ Degeneration of posterior columns
¢ Degeneration of posterior and lateral columns the proteases necessary for release of vitamin B,, from sali-
vary and gastric R proteins for absorption. A low level of free
calcium, which is necessary for calcium-dependent ileal
absorption, can cause vitamin B,, deficiency in patients with
incoordinate gait. The lateral spinal columns become involved chronic pancreatic disease.°'*
in the most severe stage of illness, with manifestations of
severe weakness and stiffness of limbs, impairment of memory,
OTHER DISORDERS Vitamin B,, deficiency has also been
and depression. Severe psychiatric symptoms are less common
reported in patients who are on hemodialysis and in patients
and include stupor, hallucinations, paranoia, and severe depres-
with human immunodeficiency virus (HIV) infection and
sion, referred to as “megaloblastic madness.” Less frequent
with acquired immunodeficiency syndrome (AIDS), espe-
neurologic problems include ophthalmoplegia. Bilateral retinal
cially in those receiving zidovudine therapy.°’
bleeding in severe anemia with thrombocytopenia has been
reported in some patients.°” Patients with SCD respond well
with proper and adequate treatment. Neurologic manifestations DRUG-INDUCED VITAMIN DEFICIENCY Other causes of
of less than 3 months’ duration are reversible; complete vitamin B,, deficiency are drugs such as alcohol, anesthetics,
improvement in 47% of patients and partial improvement in nitrous oxide (N,O), and the antituberculosis drug para-
53% have been reported.°*? However, some degree of dysfunc- aminosalicylic acid (PAS).”'*?°
tion may remain. In untreated patients, the neurologic symp-
toms are progressive, and the degree of severity is directly
proportional to the duration of symptoms.°** Folic Acid Deficiency
The basic underlying cause for the neuropathy associated
with vitamin B,, deficiency is not exactly known. However, the SOURCES AND REQUIREMENTS
impairment of methionine synthetase reaction has been indi- Folic acid, also known as folate or pteroylglutamic acid, is a
cated as the possible cause for the neuropathy. The rationale water-soluble vitamin present in a variety of foods. The high-
for this hypothesis is that the deficiency of methionine leads to est concentration is present in green leafy vegetables, fruits,
decreased production of S-adenosyl methionine (SAM), a key dairy products, cereals, and also in animal foods such as liver
intermediate in methylation reactions of myelin. The impair- and kidney. The average daily diet contains about 400 to
ment of methylation in myelin could result in demyelination 600 ug of folate; however, folate is a heat-labile vitamin and,
and, consequently, in clinical neuropathy.°** ’ therefore, is easily destroyed in overcooked vegetables.' The
recommended dietary intake of folic acid for adults is approx-
imately 5O to 100 ug/day.'° This requirement increases signif-
OTHER CAUSES OF VITAMIN B,, DEFICIENCY
icantly during infancy, pregnancy, and lactation. Folate
GASTRECTOMY Many other causes of malabsorption can
deficiency during early pregnancy (first trimester) can cause
lead to vitamin B,, deficiency (see Table 7—2). In a gastrectomy
renal tube defects in the fetus and is associated with paralysis
procedure, the IF-producing cells are removed. In the absence
and brain damage.'?*>! The body storage is about 5 to 10 mg,°
of vitamin B,, therapy, vitamin B,, deficiency develops in these
of which almost 80% is stored in the liver. Folic acid is
patients within several years. Vitamin B,, deficiency has been
absorbed through the duodenum and jejunum, and the amount
reported in 30% to 40% of patients with partial gastrectomy.°
absorbed is about 80% of intake. Folate is lost via body secre-
tions such as bile, urine, and sweat. Folic acid has a higher
BLIND LOOP SYNDROME In blind loop syndrome, an
turnover time and a higher rate of loss compared to vitamin
anatomic abnormality of the small intestine, there is an over-
B,, and, therefore, it takes only a few months to develop di-
growth of bacteria in the small bowel. These microorganisms
etary folate deficiency.
take up the vitamin B,, and make it unavailable for absorption
by the ileum. Tetracycline therapy for 10 days normalizes the
vitamin B,, level.*'? STRUCTURE
Folic acid consists of three components: pteridine, para-
FISH TAPEWORM Fish tapeworm (Diphyllobothrium latum) aminobenzoic acid, and glutamic acid (Fig. 7-12). Folic acid
is a parasite that competes for vitamin B,, by splitting B,, derived from the diet is not biologically active. Once absorbed
from IF. This type of vitamin B,, deficiency is common in through the intestinal lumen, it is hydrolyzed, reduced, and
Scandinavian countries and is reported in 1.9% to 3.0% of methylated to form methyl tetrahydrofolate (CH;THF). Other
carriers of the fish tapeworm.®!? The malabsorption type of biologically active forms of folic acid are tetrahydrofolate
vitamin B,, deficiency is normally corrected when vitamin (THF) and its coenzyme, N°,N'°-methylene tetrahydrofolate
B,, or B,, and IF are given to the patients. (N°N?°CH, THF).
146 Chapter 7 Megaloblastic Anemias

Pteroyl residue

OH 5 6

N H.| H
NEO Shs
4

eae
Hon’ SS} : las ae 75
CH2

COOH
Pteridine residue p-Aminobenzoic acid L-Glutamic
residue acid residue
A. Folic Acid

Figure 7-12 ™ Structure of folic acid and its derivative. A. Folic acid (pteroylglutamic acid). The three components are defined by vertical
lines. B. Tetrapteroyltriglutamic acid (tetrahydrofolate triglutamate), the active form of folate present in the tissues.

Serum folate is in the form of CH,THF and enters all CAUSES OF FOLIC ACID DEFICIENCY
tissue cells in this form. DIETARY DEFICIENCY The main cause of folic acid defi-
ciency is decreased dietary intake. Other causes are malab-
ABSORPTION AND METABOLISM
sorption, increased requirement, and drug-induced folate
Dietary folic acid is in the form of polyglutamic acid (many deficiencies (Table 7-4). Nutritional folate deficiency is usu-
glutamic acid residues). Once in the intestinal lumen, it is
ally a consequence of poverty, old age, alcoholism, preg-
acted on by the enzyme folate deconjugase, which is present nancy, and chronic diseases. In the United States, folic acid
in the epithelial cells of intestine, to form monoglutamic acid has been added to cereal grains, rice, and milled flour to
(single glutamic acid residue) (Figs. 7-13 and 7-14). Monog- increase the average adult’s intake by 100 ug.?! The U.S. gov-
lutamic acid is then reduced and methylated to CH;THEF." ernment urges women planning to become pregnant and in
When CH,THE is absorbed from the circulation into the tis- early pregnancy to eat a diet rich in folic acid or take vitamin
sue cells, it transfers its methyl group to homocysteine to supplements to prevent the adverse effects of folic acid defi-
form methionine and THF. The THF formed reconjugates ciency on their fetus.
with additional glutamic acid residues to form the cellular
THF (see Figs. 7-12 and 7-13). The THF is then methylated MALABSORPTION The most common causes of folate
to form coenzyme methylene THF (N°N'°CH,THEF) necessary malabsorption are tropical sprue and_ gluten-sensitive
for the formation of thymidine monophosphate from uridine enteropathy. Tropical sprue is an infection that causes
monophosphate. This is the key reaction for DNA synthesis intestinal atrophy with clinical manifestations of weakness,
(see Fig. 7-10). weight loss, and steatorrhea. Tropical sprue affects the
 Chapter 7 Megaloblastic Anemias 147

Polyglutamic acid ———» Polyglutamate Le——> CH; THE ————> CH; THF

CH3
TH

oye|O4
Glu
aseBniuooep
Folate
reductase Homocysteine THF Methionine
Monoglutamate
Glu

THF
Figure 7-13 @ Absorption and metabolism of folic acid. CH;THF = methyl tetrahydrofolate; Glu = glutamic acid; THF = tetrahydrofolate.

entire intestine and, therefore, Causes a wide variety of CLINICAL MANIFESTATIONS OF FOLIC ACID
nutritional deficiencies, including vitamin B,, deficiency.° DEFICIENCY
Affected individuals respond well to antibiotics. Clinical manifestations of folate deficiency are the same as
Gluten-sensitive enteropathy has the same clinical those for vitamin B,, deficiency, mentioned earlier. The
manifestations as those mentioned for tropical sprue. It onset of anemia is insidious, with the distinct morphology
includes both nontropical sprue and childhood celiac dis- characteristic of megaloblastic anemia in the bone marrow
ease. Affected individuals cannot digest gluten, a protein and in the peripheral blood. Although neuropathy is mainly
found in wheat and other grains. Lesions are most severe in characteristic of vitamin B,, deficiency, several cases of
the proximal intestiné. Childhood celiac disease is a malab- neurologic abnormalities, such as depression, dementia,
sorption syndrome resulting in anemia caused by iron defi- and peripheral neuropathy associated with folic acid defi-
ciency and, to a lesser degree, by vitamin B,, and folate ciency, have been reported.*° Some of these neuropathies,
deficiencies. in particular depression, have responded favorably to treat-
The requirement for folic acid also increases during ment with folate.*°***4
rapid cellular proliferation in hematologic diseases such as Folic acid and vitamin B,, are both necessary cofactors of
sickle cell anemia, thalassemia, hereditary spherocytosis, and the enzyme methionine synthase, which converts homocysteine
autoimmune hemolytic anemia. to methionine. The deficiency of these vitamins causes an

DRUG-INDUCED FOLATE DEFICIENCY Drug-induced

folate-deficient megaloblastic anemia has been reported with
a variety of drugs such as methotrexate, pyrimethamine,
phenytoin, alcohol, isoniazid, and oral contraceptives®'* (see
Table 7-4). Dietary Deficiency
Poverty
Old age
Alcoholism
Chronic diseases
Intestinal lumen Malabsorption
Tropical sprue
Gluten-sensitive enteropathy
Childhood celiac disease
Polyglutamic an 1S
© -ciu>
}
folic acid un Increased Requirement
Ke Monoglutamic Pregnancy
folic acid Infancy
Malignancy
Drugs
La Methotrexate
-Monoglutamic — GLu Pyrimethamine
- folic acid GLU GLU Phenytoin
ma GLU GLU GLU Alcohol
Isoniazid
Figure 7-14 m Intestinal absorption of the folate derivatives of Oral contraceptives
food. (From Streiff, RR: Intestinal absorption of the folate derivatives Others
of food. JAMA 214:105, 1970, with permission.)
148 Chapter 7 Megaloblastic Anemias

increased level of plasma homocysteine. Hyperhomocysteine- It is worth noting that to date, there is no standard algo-
mia is a risk factor for thrombosis.*!*° An association between rithm for investigation of vitamin B,, deficiency. Different
folate deficiency and development of leukemia in high-risk laboratories choose different algorithms based on an individ-
patients has been reported.*° ual patient, availability of the procedure, and the cost.

SERUM B,, (SERUM COBALAMIN) LEVEL

Laboratory Diagnosis of Megaloblastic The normal range for serum vitamin B,, is commonly stated as
Anemia 200 to 500 ng/L. In vitamin B,, deficiency the level is usually
less than 200 ng/L 95% to 97% of the time. The more severe the
Several important factors in differential diagnosis of mega-
vitamin B,, deficiency, the lower serum B,, level becomes.”
loblastic anemias are the patient’s physical examination, med-
Low levels of serum B,, in the absence of vitamin B,, deficiency
ical history, drug history, family history, and laboratory tests.
have been reported in patients who are pregnant or using oral
The most common laboratory screening tests and results
contraceptives, or who have folate deficiency or decreased TC I
that are used in the diagnosis of megaloblastic anemias are
level. Serum B,, level may be normal in B,, deficiency resulting
low hemoglobin level, elevated MCV, and peripheral smear
from N,O, TC II deficiency, high levels of TC I, and in patients
morphology, such as macro-ovalocytes and hypersegmented
with both folate and B,, deficiencies.'*’ Elevated serum B,, is
neutrophils. Once the diagnosis of megaloblastic anemia is
associated with myeloproliferative disorders, chronic myelo-
established, the exact cause of the anemia should be deter-
cytic leukemia, and liver disease.”'!*'* Because serum B,, level
mined for appropriate and effective treatment.
may be falsely low or normal, metabolic tests such as methyl-
malonic acid (MMA) and total homocysteine assays can be per-
Laboratory Tests for the Diagnosis formed. The levels of these metabolites are increased before
of Vitamin B,, and Folic Acid Deficiencies serum B,, level is decreased.7597

The most specific diagnostic tests used for vitamin B,, and SERUM AND RED CELL FOLATE
folate deficiencies are serum B,, and serum and red cell folate Serum and red cell folate are decreased in patients with
levels. Red cell folate is a better indicator of tissue folate levels folate deficiency. The normal range for serum folate levels
(Table 7-5). Serum cobalamin is usually measured via radioiso- is 5 to 16 ng/mL. The minimal folate level for a normal
tope dilution, or enzyme-linked immunosorbent assays which DNA synthesis is about 4 ng/mL.*’ Serum folate is sensitive
are normally automated. Serum folate may be measured by to dietary folate intake. False low serum folate levels have
microbiological assays, or by radioassay, or via an enzyme- been reported in patients taking antibiotics or certain cyto-
linked immunosorbence assay.” toxic drugs.**’° Red cell folate is a valuable test reflecting
the body folate stored. It is less affected by recent diet than
is the serum assay. In normal adults, the red cell folate con-
yEvaluation = centration ranges from 160 to 640 ug/L of packed red cells.”
In patients with a severe B,, deficiency, the serum folate
level is increased while the red cell folate level is decreased.
In this case, the increased serum folate level is caused
Screening Tests by increased CH;THF in the serum (the methyl trap
Complete blood count hypothesis), resulting from the lack of vitamin B,, necessary
WBC differential/RBC morphology
for conversion of homocysteine to methionine and methyl-
Reticulocyte index
THF to THF (see Fig. 7-10).
Serum lactate dehydrogenase (LD)
Iron studies
Bone marrow aspirate
Other Laboratory Tests
Vitamin B,, Deficiency
Serum B,, level Other laboratory tests that may support a diagnosis of vitamin
Gastric autoantibodies B,, are antibodies to IF, blood test for gastric atrophy, serum
Blood test for gastric atrophy vitamin B,, binding proteins, the Schilling test, methylmalonic
Serum cobalamin binding proteins
Serum and urine methylmalonic acid
acid (MMA) and total homocysteine assays, and the deoxyuri-
Total homocysteine dine (dU) suppression test. These laboratory tests are useful
when the other laboratory tests are inconclusive. Macrocytic
Folic Acid Deficiency anemia due to vitamin B,, and or folate deficiency should
Serum and red cell folate levels
Serum homocysteine be distinguished from macrocytosis present in patients with
myelodysplastic syndrome, hemolytic anemia, and those
Other Assays exposed to chemotherapeutic drugs (see Table 7-5).
Schilling test
dU suppression test
GASTRIC AUTOANTIBODIES
dU = deoxyuridine. Antibodies to IF are present in the serum of about 50% to
70% of patients with pernicious anemia.° Although the IF
 Chapter 7 Megaloblastic Anemias 149

antibody test is not a sensitive test, its specificity for the diag-
nosis of pernicious anemia is 95%.° Decreased serum B,, and
the presence of the antibody to IF are indicative of pernicious
anemia. Parietal cell antibody is present in 80% to 90% of
patients with pernicious anemia, however, its diagnostic value
for pernicious anemia is limited because this antibody is spe-
cific for immune gastritis.°

BLOOD TEST FOR GASTRIC ATROPHY

Gastric achlorhydria (low gastric acidity) is present in almost all
patients with pernicious anemia. Achlorhydria after histamine Normal Abnormal
stimulation supports the diagnosis of pernicious anemia. How- 1
Dietary
ever, this test is not specific, because achlorhydria has also been deficiency
reported in patients without pernicious anemia. Serum gastrin
levels are increased in 80% to 90% of patients with pernicious
anemia, reflecting gastric achlorhydria. However, elevated gas-
trin levels have many other causes such as atrophic gastritis
without pernicious anemia and food-cobalamin malabsorption,
which makes this test unreliable for pernicious anemia.° The Circulation
best test for pernicious anemia is to measure IF content in stim-
ulated gastric juice, but this test is not widely available.°

SERUM COBALAMIN BINDING PROTEINS

Transcobalamin II is the major protein carrier of vitamin B,.
Cobalamin—TC II complex (holoTC II) level in plasma is a Corrected Not corrected
sensitive measure of early vitamin B,, deficiency. The normal
level of holoTC II 1s greater than 50 pg/mL. When vitamin Pernicious Malabsorption
B,, intake is inadequate and B,, storage is depleted, the anemia
holoTCH level falls below 40 pg/mL.*’ However, the correla-
Figure 7-15 |The two-part Schilling test. IF = intrinsic factor.
tion between holoTCII level and depletion of vitamin B,, stor-
age is not yet fully proven.*’
the test. Some investigators think that the measurement of
SCHILLING TEST serum cobalamin binding protein (holoTC II) may become a
The Schilling test evaluates the pathophysiology of vitamin B,, good replacement for the Schilling test.*’
malabsorption. The test is done in two parts (Fig. 7-15).
In part I, the patient is given 0.5 to 2.0 pg of labeled SERUM AND URINE METHYLMALONIC ACID
(°’Co or *Co) vitamin B,, orally. Two hours later, a flushing Serum and urine MMA levels are both elevated in patients
dose (1000 ug) of unlabeled vitamin B,, is injected intramus- with vitamin B,, deficiency but not folate deficiency. MMA
cularly to saturate all of the circulating cobalamin binders. results from accumulation of methylmalonyl Co-A. Vitamin
The amount of the labeled vitamin B,, is then measured in a B,, in the form of adenosylcobalamin is necessary for conver-
24-hour urine collection. If IF is present and normal absorp- sion of methylmalonyl CoA to succinyl CoA (see Fig. 7-11).
tion takes place, the labeled vitamin B,, absorbed through the In the absence of vitamin B,,, the level of methylmalony]
intestine is rapidly excreted into the urine. The urinary excre- CoA is increased in the serum and consequently in the urine.
tion varies, depending on the dosage given. In normal absorp- Falsely elevated results have been reported in non—vitamin-
tion, about 5% to 35% of labeled B,, is excreted in the urine.’ deficient patients with renal disease, with inborn errors of
An abnormal result in part I indicates that B,, was not ab- metabolism, and with bacterial infection.®
sorbed through the intestine. In this case, testing proceeds to Measurement of urinary MMA is a reliable indicator of
part II to find the cause of malabsorption (see Fig. 7-15). tissue vitamin B,, deficiency in the absence of decreased
In part II, the test is repeated with the addition of IF to serum B,, and lack of clinical manifestations of megaloblas-
the oral dose to determine if malabsorption is caused by the tic anemia.'*>>
lack of IF. If the Schilling test is corrected in part II, a defi- Normal individuals excrete 0 to 3.4 mg MMA per day. In
ciency of IF is confirmed. If the Schilling test is still abnor- patients with B,, deficiency MMA excretion is highly ele-
mal, other causes of malabsorption should be investigated vated.'° Serum MMA is increased in 85% to 97% of patients
(see Fig. 7-15). Parts I and II can be done simultaneously if with vitamin B,, deficiency.'*°*’ The normal range for serum
B,, and B,, + IF are labeled with different isotopes. Reliabil- MMA is 73 to 271 ng/mL. This level increases to 2 to 100 times
ity of the Schilling test depends on normal renal function and the upper limit of normal in patients with vitamin B,, defi-
proper urine collection. The Schilling test is difficult to ciency. However, serum MMA assay is complex and expensive
perform; therefore, many hospital laboratories no longer offer and is not available in most routine clinical laboratories.*’
150. Chapter 7 Megaloblastic Anemias

SERUM HOMOCYSTEINE parenteral treatment. Several studies have shown that oral
Serum homocysteine levels are elevated in patients with both vitamin therapy can be as effective and efficient as par-
vitamin B,, and folate deficiencies, because both vitamins are enteral therapy in conducting hematologic and neurologic
necessary in the conversion of homocysteine to methionine. remissions and in maintaining normal serum B,,.°*'*° In
Serum homocysteine is also increased in a short-term folate- patients with pernicious anemia, about 1% of the oral dose
restricted diet and in patients with congenital homocystinuria is absorbed.'“° Therefore, high doses of oral vitamin (1000
and vitamin B, inhibitor.2°? to 2000 ug/day) are sufficient to replace the parenteral ther-
apy. The efficiency of the treatment should be checked by
DEOXYURIDINE SUPPRESSION TEST occasional measurement of serum B,, in patients undergoing
The dU suppression test measures the level of 5,10-CH, THF. the oral therapy.'*°
It is an indirect measurement of thymidylate synthesis in vitro Vitamin B,, is injected intramuscularly or subcuta-
and is abnormal in both vitamin B,, and folate deficiencies neously. Although the treatment protocol varies, the initial
(see Fig. 7-10). The principle of this test is based on the fact dose administered is higher in order to saturate the body stor-
that B,,- or folate-deficient cells cannot convert deoxyuridine age. Vitamin B,, may be given as 100 to 1000 ug/day for
to deoxythymidine, and therefore the radioactive-labeled 2 weeks, then weekly until hematologic values are normal-
thymidine will be incorporated into DNA. In patients with nor- ized and then monthly for life.' Vitamin B,, therapy can be
mal levels of B,, or folate, deoxyuridine is converted to thymi- monitored by reticulocyte counts.
dine, which suppresses the labeled thymidine incorporation
into the DNA.?* Specificity of the deficient vitamin can be
Folic Acid Deficiency
determined by addition of vitamin B,, or folate to the test sys-
tem and a correction of the original abnormal result. This test The recommended therapeutic dose to treat folate deficiency is
is relatively time consuming and therefore, despite its sensitiv- | to S mg/day for 2 to 3 weeks.'° Folic acid vitamin is water sol-
ity, 1s not used as a diagnostic test. uble and is given orally. Lifelong therapy is not required
The diagnosis of vitamin B,, deficiency may be com- because it is usually possible to treat folate deficiency within a
plex and inconclusive in the absence of hematologic and short period of time.'’° Folic acid may be given along with
neurologic abnormalities.** Many studies have shown that vitamin B,, when both vitamins are deficient or in a therapeutic
some patients with vitamin B,, deficiency may seek med- trial when the exact cause of megaloblastic anemia is not
ical help because of complications other than the classical known. In this case, folate therapy will correct the hematologic
features of megaloblastic anemia. Examples of these com- abnormalities in vitamin B,, patients, but the neurologic mani-
plications are retinopathy, neuropathy, vascular disorders, festations will progress.
infertility, and physical and mental growth retardations in Folic acid given as prophylaxis (0.25 to 0.5 mg/day) is
infancy.'>*°?'404445 However, these conditions have been recommended during pregnancy and dialysis and may be
reversed on early treatment with vitamin B,,. However, required in patients with hemolytic anemia and in patients
vitamin B,, deficiency should be included in the differential who are on antifolate drugs.'!?*'*? Folic acid can be injected
diagnosis in patients with neurologic and neuropsychiatric to hospitalized patients and in those who cannot take the med-
problems.** To prevent vitamin B,,-associated complica- ication by mouth.
tions, numerous attempts have been made at early diagno-
sis of vitamin B,, and folate deficiencies.
Response to Therapy
The initial sign of a positive response to therapy is an increase
Treatment
in the reticulocyte count. The number of circulatory reticulo-
Transfusion is rarely required in patients with megaloblastic cytes increases 3 to 5 days after therapy, with a peak at about
anemia unless the anemia is severe (hematocrit of less than 4 to 10 days.'* The reticulocyte count may increase to 50% to
15%) or is associated with congestive heart failure. In these 70% initially.’ The megaloblastic morphology of the bone mar-
cases packed red cells should be administered slowly to pre- row disappears within 24 to 48 hours after therapy. The hemat-
vent pulmonary edema! ocrit rises in about 5 to 7 days after therapy, reaching normal
levels in 4 to 8 weeks. Giant metamyelocytes and hyperseg-
mented neutrophils disappear within 2 weeks. The entire thera-
Vitamin B,, Deficiency peutic response process may take only 3 to 6 weeks, depending
on the severity of the disease.*!!
Most people with a vitamin B,, deficiency require lifelong
vitamin therapy. Cyanocobalamin and hydroxocobalamin are
the two therapeutic forms of vitamin B,,. Hydroxocobalamin Vitamin-Independent Megaloblastic
is preferred by some physicians because it has a longer half-
Changes
life. Cyanocobalamin is less expensive and converts to the
physiologic form. This group of disorders is characterized by the presence
Vitamin B,, can be administered orally to patients with of megaloblastic changes in the bone marrow and in the
dietary vitamin deficiency or to those who cannot tolerate peripheral blood that are refractory to vitamin B,, and
 Chapter 7 Megaloblastic Anemias 151]

folic acid treatments. The megaloblastic changes in Acquired

this group of patients may occur because of an inherited or
acquired predisposition, or they may be drug induced Megaloblastic changes may be secondary to hematologic disor-
(Table 7-6). ders such as myelodysplastic syndromes and erythroleukemia.
Megaloblastic anemia may also be associated with a variety of
Inherited drugs and toxic material.”

Orotic aciduria is a rare inherited disorder of pyrimidine me-

tabolism characterized by increased excretion of orotic acid Drug and Toxin Induced
in the urine. Lesch-Nyhan syndrome is an X-linked disorder A wide variety of drugs with differing modes of action are as-
of purine metabolism. Megaloblastic changes are also noted sociated with megaloblastic anemia. Examples of these drugs
in inherited dihydrofolate reductase deficiency, a disorder of are methotrexate, hydroxyurea, and aminopterin. They are
folate metabolism and methyl tetrahydrofolate transferase. inhibitors of dihydrofolate reductase, preventing dihydrofolate
Inherited disorders of transcobalamin II deficiency or abnor- to tetrahydrofolate reaction. This inhibition results in decreased
mal transcobalamin II molecule and congenital IF deficiency thymidine biosynthesis and DNA maturation. Drugs such as
are associated with megaloblastic anemia. In congenital 6-mercaptopurine, cytosine arabinose, and hydroxyurea inter-
dyserythropoiesis, the megaloblastic changes are limited fere with purine or pyrimidine metabolism. Nitrous oxide
only to erythrocyte precursors of.the bone marrow and red (N,O), an anesthetic gas, inactivates vitamin B,,.'°°?>*8
cells in the peripheral smear. White blood cells and platelets are Azidothymidine (AZT) is associated with megaloblastic
normal. Hereditary juvenile megaloblastic anemia is caused by changes. Exposure to toxic materials such as arsenic and chlor-
intestinal malabsorption of vitamin B,,. Grasbeck syndrome dane can also cause megaloblastic anemia.!
(IGS) is associated with total absence of vitamin B,, absorption
which is not corrected by the administration of IF.*’ There are
other inherited disorders that are associated with methyl- Macrocytic Nonmegaloblastic Anemias
malonic aciduria, homocystinuria, or both. In these disorders,
pancytopenia is present with or without megaloblastic Macrocytic anemias may be megaloblastic or nonmega-
pictures.° loblastic. Differentiation between the two is important. In
macrocytic, normoblastic anemias, the MCV is more than
100 fL, but not as high as in megaloblastic anemias (where
the MCV is more than 110 fL). The red cells on the periph-
eral blood smear appear large and round, but not oval. The
neutrophils are not hypersegmented. The bone marrow is
Oe isdene coe normocellular or hypercellular with erythroid hyperplasia.
The red cell precursors in the marrow are normoblastic
Inherited and not megaloblastic. The mechanism responsible for the
Orotic aciduria
Lesch—Nyhan syndrome
macrocytic morphology may be associated with an increase
Dihydrofolate reductase deficiency in both red cell membrane cholesterol and phospholipid.
Methy] tetrahydrofolate transferase deficiency Increased lipid deposition onto the red cell membrane and
Transcobalamin deficiency altered maturation time of the red cell precursors are among
Abnormal cobalamin molecule the possible causes.'°°°
Congenital IF deficiency
Methylmalonic aciduria
Homocystinuria Causes of Macrocytic Nonmegaloblastic
Congenital dyserythropoiesis
Grasbeck syndrome Anemias
Acquired The most common causes of macrocytic anemia are chronic
Myelodysplastic syndrome liver disease and alcoholism. In 40% to 96% of alcoholics,
Erythroleukemia macrocytosis is present in the absence of anemia, and in this
Drugs case, alcohol has a direct toxic effect on the red cells rather
Folate antagonist (methotrexate) than causing folate deficiency. The finding of macrocytosis is
Purine or pyrimidine antagonists (6-mercaptopurine, a valuable screening test for early detection of alcoholism.®!*
cytosine arabinoside, and hydroxyurea) Megaloblastic changes have been reported in 20% to 40% of
Nitrous oxide
alcoholics.*'® Liver function tests such as serum LD and
Azidothymidine (AZT)
Others bilirubin are helpful in the diagnosis. Although 50% of
patients with liver disease have macrocytic anemia, normo-
Toxic Materials cytic and microcytic anemias have also been reported.'°
Arsenic
Macrocytic anemia with an elevated reticulocyte count is
Chlordane
Others
associated with hemolytic anemia or acute blood loss. Macro-
cytic anemia may also be associated with aplastic anemia,
152. Chapter 7 Megaloblastic Anemias

Table 7-7 Causes of Macrocytic QUESTIONS

Nonmegaloblastic 1. These laboratory findings are representative of what type
Anemias of anemia?
i) . How would you classify this anemia based on the RBC
Chronic liver disease
indices?
Alcoholism
3. What does the decreased reticulocyte count represent?
Acute hemorrhage
Hypothyroidism
4. What is the cause of this anemia?
Hematologic disorders 5. What test is used to evaluate vitamin B,, malabsorption?
Hemolytic anemia
Aplastic anemia COMMENTS
Chronic myeloproliferative disorders
Immunosuppressive drugs The diagnosis of megaloblastic anemia was made based on the
Arsenic and chlordane intoxication patient’s physical examination and the highlights of the labo-
ratory results. Physical findings reveal that the patient had the
signs and symptoms of anemia, with the “lemon-yellow skin”
caused by anemia and jaundice. The low hemoglobin and
chronic myeloproliferative disorders, and sideroblastic ane- hematocrit levels in combination with an elevated MCV and
mia. In sideroblastic anemia, a dimorphic red cell population normal MCHC are suggestive of macrocytic, normochromic
may be observed. Bone marrow examination is often required anemia, a characteristic of megaloblastic anemia. The macro-
for differential diagnosis (see Table 7-5). ovalocytes and hypersegmented neutrophils present on the
Macrocytosis has also been reported in patients taking peripheral smear are striking features of megaloblastic anemia.
immunosuppressive drugs as well as arsenic and chlordane Howell—Jolly bodies are the red cell inclusions commonly
intoxificauon. Causes of macrocytic nonmegaloblastic ane- seen in this type of anemia. The low reticulocyte value is
mia are summarized in Table 7—7. indicative of ineffective erythropoiesis. The white blood cell
(WBC) and platelet counts are normal in this patient, as is
expected in early stages of megaloblastic anemia.
The elevated bilirubin, serum LDH, and serum iron levels
can all be attributed to red cell hemolysis. In megaloblastic
Case Study anemia, the red cells are fragile and have a shortened life
span, and are thus prematurely destroyed in the bone mar-
A 50-year-old woman visited her physician because she had row and in the peripheral blood.
experienced weakness, fatigue, and shortness of breath for The most common causes of megaloblastic anemia are
the past few months. Physical examination revealed a tall, vitamin B,, and folate deficiencies. In this patient, the serum
slender woman with “lemon-yellow skin” and a smooth, red B,, level was decreased, whereas the red cell folate level
tongue. No hepatosplenomegaly was noted. She had experi- was normal. These findings indicate that B,, deficiency was
enced a loss of vibratory sense and had some problems with the probable cause of the megaloblastic anemia. The patient
gait coordination. Her family history and her past medical was eating a balanced diet and had no past medical history.
history were unremarkable. The patient was not taking any Vitamin B,, deficiency because of malabsorption was sus-
medications. pected, and a two-part Schilling test was ordered to evaluate
A complete blood count with WBC differential was ordered the cause of B,, malabsorption. In part I, the patient excreted
and the results were as follows: WBC = 7.4 X 10°/L (N: 4.4 to 2% labeled B,, in her 24-hour urine, and in part II, she
11); RBC = 2.2 X 10°/L(N: 4.1 to 5.1); Hgb = 8.5 g/dL (N: excreted 8%. This result supported the diagnosis of perni-
12.3 to 15.3); Hct = 25% (N: 36 to 45); MCV = 114 fL cious anemia.
(N: 87 to 98); MCHC = 34% (N: 32 to 36); RDW = 15.2% The initial doses of hydroxocobalamin were administered
(N: 11.5 to 14.5); Platelet = 170 x 10°/L (N: 150 to 400). intramuscularly for several weeks, followed by maintenance
Differential: 67% segmented neutrophils (5% hyperseg- doses. After 2 months of therapy, the patient had completely
mented); 25% lymphocytes; 5% monocytes; 3% eosinophils. recovered. Her hemoglobin and hematocrit had returned to
RBC morphology: macrocytic with few macro-ovalocytes normal and her abnormal blood cell morphologies had dis-
and few schistocytes. RBC inclusions: few Howell—Jolly bod- appeared. The patient was advised to continue the vitamin
ies. Reticulocytes = 0.2% (N: 0.5 to 1.5). B,, therapy to prevent relapse of the symptoms, because
Chemistry test results were as follows: total bilirubin = lack of IF in patients with pernicious anemia prevents
4.0 mg/dL (N: 0.2 to 1.0); indirect bilirubin = 2.9 mg/dL (N: absorption of dietary B,,.
0.2 to 0.8); serum LD = 720 U/L (N: 100 to 190); serum
iron = 220 ug/dL (N: 50 to 170); TIBC = 215 ug/dL (N: 250
to 450); serum B,, = 50 pg/mL (N: 100 to 700); red cell
folate = 200 ng/mL (N: 130 to 628).
continued

N = normal range.
 Chapter 7 Megaloblastic Anemias 153

th ophysiology of megaloblastic anemia is: 7. The glycoprotein necessary for absorption of vitamin B,, is:
a. Defective RNA synthesis and abnormal cytoplasm a. Albumin
maturation b. Transcobalamin II
b. Defective DNA synthesis and abnormal nuclear matu- c. Haptoglobin
ration d. Intrinsic factor
c. Defective RNA synthesis and abnormal nuclear matu-
. Which of the following are clinical manifestations of
ration
both B,, deficiency and folate deficiency?
d. Defective DNA synthesis and abnormal cytoplasm
a. Anemia and jaundice
maturation
b. Thrombocytosis
. Which of the following laboratory findings coincide with c. Hemoglobinemia
megaloblastic anemia? d. Hemoglobinuria
a. Increased serum iron and serum bilirubin
. Which of the following Schilling test results corresponds
b. Decreased serum iron and serum bilirubin
to a diagnosis of pernicious anemia?
c. Decreased serum muramidase
a. Part I abnormal, part II not corrected
d. Increased haptoglobin b. Part I abnormal, part II corrected
. Megaloblastic anemia is associated with: c. Part [ and part IT abnormal
a. Ineffective erythropoiesis and increased reticulocytes d. Part I normal, part IH corrected
b. Ineffective erythropoiesis and decreased reticulocytes 10. Which of the following is not a cause of vitamin B,,
c. Ineffective erythropoiesis and decreased LDH deficiency?
d. Ineffective erythropoiesis and decreased erythropoietin a. Atrophic gastritis
. According to the morphological classification of ane- b. Total gastrectomy
mias, megaloblastic anemia is a: c. Blind loop syndrome
a. Macrocytic, hypochromic anemia d. Chronic glossitis
b. Macrocytic, hyperchromic anemia . Macrocytosis associated with acute blood loss is charac-
c. Macrocytic, normochromic anemia terized by:
d. Normocytic, normochromic anemia a. Decreased reticulocyte count
. Which of the following are not seen on the peripheral b. Increased reticulocyte count
smear of a patient with megaloblastic anemia? c. Pancytopenia
a. Macro-ovalocytes d. Macro-ovalocytes
b. Hypersegmented neutrophils . Which of the following is associated with pernicious
c. Hyposegmented neutrophils anemia and not macrocytic anemia due to liver disease?
d. Howell—Jolly bodies a. Increased LD
. Which of the following are the characteristic findings b. Increased bilirubin
of the bone marrow in a patient with megaloblastic c. Increased MCV
anemia? d. Hypersegmented neutrophils
a. Hypocellular with low M:E ratio
b. Hypercellular with high M:E ratio See answers at the back of this book.
c. Hypocellular with high M:E ratio
d. Hypercellular with low M:E ratio
154. Chapter 7 Megaloblastic Anemias

m Other causes of vitamin B,, deficiency are dietary malab-

“-') SUMMARYC sorption secondary to diseases and drugs.
m The main cause of folic acid deficiency is a poor diet.
= Megaloblastic anemia is a macrocytic anemia character-
ized by defective nuclear maturation caused by impair- w Other causes of folic acid deficiency are malabsorption,
ment of DNA synthesis. increased requirement, and drugs.
= Megaloblastic anemia is associated with ineffective ery- = Clinical manifestations associated with folic acid defi-
thropoiesis, ineffective granulopoiesis, and ineffective ciency are similar to those in vitamin B,, deficiency, with
thrombopoiesis. neuropathies not being the prominent features.
mw The bone marrow of patients with megaloblastic anemia w Laboratory tests used for the differential diagnosis of
is hypercellular with a low MEE ratio (1:1 to 1:3), high vitamin B,, and folate deficiencies are serum B,,, and
number of megaloblasts, and giant bands and metamyelo- serum and red cell folate.
cytes. mwOther laboratory tests that may be useful are gastric
m The peripheral blood is characterized by pancytopenia, achlorhydria, antibodies to intrinsic factor, Schilling test,
macrocytes, macro-ovalocytes, and hypersegmented neu- serum and urine methylmalonic acid serum and urine
trophils. homocysteine, and deoxyuridine suppression test.
mwOther biochemical changes are increased levels of w Vitamin B,, deficiency can be treated with cyanocobal-
LDH, indirect bilirubin, serum iron and ferritin, and amin or hydroxocobalamin, and folate deficiency can be
erythropoietin. treated with folic acid supplementation.
m The major causes of megaloblastic anemias are vitamin mw The initial response to therapy is increased reticulocyte
B,, deficiency, folic acid deficiency, or both. counts.

mw Vitamin B,, and folic acid in the form of cofactors are w Vitamin-independent megaloblastic anemias can be inher-
essential for two key reactions in the body. ited or acquired.
@ Intrinsic factor enhances vitamin B,, absorption through gs Macrocytic nonmegaloblastic anemias are characterized
the receptors present on the brush borders of the ileum. by a high MCV, macrocytes in the peripheral blood, and
w Transcobalamin II transfers vitamin B,, to the tissues. normocellular or hypercellular bone marrow with ery-
throid hyperplasia.
w The main cause of vitamin B,, deficiency is pernicious
anemia. a The most common causes of macrocytic anemia are liver
disease and alcoholism.
gw Pernicious anemia is the lack of intrinsic factor.
Clinical manifestations that are often associated with
vitamin B,, deficiency are anemia, fever, glossitis, and
neurologic symptoms.
 Chapter 7 Megaloblastic Anemias = 155

REFERENCES the autosomal recessive megaloblastic ieS)Cn. Ballas, SK, and Saidi, P: Thrombosis,
anemia (MGA1) region. Blood 91:3593 megaloblastic anemia, and sickle cell
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1998, disease: A unified hypothesis.

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2006, pp 477-499.
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into DNA. Blood 83:1656, 1994.
Vitam Nutr Res 63:283, 1993. Blood 90:4054, 1997.
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Neuroradiology 40:398, 1998. Management, ed 4. McGraw-Hill, New
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. Stabler, SR: Vitamin B,, deficiency in older York, 2005, pp 95-108.
Koury, MJ, et al: Apoptosis of late-
people: Improving diagnosis and prevent- 38. Daniel, P, et al: Steroid-responsive func-
erythroblasts in megaloblastic anemia.
ing disability. JAGS 46:1317, 1998. tional B,, deficiency in association with
Association with DNA damage and
. Hoggarth, K: Macrocytic anemia. Practi- transcobalamin II polymorphism
macrocyte production. Blood 89:4617,
tioner 237:331, 1993. 776C — G. Eur J Haematol 76(1):75,
1997.
. Hemmer, B, et al: Subacute combined 2005.
Nn . Ingram, CF, et al: Evaluation of DNA
degeneration: Clinical, electrophysiolog- . Joosten, E, et al: Is metabolic evidence
analysis for evidence of apoptosis in
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megaloblastic anemia. Br JHaematol
findings. J Neurol Neurosurg Psychiatry more frequent in elderly patients with
96:576, 1997.
65:822, 1998. Alzheimer’s disease? J Gerontol Med
Lee, GR, et al: Wintrobe’s Clinical
24. Metz, J: Pathogenesis of cobalamin neu- Sci 52A:M76, 1997.
Hematology, ed 11, vol 1. Lea & Febiger,
ropathy: Deficiency of nervous system AO. Ubbnik, JB, et al: Vitamin B,,, vitamin Be,
Philadelphia, 2004, pp 1367-1388.
S-adenosylmethionine. Nutr Rev 51:12, and folate nutritional status in men with
~ . Nathan, DG, and Oski, AF: Hematology
1993. hyperhomocysteinemia. Am J Clin Nutr
of Infancy and Childhood, ed 6, vo! 1.
. Hoffbrand, AV, et al: Post graduate 57:47, 1993.
WB Saunders, Philadelphia, 2003, 4 i . Savage, DG, et al: Sensitivity of serum
Hematology, ed 5. Blackwell, Oxford,
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2005, pp 60-81. methylmalonic acid and total homocys-
(o,e) . Bell, A, and Sallah, S: The Morphology of
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Human Blood Cells, ed 7. Abbott Diag-
depression. The role of folate. Nutr Rev cobalamin and folate deficiencies. Am J
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Sse), WOT. Med 96:239, 1994.
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St. Louis, 1993, p 460.
depletion. Neuropediatrics 28:126, 1997. folate, and vitamin B6 occur commonly
. McKenzie, SB. Clinical Laboratory
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ment. Br J Obstet Gynaecol 100:307, 58:468, 1993.
Upper Saddle River, NJ, 2004,
1993. 43. Miller, A: Food-bound B,, absorption
pp 264-284
Pg). Wald, NJ, and Bower, C: Folic acid, per- and serum total homocysteine in patients
. Babior, BM, and Stossel, TP: Hematol-
nicious anemia, and prevention of neural with low serum B,, levels. Am J Hema-
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ed 3. Churchill Livingstone, New York, 30. Lockith, G: Handbook of Diagnostic 44. Rees, MM, and Rodgers, GM: Homocys-
1994, p 87. Biochemistry and Hematology in Nor- teinemia: Association of a metabolic
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mal Pregnancy. CRC Press, Ann Arbor, disorder with vascular disease and
mentals of Clinical Hematology, ed 3. QI. joy 2S. thrombosis. Throm Res 71:337, 1993.
Johns Hopkins University Press, Balti- WwW — . Morgan, SL: Folic acid supplementation 45. Menachem, Y, et al: Cobalamin defi-
MOre OOS pri prevents deficient blood folate levels and ciency and infertility. Am J Hematol
. Simmons, A: Hematology, A Combined hyperhomocysteinemia during long- 469:152, 1994.
Theoretical and Technical Approach, term, low-dose methotrexate therapy for . Kuzminski, AM: Effective treatment of
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1997, p 48. cardiovascular disease prevention. amin. Blood 92:1191, 1998.
14. Herbert, V: Staging vitamin B,, (cobal- J Rheumatol 25:441, 1998. 47. Tanner, SM, et al: Hereditary juvenile
amin) status in vegetarians. Am J Nutr . Wevers, RA, et al: Folate deficiency in cobalamin deficiency caused by muta-
59:1214S, 1994. cerebrospinal fluid associated with a tions in the intrinsic factor gene. Proc
. Graham, SM, et al: Long-term neuro- defect in folate binding protein in the Natl Acad Sci USA 102(11):4130, 2005.
logic consequences of nutritional central nervous system. J Neurol 48. Cohen, PR, et al: Pancytopenia after a
vitamin B,, deficiency in infants. Neurosurg Psychiatry 57:223, 1994. single intradermal infiltration of methotrex-
ibediatmle/MOMIOO7: . Crellin, R, et al: Folate and psychiatric ate. J Drugs Dermatol 4(5):648, 2005.
. Pippard, MJ: Megaloblastic anemia. disorders, clinical potential. Drugs
Geography and diagnosis. Lancet 344:7, 45:624, 1993.
1994. . Skerritt, UM: A prevalence study of
. Kozyraki, R, et al: The human intrinsic folate deficiency in a psychiatric inpa-
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Molecular characterization and chromo- Scan 97:228, 1998.
somal mapping of the gene 10p within
 Chapter ‘ ,

Aplastic Anemia
Including Pure Red Cell
Aplasia, Congenital
Dyserythropoietic
Anemia, and Paroxysmal
Nocturnal
Hemoglobinuria
Sherrie L. Perkins, MD, PhD

Definition OBJECTIVES
Pathogenesis At the end of this chapter the learner should be able to:
Etiology
1. Define aplastic anemia.
Acquired Aplastic Anemia
2. Understand the etiologic subclassification of aplastic anemia.
Idiopathic or Primary
Secondary Causes 3 . List four causes of acquired aplastic anemia and identify the most common cause.
Congenital Aplastic Anemia 4. Name the most common congenital and acquired disorders associated with the
Fanconi’s Anemia development of aplastic anemia.
Clinical Manifestations of
Nn . Describe the clinical and laboratory features associated with aplastic anemia.
Aplastic Anemia
Laboratory Evaluation 6. Describe the bone marrow findings in aplastic anemia.

Treatment, Clinical Course, 7. List the treatment modalities used in aplastic anemia and identify the best therapy for
and Prognosis younger patients.

Related Disorders 8. List clinical and laboratory features associated with pure red cell aplasia.
Pure Red Cell Aplasia
9. Describe the common characteristics seen in congenital dyserythropoietic anemias
Congenital
(CDAs).
Dyserythropoietic
Anemias 10. Describe the clinical and laboratory features associated with paroxysmal nocturnal
Paroxysmal Nocturnal hemoglobinuria (PNH).
Hemoglobinuria
Laboratory Evaluation of
PNH
Case Study 1
Case Study 2

156
 Chapter 8 Aplastic Anemia = 157

Definition
“ ‘Table é
e-1 Postulated Pathogenic
Aplastic anemia is a bone marrow failure disorder (or group 3 _ Mechanisms in 3
of disorders) characterized by cellular depletion and fatty Laan of Aplastic
replacement of the bone marrow. The concomitant decreases
Anemia — ‘
in hematopoietic progenitors lead to diminished production of
erythrocytes, leukocytes, and platelets and development Tee or Unknown
of peripheral blood cytopenias or pancytopenia. The loss Direct Bone Marrow Toxic Effects
Radiation
of functional bone marrow may occur following a variety of
Drugs (i.e., chemotherapy drugs, other drugs)
bone marrow insults that include drugs, chemicals, irradia- Benzene
tion, infections, and immune dysfunction. Though the inciting Toxins or chemicals
mechanisms vary, all lead to the loss of bone marrow precur- Starvation
sor cells or damage of the bone marrow microenvironment Some bone marrow infections
required to sustain bone marrow cell growth and differentia- Immune-Mediated Bone Marrow Damage
tion. Thus, the hematopoietic progenitor cells that give rise to Drug-induced autoantibodies
the various peripheral blood elements lose their ability to self Autoimmune disorders
renew and produce progeny. This leads to a loss of bone Pregnancy
Some bone marrow infections
marrow cellular mass and bone marrow failure. Clinical crite-
ria that have been used to define aplastic anemia include Congenital Bone Marrow Defects
(1) marrow of less than 25% normal cellularity and (2) at least Fanconi’s anemia
two blood cytopenias defined as neutrophil count less than Dyskeratosis congenita
500 per microliter or platelets less than 20,000 per microliter
or anemia with corrected reticulocyte count of less than 1%.'

hematopoiesis, it has been classified as a refractory or a

Pathogenesis regenerative process. No clear-cut cause for the loss of blood
The basic defect in aplastic anemia 1s a failure of blood cell cell production is applicable to all causes of aplastic anemia.
production by the bone marrow, involving erythrocytes,
Postulated mechanisms for the development of aplastic
anemia include direct toxic effects on the bone marrow,
leukocytes, and platelets. Blood cell production within the
immune-mediated destruction of the marrow stem cells, and
bone marrow is dependent on the growth, differentiation, and
congenital disorders. However, in most cases a clear-cut
self-renewal of a common, pluripotential stem cell (CFU-S).?
mechanism for the development of bone marrow failure can-
To proliferate and differentiate into mature blood elements,
not be identified (Table 8—1).'*°
the CFU-S responds to cytokines and other growth factors
produced in the bone marrow microenvironment.’ Thus, bone
marrow failure may develop as a consequence of decreased Etiology
hematopoietic stem cells resulting from decreased self-
renewal or cellular destruction. Alternatively, a disruption of Clinically, it is useful to divide aplastic anemia into acquired
the bone microenvironment, leading to decreased signal for or congenital (hereditary) types (Table 8—2).! The vast major-
cellular proliferation and differentiation, could lead to bone ity (more than 95%) of cases are acquired. Of these cases,
marrow aplasia (Fig. 8-1). most (40% to 70%) are considered primary or idiopathic in
Most studies to date show decreases in bone marrow nature, because no clear-cut etiologic agent can be identified.
stem cells rather than a defective microenvironment to be the The remaining acquired cases of aplastic anemia are consid-
underlying defect in development in most cases of aplastic ered to be secondary, resulting from documentable exposure
anemia.!+ Because the bone marrow is unable to respond to
the developing peripheral blood cytopenias by increased

BONE MARROW
Self-renewal
1

Red blood @@® Figure 8-1 m@ Schematic representation of possible defects

Proliferation and Differentiation eels _ in hematopoiesis that may give rise to aplastic anemia. It is
White blood be) postulated that decreased numbers of bone marrow stem
2 ; cells cells and/or changes in the bone marrow microenviron-
Bone marrow <5 WP
growth ass
factors Platelets © a)8
@ = ment that alter cytokine levels, ' or both, ’ may cause aplasia
stem cell > to develop. Most evidence points to decreased stem cells
vy caused by the lack of self-replication (1) or direct destruction
i of stem cells (2), rather than changes in the bone marrow
Stromal cells and bone microenvironment (3), as pathogenetic mechanisms for
marrow microenvironment development of aplastic anemia.
158 Chapter 8 Aplastic Anemia

table 8-2 Etiologies Associated Table

8-5 Caus
with Development of
Aplastic Anemia — Chemical Agents
Benzene
Acquired (>95%)
Insecticides
Idiopathic or primary (40%-70%)
Weed killers
Secondary
Chemical agents Drugs
Drugs Chloramphenicol
Ionizing radiation Phenylbutazone
Infections Anticonvulsants
Miscellaneous causes
Sulfonamides
Congenital (Hereditary) (>5%) Gold
Chemotherapeutic agents
Fanconi’s anemia
Dyskeratosis congenita Ionizing Radiation
Infections
Hepatitis
Epstein-Barr virus
to chemicals, drugs, irradiation, or infection. In addition, some Cytomegalovirus
cases of aplastic anemia have been attributed to immune dys- Miscellaneous Causes
function, leading to immunologic attack on and ultimate “rejec- Pregnancy
tion” of the marrow. The antibodies against bone marrow cells Malnutrition
may be induced by drugs, some infections or states of altered Immunologic dysfunction
immunity, such as pregnancy or collagen vascular disorders.°
Hereditary cases of aplastic anemia are extremely rare, with the
most common group designated as Fanconi’s anemia.*» description by Santesson of four cases of fatal aplastic
anemia occurring in workers in a bicycle tire factory.” Ben-
zene has a variety of industrial applications. These include
Acquired Aplastic Anemia use as a solvent for rubber, fats, and alkaloids, and the man-
Idiopathic or Primary ufacture of drugs, dyes, and explosives. Because most ben-
zene compounds are volatile, they are easily absorbed by
Aplastic anemia is most often thought to be idiopathic in inhalation.’
nature, because no clear-cut cause of the bone marrow failure Benzene may induce a wide spectrum of bone marrow
can be identified despite a careful search. Idiopathic aplastic suppression, ranging from mild anemia or thrombocytope-
anemia makes up about 40% to 70% of the cases of nonhered- nia to fatal pancytopenia. There is wide diversity in individ-
itary aplastic anemia seen in western populations. ual susceptibility to benzene compounds. Bone marrow
suppression occurring shortly after initial exposure in some
people, or as long as 10 years after exposure in others.
Secondary Causes
Often bone marrow suppression is reversible after discon-
A wide variety of chemical, physical, and infectious agents tinuation of benzene exposure.'° It is thought that benzene
have been associated with the development of aplastic anemia acts to inhibit synthesis of DNA and RNA, inhibiting cellu-
(Table 8-3). Usually these agents are divided into those that lar proliferation and differentiation of bone marrow
regularly produce bone marrow aplasia upon sufficient expo- cells.”''!* Benzene has also been associated with accumula-
sure (e.g., benzene, irradiation, and chemotherapeutic agents) tion of chromosomal abnormalities and development of
and those for which development of aplasia is considered a acute leukemia in some patients.'*
rare or idiosyncratic event (e.g., chloramphenicol, phenylbuta-
zone) (Table 8-4).'*° DRUGS
A wide variety of drugs have been associated with develop-
CHEMICAL AGENTS ment of aplastic anemia. These are often the result of a non-
Some of the chemical agents linked with bone marrow failure predictable or idiosyncratic reaction to a drug.'*°'4 As
or aplastic anemia include benzene, trinitrotoluene, arsenic, in- discussed previously, this may be a result of direct toxicity or
secticides, and weed killers. Many of these compounds have a development of an abnormal immune reaction whereby anti-
benzene ring as part of their chemical structure. Modification bodies against a drug cross-react with bone marrow cells. The
of the benzene ring moiety with a nitroso or nitro group is antibiotic chloramphenicol and the anti-inflammatory drug
highly associated with development of aplastic anemia.’* phenylbutazone are probably the best documented examples
Benzene has been known to cause varying degrees of of drugs causing aplastic anemia. The toxicity associated with
bone marrow failure for nearly 100 years, since the original these drugs is usually not related to the total dosage of the
 Chapter 8 Aplastic Anemia 159

Agents That Regularly Produce Bone Marrow Hypoplasia with Sufficient Doses
Ionizing radiation
Benzene and benzene derivatives
Chemotherapeutic agents (e.g., busulfan, vincristine)

Drugs That Produce Bone Marrow Hypoplasia in an Idiosyncratic Manner

Type of Drug Relatively Frequent Rare

Antimicrobials Chloramphenicol Streptomycin

Penicillin, tetracycline Amphotericin B
Sulfonamides
Anticonvulsants Methylphenylethylhydantoin Methylphenylhydantoin
Trimethadione Diphenylhydantoin
Primidone
Analgesics Phenylbutazone Aspirin
Tapazole
Hypoglycemic agents Tolbutamide
Chlorpropamide
Insecticides Chlorophenothane
Parathion
Miscellaneous Colchicine
Acetazolamide
Hair dyes

drug received, and drug-induced antibodies have been identi- incidence and predictability of bone marrow suppression vary
fied in only a few patients. Thus, the association between the with the type of drug (see Table 8-4). For example,
drug and development of aplastic anemia is dependent on epi- chemotherapeutic agents are well known to regularly cause
demiologic data and a temporal relationship to drug ingestion bone marrow hypoplasia in a dose-related manner. Other
and development of bone marrow failure.'* The mechanism of drugs (antibiotics, anticonvulsants, analgesics) are much less
drug-induced bone marrow failure suppression is usually predictable. Knowledge of potential bone marrow side effects
unknown, and it is impossible to identify which patients will must be kept in mind when using these drugs and appropriate
react adversely to a drug. Luckily, such idiosyncratic reac- monitoring of peripheral blood indices performed. Often,
tions to drugs are relatively rare. It is estimated that 1 person drug-induced bone marrow hypoplasia is fully reversible on
in 20,000 to 30,000 may have an idiosyncratic reaction to removal of the drug. A small minority of patients may develop
chloramphenicol, which is about 10 times the incidence of irreversible damage.*°"*
developing aplastic anemia for the general population not
taking chloramphenicol.'°
Chloramphenicol has been shown to cause two types
of bone marrow effects.'° The most common reaction is a
reversible bone marrow suppression that occurs while the
patient is receiving the drug and is associated with vacuoliza-
tion of bone marrow precursor cells (Fig. 8—2) and increased
serum iron levels, reflecting ineffective erythropoiesis. The
second reaction seen is development of an irreversible aplas-
tic anemia that occurs weeks to months after drug exposure.
This more severe reaction is not predictable on the basis of
the dose, duration, or route of administration of the drug.
Because of the strong association with development of aplas-
tic anemia, chloramphenicol use has decreased. Currently the
drug is administered only for specific indications when no
other reasonable alternative exists.'>'°
A wide variety of other drugs have been implicated as Figure 8-2 @ Vacuolization of bone marrow hematopoietic
direct suppressors of hematopoiesis and are occasionally precursor cells indicating toxicity in a patient being treated with
associated with development of bone marrow aplasia. The chloramphenicol. (Wright-Giemsa stain, X 1000 magnification)
160 Chapter 8 Aplastic Anemia

IONIZING RADIATION nervosa’ or pancreatic insufficiency (Schwachman-Diamond

[t is well known that ionizing radiation has an acute destruc- syndrome).*’ These are associated with stem cell necrosis and
tive effect on the rapidly dividing cells of the bone marrow, degenerative changes within the bone marrow stromal cells,
which is predictable based on the radiation dosage.'’ High termed gelatinous transformation.°
doses of radiation, in the range of 300 to 500 rads, lead to
complete loss of hematopoietic cells that is irreversible and
lethal. Lesser doses lead to reversible anemia, leukopenia,
Congenital Aplastic Anemia
and thrombocytopenia with full recovery of counts in 4 to 6 Fanconi’s Anemia
weeks. Hematopoietic cells are most susceptible to penetrat-
ing forms of radiation, such as those found in gamma rays and Congenital aplastic anemia is characterized by hematologic
x-rays. However, chronic ingestion of lower-energy radiation abnormalities that have been present since birth, a familial
sources (such as was seen in watch dial painters who ingested inheritance pattern, and the frequent presence of associated
radium by wetting their brushes in their mouths and inhaling congenital defects. Several identifiable clinical entities are
radium dust) may also cause deleterious bone marrow known, of which the best described is Fanconi’s anemia.>*!
elects Fanconi’s anemia is a rare disorder, with more than 500 cases
Ionizing radiation affects bone marrow and other reported in the literature that show variable clinical features
rapidly proliferating cells by disrupting chemical bonds. and a frequent autosomal recessive inheritance pattern. Often
This leads to the formation of free radicals and other bio- these patients have a variety of associated developmental
logically active compounds. These interact with DNA to abnormalities, including one or more of the following: skeletal
cause breaks or cross-linking of the DNA strands. This defects (usually aplasia or hypoplasia of the thumb), cutaneous
results in cellular death or acquisition of lethal or nonlethal hyperpigmentation, renal abnormalities, microcephaly, mental
genetic abnormalities.'” In addition to the immediate dose- retardation, and poor growth. Patients develop pancytopenia
dependent effects of irradiation on hematopoietic cells, that progresses with age and is usually symptomatic within
there may also be delayed or long-term effects that are less 5 to 10 years after birth. Anemia (usually macrocytic or nor-
predictable. Aplastic anemia may occur months to years mochromic and normocytic) and thrombocytopenia usually
after radiation exposure, presumably secondary to long- precedes development of leukopenia. There may be increased
lasting genetic alterations in hematopoietic stem cells, expression of antigen on red cells, increased fetal hemoglobin
although development of bone marrow dysplasia and acute levels, and elevated erythropoietin levels. These suggest a
leukemias is more common.°”° stress erythropoiesis pattern. The bone marrow may be origi-
nally normocellular or hypercellular, but over time hypoplasia
INFECTIONS develops.*'** These patients also have an increased suscepti-
Many infections have suppressive effects on the bone marrow. bility to development of cancer.*?
Acute, self-limited infections may suppress bone marrow Fanconi’s anemia is characterized by a number of
activity for 10 to 14 days with minor effects on the peripheral genetic abnormalities involving at least nine different genes
blood counts. Chronic infections may have more severe effects that interact physically or functionally in cell cycle control
on hematopoiesis. Several viral infections, including hepatitis,”! and DNA repair.**** This may be seen functionally by special
Epstein-Barr virus,?> and cytomegalovirus,*** have been studies that show a high level of chromosomal breakage,
associated with the development of aplastic anemia. Of these, DNA cross-linking, and defective DNA repair in Fanconi
hepatitis from an uncharacterized hepatitis virus (i.e., non-A, patient cells treated with cross-linking agents or genotoxic
non-B, non-C type) has the strongest association with the de- stresses.** It is therefore not surprising that these patients may
velopment of a refractory aplastic anemia.?! The mechanism also have an increased incidence of acute myelogenous
whereby viruses cause aplastic anemia is unknown. It has been leukemia and other malignancies, leading to characterization
suggested that the virus may directly infect the hematopoietic as a genetic-cancer syndrome.*!“? Nine different Fanconi’s
stem cell or induce an autoimmune reaction against bone anemia-associated genes have been cloned, which are all
marrow hematopoietic stem cells.'*° Other systemic infec- linked to DNA damage response. This pathway is regulated
tions, such as miliary tuberculosis,”> have also been associated by protein ubiquitination and may affect p53-mediated apop-
with the development of aplastic anemia or bone marrow tosis, leading to development of the clinical features of the
dysfunction. disease.**
Untreated, patients with Fanconi’s anemia usually die
MISCELLANEOUS CAUSES from infections or hemorrhage secondary to blood cytopenias
Aplastic anemia often appears to have an immune-based com- or development of malignancy. Currently, most patients
ponent and many patients with aplastic anemia will respond to are treated with allogeneic bone marrow transplantation,
immunosuppressive therapy, although little is known about although chemotherapy or irradiation conditioning regimens
mechanisms or inciting antigens.”° States of altered immune are associated with high levels of toxicity as a result of the
functions, such as pregnancy”’ or graft versus host disease post DNA repair defects, so that a reduced intensity conditioning
bone marrow transplant** have been associated with develop- approach may be preferable.** The development of other pos-
ment of aplastic anemia. Other cases of aplastic anemia have sible treatment approaches, such as gene therapy, may offer
been associated with malnutrition, such as in cases of anorexia hope to these patients in the future.*!
 Chapter 8 Aplastic Anemia 16]

Other congenital causes of aplastic anemia are much

more rarely seen than Fanconi’s anemia. Dyskeratosis con- 4 Table 8-5 Laboratory Evaluation
genita is an X-linked disorder in which approximately half of , for Aplastic Anemia
the affected patients develop aplastic anemia. It is not associ-
ated with chromosomal instability, but appears to arise due to Test Purpose
defects in gene encoding the protein dyskerin. Dyskerin is
CBC and differential Establish severity of
known to be involved in telomere structure and maintenance.
cytopenias
Patients with dyskeratosis congenita have excessively short Peripheral blood Exclude malignancy and
telomeres, which are hypothesized to result in bone marrow examination other causes of cytopenias
failure.*° Reticulocyte count Establish decreased
marrow regeneration
Bone marrow examination Rule out leukemia, other
causes of cytopenias (i.e.,
Clinical Manifestations of Aplastic myelodysplasia, storage
Anemia disorder, metastatic
diseases, granulomas,
Aplastic anemia often presents as an insidious process, owing fibrosis); establish
to the gradual decrease in bone marrow production of erythro- hypoplasia of bone marrow
cytes, leukocytes, and platelets. It may occur in all age Biochemical testing Liver function, renal function
Cultures Document possible infection
groups. Most patients present with symptoms of progressive Serologic testing Document infection
fatigue, dyspnea, and palpitations. However, bleeding or
infection may also be seen. Physical examination may reveal
pallor secondary to the anemia, or evidence of thrombocy-
topenia (including petechiae, purpura, ecchymoses, and
red cells seen. White cells usually show a relative lymphocy-
mucosal bleeding). Signs of infection, resulting from the
tosis of up to 70% to 90%, reflecting decreased numbers of
decrease in leukocytes, are usually late manifestations of
myeloid and monocytic cells. If the white cell count falls
the disease. Other physical findings are minimal. Mild
below 1.5 < 10°/L, an absolute lymphopenia may also be pre-
lymphadenopathy may be seen, but splenomegaly is unusual.!
sent. Rarely, immature myeloid cells such as myelocytes and
A detailed history of drug ingestion, toxic exposure, or infec-
metamyelocytes are seen, but large numbers of these cells
tion is essential, as well as a family history of similar hema-
would call into doubt the diagnosis of aplastic anemia.
tologic problems.'>° Platelets are usually decreased, and it is unusual to find large
or abnormal forms. When thrombocytopenia is present,
Laboratory Evaluation bleeding time is prolonged and clot retraction poor.
Because of the lack of specific features in the CBC data
The laboratory studies to evaluate a patient with aplastic and peripheral smear, a wide differential diagnosis for causes
anemia are aimed at defining the degree of bone marrow dys- of pancytopenia must usually be considered (Table 8—7), and
function and at excluding other possible causes of cytopenias a bone marrow examination must be performed to arrive at a
for which other therapies are available.'*° Laboratory evalu- diagnosis. The bone marrow aspiration is often markedly
ation usually involves a complete blood count (CBC) and hypocellular or is a dry tap (Fig. 8-3). Small numbers of lym-
reticulocyte count, peripheral smear, and bone marrow exam- phocytes, plasma cells, and rare hematopoietic precursor cells
ination. Ancillary biochemical tests to evaluate renal and are seen. The bone marrow biopsy specimen most commonly
hepatic function, as well as cultures or serologic tests looking
for infectious agents are also necessary (Table 8-5). The CBC
shows pancytopenia of varying degrees, often with anemia —

being the most notable (Table 8—6). The hemoglobin concen- Table 8-6 Characteristic Abnormal
tration is usually 70 g/L or lower, and the anemia is usually
CBC Values Seenin
normochromic and normocytic, although the cells may
occasionally be macrocytic with moderate anisocytosis and
Severe Aplastic Anemia
poikilocytosis. The corrected reticulocyte count is character- Red Blood Cells
istically low (less than 1%, or less than 25 Xx 10°/L absolute Hematocrit = 0.20-0.25 (L/L) or 20%-25%
reticulocyte count), reflecting the lack of bone marrow regen- Hemoglobin concentration = 70 g/L
erative activity. The white cells, in particular the myeloid and Absolute reticulocyte count = 25 = 10°/L
Corrected reticulocyte count < 1%
monocytic cells, as well as platelets are decreased. Lympho-
cytes may be normal or decreased in number. White Blood Cells
There is no specific morphological abnormality of the Total leukocyte count S$ 1.5 x 10°/L
Absolute neutrophil count = 0.5 x 10°/L
blood smear that is diagnostic of aplastic anemia. Examina-
tion of the blood smear confirms a normochromic, normo- Platelets
cytic anemia with little or no evidence of regeneration such as Platelet count = 20-60 x 10°/L
polychromatophilic cells, basophilic stippling, or nucleated
162 Chapter 8 Aplastic Anemia

Infiltration of the Bone Marrow

Tumors—leukemias, lymphomas, metastatic disease
Fibrosis—myelofibrosis
Granulomatous infections—mycobacteria, fungi
Other processes—sarcoid, storage disorders such as
Gaucher’s disease

Inhibition of Hematopoiesis or Ineffective Hematopoiesis

Myelodysplasia or paroxysmal nocturnal hemoglobinuria
Vitamin B,, or folate deficiency
Myelosuppressive drugs

Increased Splenic Activity (Hypersplenism)

Congestion
Splenic infiltrative disorders—Gaucher’s disease,
Niemann-—Pick disease
Infections
Primary hypersplenism
Aplastic anemia

shows a very hypocellular bone marrow with marked reduc-

tions in the myeloid, erythroid, and megakaryocytic lineages.
Compared to a normocellular bone marrow biopsy specimen
(Fig. 8-4A), a markedly hypocellular bone marrow biopsy
sample shows only residual stroma and fat (Fig. 8-4B). Scat-
tered lymphocytes and plasma cells (Fig. 8-5) or occasional
lymphoid aggregates (Fig. 8-6) may be seen. It should be noted
that patients with aplastic anemia often have residual islands of
normal marrow or focal areas of bone marrow hyperplasia ad- Figure 8-4 @ A. Normocelluiar bone marrow. B. Markedly hypocel-
jacent to hypoplastic areas that may mimic the findings of lular bone marrow biopsy specimen from a patient with aplastic
anemia. (H & E stain, 500 magnification)
myelodysplasia or other processes, further complicating the
morphologic diagnosis of aplastic anemia (Fig. 8-7). It is cru-
cial to remember that there may be variations within each bone
marrow biopsy specimen and between different biopsy sites in
aplastic anemia, perhaps necessitating multiple sequential
biopsies to establish the diagnosis.

Figure 8-5 m@ Residual lymphocytes, plasma cells, and bone

Figure 8-3 @ Hypocellular bone marrow aspirate containing marrow stroma with marked decrease in hematopoietic cells in a
primarily lymphocytes and plasma cells, reflecting bone marrow bone marrow biopsy specimen from a patient with aplastic anemia.
aplasia. (Wright-Giemsa stain, 500 magnification) (H & E stain, X200 magnification)
 Chapter 8 Aplastic Anemia 163

available. A reduced intensity conditioning regimen may be

used because of the impairment of the recipient’s immune
system inherent in aplastic anemia.** Long-term survival rates
of 65% to 85% have been reported following bone marrow
transplantation, usually with full functional bone marrow
recovery.':!?¢389 Jt should be noted that the chances of suc-
cessful transplantation diminish in patients who have received
multiple transfusions (more than 20), probably as a result of
autoimmunization, which increases the chance of graft rejec-
tion. Thus, to minimize the numbers of supportive transfu-
sions, many patients receive bone marrow transplantation
early in the course of the disease.**~?
For patients who are unable to receive bone marrow
transplantation because of older age or lack of a suitable
donor, immunomodulatory therapy has been utilized.*’ This
Figure 8-6 = Lymphoid aggregate seen in a bone marrow biopsy type of therapy makes use of antithymocyte or antilymphocyte
specimen from a patient with aplastic anemia. (H & E stain, <100 globulin, cyclosporine, or cyclophosphamide in an attempt to
magnification)
inhibit a presumed immune attack on the bone marrow stem
cell. Up to 75% survival after 5 years has been seen with this
therapy, although relapses may occur.*°“° Other alternative
approaches include long-term stimulation with hematopoietic
growth factors, usually in association with immunosuppres-
sive therapy. There is a variable increase in neutrophil count,
often dependent on the severity of the bone marrow aplasia.
Often this is a much less effective stimulation of erythrocyte
or platelet production.*°**!
The prognosis for patients with aplastic anemia has
markedly improved with the advent of such therapeutic
options as bone marrow and stem cell transplantation. The
outcome is still variable and depends primarily on the sever-
ity of the anemia at the time of presentation, supportive care
such as blood product transfusions, and the treatment modal-
ity employed.)°**3**!

Figure 8-7 © Focal area of bone marrow hyperplasia adjacent to a Related Disorders
hypoplastic area in early aplastic anemia. (H & E stain, 100
magnification) Pure Red Cell Aplasia
Pure red cell aplasia is an uncommon disorder in which the
erythroid cells in the bone marrow are selectively destroyed.
Treatment, Clinical Course, This gives rise to an anemia without other associated cytope-
nias. More than 900 cases have been reported in the world’s
and Prognosis literature. This may be an acquired or congenital process
Untreated aplastic anemia has an extremely poor prognosis, as (Table 8-8). Pure red cell aplasia is characterized by severe,
the patients undergo progressive decreases in blood counts and chronic, normocytic to slightly macrocytic anemia. Reticulo-
subsequent lethal infection or bleeding.'*°*° However, some cytes are decreased and may be absent. There is no evidence
patients recover spontaneously. Until the early 1970s, patients of hemolysis or hemorrhage. White cells and platelets are
with severe aplastic anemia were treated via supportive trans- normal. A bone marrow biopsy usually demonstrates normal
fusions, possible treatment with androgens or anabolic steroids cellularity with a notable absence of erythroid precursor cells.
to stimulate hematopoiesis, and an extensive search for the Erythropoietin levels are often markedly increased as the
possible etiologic agent to prevent further exposure. Even so, body attempts to compensate for the profound anemia,
the course was usually progressive, with a 5-year mortality although cases arising secondary to development of anti-
rate of about 70% and only 10% of patients fully recovering.”’ erythropoietin antibodies (discussed further later in this chap-
Currently, the treatment of choice for aplastic anemia in ter) may have low or absent erythropoietin levels.****
patients younger than 50 years of age is allogeneic bone Acquired causes of pure red cell aplasia are most
marrow or peripheral stem cell transplantation. This therapy common. These may be short-lived acute illnesses or have a
is optimal if bone marrow from an HLA-matched sibling is more prolonged chronic course. Viral illnesses have been
used, although unrelated donors are becoming more readily associated with development of a transient cessation of red
164 Chapter 8 Aplastic Anemia

Another frequent cause of pure red cell aplasia is an idio-

Table 8-8 Causes of Pure Red Cell syncratic reaction to a drug. Several drugs have been implicated,
Aplasia and these are usually the same drugs that give rise to aplastic
anemia, including phenytoin, isoniazid, and azathioprine. Both
Acquired direct inhibition of erythroid cell proliferation and differentia-
Infections—parvovirus B19
Aplastic crisis in patients with hemolytic disorders
tion by the drug and development of drug-induced antibodies
Immunocompromised patients that lead to destruction of red cell hematopoietic precursors in
Malnutrition the marrow have been implicated as pathogenetic mecha-
Drugs nisms.*> There has been a recent increase in pure red cell aplasia
Direct toxicity (more than 200 cases) in patients (particularly with renal dis-
Development of antibodies to red cell precursors or
ease) receiving recombinant erythropoietin that was associated
erythropoietin
Thymoma or lymphoid neoplasms with a new formulation of the recombinant cytokine and use of
Idiopathic subcutaneous administration. These patients developed neutral-
izing anti-erythropoietin antibodies that led to development
Congenital
of pure red cell aplasia with low erythropoietin levels. The neu-
Diamond—Blackfan anemia
tralizing antibodies can be identified in the patient’s serum.
Patients often improve with immunosuppressive therapy and
discontinuation of erythropoietin administration. Other etiolo-
cell production and disappearance of erythroblasts from the gies associated with development of pure red cell aplasia
bone marrow. In most patients this would be relatively include malnutrition, chronic infections, vitamin deficiency in
asymptomatic; however, in patients with long-standing children, and the presence of a thymoma in adults. In adults with
hemolytic disease and rapid cell turnover (e.g., sickle cell dis- persistent red cell aplasia, more than 50% have a thymoma,
ease, hereditary spherocytosis), this loss of red cell produc- although it may not be detected until several years after the
tion causes a precipitous decrease in hematocrit or an aplastic development of anemia. Removal of the thymic tumor causes a
crisis. In some cases, aplastic crisis has been associated with resolution of the anemia in about one-third of the patients.*° Pure
parvovirus B19 infection (the causative agent of erythema cell aplasia has also been associated with B- or T-cell lympho-
infectiosum, or fifth disease, observed in children). Par- proliferative disorders, such as chronic lymphocytic leukemia or
vovirus B19 selectively infects red blood cell precursors, T-cell large granular lymphocytosis. This association of pure red
leading to an absence of late erythroblasts and subsequent cell aplasia with these neoplasms is usually a result of produc-
stages of erythroid differentiation. Some erythroblasts may con- tion of antibodies that cross-react with erythroid precursors or
tain viral inclusions (Fig. 8-8). Parvovirus B19 infection may production of cytokines that inhibit erythropoiesis. Response to
also cause a transient anemia in immunocompromised patients immunosuppressive therapies or treatment of the underlying
or patients with severe malnutrition. Usually the patient recovers disease process has been noted in some of these patients.*7“*
erythropoietic capacity, as evidenced by the appearance of retic- A congenital form of pure red cell aplasia was described
ulocytes, within 7 to 10 days. Chronic severe anemia may persist in 1938 and bears the name Diamond—Blackfan anemia. It is
in some immunocompromised patients as they are unable to characterized by a chronic, moderate to severe anemia that
clear the parvovirus infection. Most immunocompetent patients, usually manifests early in infancy and is associated with
without conditions of rapid cell turnover, experience only a brief, normal numbers of white cells and platelets. The anemia is
transient fall in hematocrit.?* usually macrocytic, but may occasionally be normocytic. The
reticulocyte count is decreased. Bone marrow examination
shows a normocellular bone marrow with erythroid hypopla-
sia. Usually normal or increased numbers of proerythroblasts
are present in the marrow with marked decreases in the more
mature stages of differentiation in the erythroid cells. Minor
congenital abnormalities of the head and upper limbs may be
present, similar to Fanconi’s anemia. The mode of inheritance
is uncertain, with sporadic cases making up 75%. Both reces-
sive and dominant inheritance patterns have been described.
There is a wide variation in the age at onset, severity, and
natural course of the disease. Spontaneous remissions have
been noted in up to 25% of patients, often following many
years of anemia.****? Most patients respond to steroids and
are able to maintain adequate hemoglobin levels. Some
patients may become transfusion dependent and develop pos-
sible iron overload from long-term transfusion therapy. Bone
marrow transplantation is curative.’ There is a marked
Figure 8-8 @ Erythroid precursors containing parvovirus B19 viral increase in the incidence of acute myelogenous leukemia
inclusions. (Wright-Giemsa stain, 500 magnification) late in the course of the disease. Although the pathogenetic
 Chapter 8 Aplastic Anemia 165

mechanism underlying Diamond-Blackfan anemia is not

clear, most studies point to a molecular defect in signa]
transduction in the erythroid bone marrow progenitors that
leads to decreased responsiveness to erythropoietin or other
cytokines.*74?

Congenital Dyserythropoietic Anemias

Congenital dyserythropoietic anemias (CDAs) are a rare group
of familial disorders in which anemia and ineffective erythro-
poiesis are associated with bizarre binuclear and multinuclear
erythroblasts (Fig. 8-9). Three types of CDA have been well
described (Table 8-9), with an additional four recognized
types that are less well characterized (Table 8-10). In addition,
some case reports of congenital dyserythropoietic processes
that do not fit into these seven recognized categories are in the Figure 8-9 ® Multinucleated erythroid precursors in type 2
literature. All of the CDAs present clinically with anemia, ery- congenital dyserythropoietic anemia (HEMPAS). (Wright-Giemsa
stain, “1000 magnification)
throid hyperplasia with variable degrees of dyserythropoiesis,
and indirect hyperbilirubinemia or mild jaundice.*?>”!
Type 1 CDA is characterized by a mild to moderate water test, nor do they have the characteristic flow cytometric
macrocytic anemia with prominent anisocytosis and poikilo- finding of decreased levels of CD55 and CD59.” In addition,
cytosis. Bone marrow erythroblasts show inegaloblastic mat- type 2 CDA cells have been found to be strongly agglutinated
uration and the presence of a small number (1% to 3%) of by anti-i, and the i antigen is persistently expressed on all of
marrow erythroblasts that are binucleated or contain chro- the erythrocytes in HEMPAS.
matin bridges (thin, fiberlike connections between the nuclei) Type 3 CDA presents as a mild to moderate macrocytic
(Fig. 8-10). It is inherited as an autosomal recessive trait.*! anemia. It differs from type 1 CDA by having as many as 30%
Type 2 CDA gives rise to a mild to severe normocytic multinucleated bone marrow erythroid cells, some containing as
anemia in which 10% to 50% of the erythroblasts in the mar- many as 12 nuclei (gigantoblasts). This disorder appears to be
row are binucleated or multinucleated (see Fig. 8-9). This inherited in an autosomal dominant fashion. The anemias asso-
form is also characterized by an autosomal recessive mode of ciated with the well-described types of CDA are mild to moder-
inheritance and is the most commonly seen form of CDA. ate, and most patients may be treated with transfusions and
Type 2 CDA red cells will lyse in acidified serum (Ham’s supportive therapy. Some patients may develop splenomegaly
test), giving rise to the alternative name for this disorder of requiring a splenectomy.*””!
HEMPAS (hereditary erythroblast multinuclearity with a pos-
itive acid serum test). Because of red cell lysis in an acidified Paroxysmal Nocturnal Hemoglobinuria
serum test, a diagnosis of paroxysmal nocturnal hemoglobin-
uria (PNH) (discussed later) may be entertained. However, Paroxysmal nocturnal hemoglobinuria (PNH) is a rare
unlike PNH, the cells of CDA type 2 do not lyse in a sugar acquired stem cell disorder that results in abnormalities of the

ong niitalDyse16
onge etic Anem jas
rythropcDie

Typeii
Inheritance cn Autosomal recessive Autosomal recessive Familial: autosomal dominant
Sporadic: variable
Red cells Macrocytic Normocytic Macrocytic
Anemia Mild to severe Mild to moderate Mild to moderate
Light microscopy-marrow Intranuclear chromatin bridges Binucleation Giant erythroblasts
Electron microscopy “Swiss cheese” nuclei Peripheral cisternae No specific findings
Acidified serum lysis Negative Positive Negative
Genetic localization 15q15. -15.3 20q11.2 Familial: 15q22
Sporadic: variable
Postulated biochemical defect Unknown Underglycosylation of band 3 Unknown
Associated abnormalities Skeletal defects, skin None Familial: visual defects
hypopigmentation monoclonal gammopathy
Sporadic: association with
lymphoma, mental
retardation
166 Chapter 8 Aplastic Anemia

Type IV Type V Type VI Type VII

Anemia Severe; requires Mild to absent Mild to absent Severe; requires
transfusion transfusion
RBCs Normocytic Mildly macrocytic Marked megaloblastic Normocytic
Normoblasts Irregular or Little dysplasia Florid megaloblastic Irregular nuclear
karyorrhectic nuclei maturation shapes;
intracytoplasmic
inclusions

red cell membrane. This causes the red cells to be highly sen- marrow hypoplasias.**° Usually patients presenting with
sitive to complement-mediated hemolysis. As this is a stem hemolytic type PNH have small base pair deletions (1 to 14 base
cell disorder, abnormalities are seen in leukocytes and pairs) or insertions (1 to 8 base pairs) or substitutions that lead
platelets, as well as in red cells. PNH is characterized by to DNA reading frameshifts. These may occur throughout the
recurrent, episodic intravascular hemolysis, hemoglobinuria, PIG-A_ gene.’ PNH arising in association with aplastic
and venous thrombosis. Hemolysis may be worse at night due anemia will have similar mutations but may also demonstrate
to decreased plasma pH, or may be more chronic in nature.°?*? tandem duplications and insertions that are significantly
PNH is also strongly associated with aplastic anemia. In larger in size than those seen in PNH.***° Patients with
aplastic anemia atypical red cell clones with a PNH pheno- combined aplastic anemia and PNH are more likely to have
type may develop and many cases of PNH may evolve into multiple mutations at many sites and a variety of different
aplastic anemia or bone marrow failure.°°**° mutational types.°°°’ Within the bone marrow, the cells con-
PNH arises secondary to a somatic mutation in the bone taining the P/G-A mutation may be a minority of bone
marrow pluripotential stem cell that gives rise to complete or marrow cells or the mutation may be expressed in nearly all
partial deficiencies of cell surface glycophosphatidylinositol hematopoietic cells.°*°
(GPI) anchor proteins. The mutated gene is phosphatidylinos- The GPI anchoring defect seen in PNH causes variable
itolglycan A (PIG-A), which is located on the X chromosome. deficiencies in at least 17 cell surface proteins that may affect
Similar mutations are seen in patients with bone marrow the function of hematopoietic cells (Table 8-11). Included in
failure syndromes, such as aplastic anemia, or other bone these proteins are two to three membrane glycoproteins that nor-
mally regulate complement fixation and activation on the cell
surface. These proteins include decay-accelerating factor (DAF
or CD55), which regulates the activity of C3 convertase, homol-
ogous restriction factor (HRF), which regulates C9 binding and
activity; and membrane inhibitor of reactive lysis (MIRL or
CD59), which modulates complement mediated red blood cell
lysis. A deficiency in one or more of these proteins leads to
enhanced formation of membrane complement lytic com-
plexes on the cell surface and increased complement-mediated
hemolysis (Fig. 8-11). White blood cells and platelets also
demonstrate abnormal cell surface GPI anchoring protein
levels; however, it is the red cell deficiency that gives rise to the
classic episodic intravascular hemolysis. In all cell types there
may be variable expression of the abnormal phenotypes, per-
haps reflecting genetic mosaicism. Thus some cells express
normal levels of the GPI anchoring proteins and other cells
express lesser levels of these proteins or they may be completely
absent. This has led to description of three different subtypes of
PNH cells (Table 8—12), and the relative numbers of each PNH
cell type will correlate with disease severity. A patient may have
some or all of these cellular subtypes present and the numbers
of each subtype may shift over the course of the disease.5?4
Clinically PNH has an estimated prevalence of 1 to
10 cases per million population. It is seen primarily in adults but
Figure 8-10 @ Binucleated erythroid precursor with chromatin has also been described in children and adolescents.>25* The dis-
bridge seen in a patient with type | congenital dyserythropoietic ease displays a wide spectrum of symptomatology and usually
anemia. (Wright-Giemsa stain x 1000 magnification) begins as an insidious onset of anemia. Virtually all patients are
 Chapter 8 Aplastic Anemia 167

Panel A Panel B

cg
complement
complex

Complement Regulatory Proteins

cg
Decay accelerating factor (CD55)
complement
Homologous restriction factor complex
Membrane inhibitor of reactive lysis (CD59)

Proteins Associated with Immune Function

Lymphocyte function antigen-3 (LFA-3, CD58) Hemolysis
Fe receptor gamma III (CD16)
Endotoxin binding protein receptor (CD14)

Other Receptors
Urokinase receptor
EONS sis Figure 8-11 @ A. The normal cell is protected from C3b binding
Enzymes and complement-mediated hemolysis because the PIG-A anchored
Alkaline phosphatase proteins CD59 and CD55 are present and thus complement fixation
Acetylcholinesterase cannot occur. B. The PNH cell has decreased CD55 and CD59 pro-
5’-Ectonucleotidase tein on the cell surface due to lack of GP! anchoring proteins,
allowing for C3b binding and complement fixation that leads to red
Other Proteins cell lysis. (Adapted from Kinoshita, T, and Inoue, N: Relationship
CD24 between aplastic anemia and paroxysmal nocturnal hemoglobinuria.
CD48 Int |Hematol 75:117, 2002.)
CD52 (Campath-1)
CD66c 2
CD67 anemic, often with severe anemia. The classically described pat-
JMH-bearing protein tern of episodic hemolysis at night causing dark urine upon
awakening is frequently not seen. Hemolysis is more likely to
occur in an irregular fashion, precipitated by infection, surgery,
and transfusion. Hemolysis may also be occurring chronically
throughout the day. Hemoglobinuria or dark urine may be pre-
sent or absent. Chronic urinary iron loss or hemosiderinuria is a
constant feature and may lead to an iron-deficiency anemia.

‘Table 8-12 TTypesof Cells

PoiaeN aaanS ER a a

Observed in Paroxysmal Nocturna Ve eee

PNH Sensitivity to Observed

Ceil Complement Complement GPI Protein Associated PIG-A
Type Lysis Pathway Defects Expression Mutations

I Normal to Near normal Near normal to None or single

near normal lytic behavior mild deficiency; point mutation
partial lack of
DAF (CD55) and/or
MIRL (CD59)
Il Intermediate Increased C, binding to Partial lack of DAF Missense
(10-15 times cell; increased C,/C; (CD55) and MIRL (partial)
more sensitive) convertase activity (CD59) (DAF deficiency
most significant)
Ul Highly sensitive Increased binding of C; Near total lack of DAF Nonsense
(25 times more to cell; increased C,/C; (CD55), MIRL (CD59), mutation
sensitive) convertase activity; HRF frameshift,
increased binding of deletion or
C5467 complexes; insertion
increased C, binding causing gene
inactivation

PNH = paroxysmal nocturnal hemoglobinuria; GPI = glycophosphotidylinositol; PIG-A = phosphatidylinositolglycan A;

DAF = decay accelerating factor, MIRL = membrane inhibitor of reactive lysis; HRF = homologous restriction factor.
168 — Chapter 8 Aplastic Anemia

Chronic hemolysis may also result in chronic renal failure due erythrocytes present. Thus hemoglobin levels may vary from
to renal tubular damage.**°° normal to <6 g/dL. Morphologically the red cells appear
Abnormal platelet function in PNH patients is frequently normochromic and normocytic without significant abnormal-
associated with venous thromboses. These are a common ities. Occasionally slight macrocytosis and polychromasia
cause of death in this patient population, and may affect up to may occur owing to increased reticulocytes or red cells may
one third of patients in a significant manner. Thrombotic be hypochromic and microcytic due to iron deficiency sec-
events may cause severe abdominal or back pain or severe ondary to hemosiderinuria or hemoglobinuria (Fig. 8-12).
refractory headaches. Development of Budd-Chiari syndrome The increased reticulocytosis noted in association with PNH
(hepatic vein thrombosis), mesenteric vein, or cerebral vein is in direct contrast to the hypoproliferative response seen
thromboses are most common. Thrombophlebitis may also with aplastic anemia and reflects the marrow response to
occur, leading to pulmonary thromboembolism. Arterial hemolysis. However, although the reticulocyte count is usu-
thromboses are rare. Rare patients may experience bleeding ally elevated (5% to 10%), the absolute reticulocyte count
due to poor platelet function.**°* may be low for the degree of anemia, reflecting the abnormal
Patients are often leukopenic at some time during the hematopoietic state. Occasional nucleated red blood cells
course of their disease. This will lead to increased suscepti- may be seen, particularly in episodes of severe hemolysis.
bility to infection and sepsis. Often leukocytes will show Spherocytes are typically absent, although occasional schisto-
decreased leukocyte alkaline phosphatase (LAP) activity. In cytes may be seen on the smear, particularly in the presence
addition to functional defects in the leukocytes, patients of intravascular thrombosis.°**!
also develop leukopenia in addition to thrombocytopenia White blood cells, in particular granulocytes, and platelets
and anemia. Treatment with cytokines or transfusions is have the same membrane defects seen in the red cells that will
usually indicated. render them more sensitive to complement lysis. Granulocytes
As noted in the preceding text, aplastic anemia may pro- appear morphologically normal. However, neutropenia is often
ceed or coexist with PNH.'°*°S Patients with aplastic anemia observed and the neutrophils will have decreased leukocyte
often develop abnormal hematopoietic cell clones that have a alkaline phosphatase activity. Platelet counts also vary in
PNH phenotype and are associated with mutations in the P/G- PNH with mild to moderate thrombocytopenia and platelet
A gene. It should be noted that small numbers of abnormal, counts ranging from 50 to 100 x 10°/L commonly observed.
PIG-A-deficient clones may also be seen in normal marrows. Although thrombocytopenia is often present, the most common
This suggests the possibility of a survival advantage for these platelet dysfunction is abnormal clotting with increased venous
abnormal hematopoietic cells in a patient with aplastic thromboses.°**!
anemia. It has been hypothesized that the PNH phenotype The bone marrow often shows erythroid hyperplasia in
may allow the hematopoietic precursor to escape immune- response to chronic hemolysis (Fig. 8-13). As noted in the
mediated destruction. In contrast, the P/G-A—deficient clones preceding text, some patients with PNH will develop
do not have a survival advantage over normal hematopoietic hypoplastic or aplastic marrows. Often adequate numbers of
cells, resulting in the mosaicism characteristically seen in myeloid and megakaryocytic precursors are present. Bone
PNH. A recent study showed up to 89% of patients with aplas- marrow iron stains usually demonstrate decreased iron.°!
tic anemia had some number of PNH-phenotype granulocytes Diagnostic testing for PNH includes demonstration of
present at the time of diagnosis.°’ Similar other studies have hemosiderin in the patient’s urine by staining with Prussian
shown 50% to 60% involvement by the abnormal PNH-like
clone in newly diagnosed aplastic anemia.°»*>>
In addition to the linkage between aplastic anemia and
PNH, there also appears to be some overlap with large gran-
ular lymphocytic leukemias. Often PNH patients will show
clonal large granular lymphocyte expansions, and this may
reflect the abnormal immune system that is associated in this
disorder. Large granular lymphocyte expansions are also seen
in aplastic anemia, and it has been recently hypothesized that
the expansion of the GPI deficient clones may be due to an
attempt by the marrow progenitor cell to escape antigen
driven immune attack.°’° Thus, the pathophysiologic under-
pinnings of aplastic anemia, PNH, and other bone marrow
failure syndromes appear to be closely related and may have
similar or overlapping pathophysiologic features.°°!

Laboratory Evaluation of PNH Figure 8-12 ® Peripheral blood smear from a patient with PNH
(Wright-Giemsa stain, x600 magnification) demonstrating
Characteristic laboratory findings in PNH include anemia,
normochromic, normocytic red cells, as well as occasional
leukopenia, and thrombocytopenia. The anemia may be mild hypochromic, microcytic, and polychromatophilic cells. A nucleated
to severe depending on the number and subtype of PNH type red cell is also seen.
 Chapter 8 Aplastic Anemia 169

than 5% lysis is usually considered to be a negative screening

test, although a small degree of lysis (usually less than 5%)
has been described in patients with megaloblastic anemia,
autoimmune hemolytic anemia, and some leukemias. All pos-
itive sucrose hemolysis tests should be confirmed with
the Ham’s test or flow cytometry for definitive diagnosis
COHEN Eee
The Ham’s test or acidified serum lysis test utilizes acidi-
fied serum which activates complement via the alterative path-
way and allows binding of C3 to the red cell membrane. PNH
erythrocytes will then lyse, as they are deficient in the GPI-
anchor proteins, making them more sensitive to complement
lysis. Normal erythrocytes will be unaffected in this test. A pos-
itive Ham’s test must demonstrate the following characteristics:

Figure 8-13 @ Bone marrow aspirate smear from a patient with 1. Hemolysis occurs with the patient’s cells and not with con-
paroxysmal nocturnal hemoglobinuria demonstrating erythroid trol cells.
hyperplasia. (Wright-Giemsa stain, x 500 magnification) NO. Hemolysis is enhanced by acidified serum and does not
occur with heat-inactivated serum (heating to 56°C for
30 minutes to inactivate complement) (Table 8-13 and
blue. Hemoglobinuria, when present, will be detected by dip-
Fig. 8—15).°*°!
stick. This must be coupled with microscopic examination to
determine that intact red cells are absent. Occasionally patients A very specific test for the diagnosis of PNH is the use of flow
will show hemoglobin casts, and this is often associated with cytometry to assess the presence or absence of GPI-anchoring
renal failure. Because of ongoing hemolysis, indirect bilirubin proteins. This method is very sensitive and specific, utilizing
will often be increased, as will plasma hemoglobin, while hap- monoclonal antibodies against CD55 and CD59 proteins that
toglobin is decreased. The direct anti-globulin test (Coombs’ modulate complement activity. Detection of decreased or
test) will be negative.*°! absent expression of these proteins by flow cytometric analy-
Several diagnostic tests have been developed for the sis 18 diagnostic of PNH. CD59 is usually present in high lev-
determination of PNH. Initial screening is usually done with a els on the red cell membrane with somewhat lower levels of
sugar water test (sucrose hemolysis test) when a diagnosis of CD55. The patient’s red blood cells can be stained with
PNH is considered. Placing blood into a sucrose-containing fluorochrome-labeled anti-CD59 or anti-CD55 for flow cyto-
medium of low ionic strength will promote the binding of metric analysis. Patients with PNH will show variably
complement, especially C3, to the red cell membrane. This decreased to absent expression of CD59 and/or CD55, which
low ionic strength solution will activate the classic or alterna- is manifested by a shift in the peak channel of fluorescence.
tive complement lysis pathways leading to red cell hemolysis. In most patients, a population of normal cells is often com-
Normal cells should be unaffected (Fig. 8-14). Most monly seen, reflecting phenotypic mosaicism (Fig. 8-16).
patients with PNH will show 10% to 80% red cell lysis. Less The degree of CD55 or CD59 deficiency is often associated

Figure 8-15 @ Ham’s test. Positive results occur in patients with

Figure 8-14 m Sugar water test. The tube on the left represents PNH. A positive test is reported when hemolysis occurs in tube 1,
the control (C) and the tube on the right represents the patient (P) containing fresh normal serum and patient cells; tube 2, containing
with a positive sugar water test demonstrating 10% to 80% hemol- acidified normal serum and patient cells; and tube 3, containing
ysis associated with PNH. acidified patient serum and patient cells.
170. Chapter 8 Aplastic Anemia

Fresh normal serum

Patient’s serum

Heat—inactivated normal serum

0.2 N HCl

50% Patient’s red cells

50% Normal red cells

Pattern of lysis in positive test Trace

CD55 CD59
300 300

240 240

S 180 Sa 100
PNH 8 8
S 120 | M1 8 120 | M1

60 60

0 0
100 7) 10%. "O02 9 "102% ~ 104 10° 101 102 103 104
A Log fluorescence B Log fluorescence

300 300

240 240

S 180 § 180
Normal & 8
S 120 M1 S 120 M1

60 60

0 0
10° 10! 10? 103 102 10° 10! 10° 108 104
C Log fluorescence D Log fluorescence

Figure 8-16 @ Flow cytometry diagnosis of PNH. The flow cytometric histograms from a patient with PNH (A, B) show decreased expression
of CDSS (A, arrow) and CD59 (B, arrow) in addition to populations of red cells containing relatively normal levels of CD55 and CD59. For
comparison, a normal control showing single red cell populations for both CD55 (C) and CD59 (D). (From Smith, LJ: Paroxysmal nocturnal
hemoglobinuria. Clin Lab Sci 17:172, 2004, with permission.)
 Chapter 8 Aplastic Anemia ‘1171

with the severity of disease. Flow cytometry may also be per-

formed on granulocytes, but is technically more challenging. following conditioning with cyclophosphamide to deplete
Flow cytometry has the added advantage of detecting very the patient’s immune system. The patient had one bout
small numbers of PNH clones (1% to 5% deficient cells) and of acute graft-versus-host disease approximately 35 days
after transplantation that was successfully treated with
is a sensitive manner to detect early PNH, as well as emer-
cyclosporine therapy. Her counts | year after the transplan-
gence of PNH clones in aplastic anemia. Flow cytometry is
tation showed a hematocrit of 42%, WBC of 6.0 x 10°/L,
also useful after a recent hemolytic episode when the number and platelets of 210 < 10°/L. The WBC differential showed
of abnormal red cells may be decreased.53? 71% neutrophils, 21% lymphocytes, 6% monocytes, and
Treatment of PNH is usually supportive, but develop- 2% eosinophils. A bone marrow biopsy specimen showed a
ment of specific inhibitors of complement may aid in manage- bone marrow cellularity of 50%, with all cell lineages pre-
ment of these patients. Bone marrow transplant is considered sent and maturing normally. The patient has had no further
curative, but patients often have complicating factors related episodes of graft-versus-host disease, bleeding, or infection.
to long-term transfusions and comorbid conditions that make
transplantation difficult. Gene therapy, allowing for transduc- QUESTIONS
tion of hematopoietic stem cells with a functional PIG-A 1. What is the most likely diagnosis?
gene, or autotransplantation using the patient’s stem cells, 2. What are possible etiologies of this disorder?
which have been sorted to select those that do not express the 3. What is the clinical approach to therapy?
PNH phenotype (as determined by levels of GPI-anchored
proteins), may be possible future approaches. Usually PNH is ANSWERS
considered to be a chronic disease. Many patients survive
20 to 40 years after diagnosis, although average survival is . Aplastic anemia
2. Anumber of causes of aplastic anemia including drugs,
10 years. The most common cause of death is thromboem-
infections, irradiation, and chemical exposures are iden-
bolism. Some patients with bone marrow hypoplasia may suf-
tified, but most cases are idiopathic and are thought to
fer from severe infections or hemorrhage. Some patients may arise secondary to immune destruction of bone marrow
develop clinical remission, and a small number of patients precursors. A minority of cases may be congenital.
may progress to acute myelogenous leukemia.°° 3. Treatment with immune suppression or bone marrow
transplant are most likely approaches. If a patient does
not respond to immune suppression and has a bone mar-
row donor available, most will be transplanted as this is

Case Study 1 the patient’s best chance for cure.

A 20-year-old woman was seen by her physician for fatigue,

pallor, and easy bruising. Physical examination was unre-
markable except for pallor, widespread petechiae, ecchymo-
sis, and bleeding gums. The spleen was not enlarged. There

Case Study 2
was no history of recent exposure to drugs, toxins, or radia-
tion. There was no history of any other illness, and she had
been in good health until the past 2 weeks.
Laboratory data revealed a normochromic, normocytic A 43-year-old man presented to his physician with com-
anemia with a hematocrit of 20%, WBC of 20 x 10°/L, and plaints of lower back pain, fatigue, easy bruising, and a sud-
platelets of 20 X 10°/L. The peripheral blood showed den onset of dark urine on arising in the morning. Laboratory
9% neutrophils, 90% lymphocytes, and 1% monocytes. The studies and a bone marrow aspirate were ordered by his
corrected reticulocyte count was 0.5%. A bone marrow aspi- physician. The CBC revealed an anemia (hematocrit, 28%),
rate and biopsy specimen was markedly hypocellular, with leukopenia (white blood cell [WBC] count 33 x 10°/L), and
less than 5% cellularity composed of lymphocytes and thrombocytopenia (platelets, 76 * 10°/L). The reticulocyte
plasma cells admixed with a rare myeloid or erythroid precur- count was 5.1%, (3.2% corrected). The RBCs showed mod-
sor. No megakaryocytes were noted. No dysplastic changes erate polychromasia, slight anisocytosis, and poikilocytosis,
were noted in the few hematopoietic cells seen, and there was and | nucleated RBC per 100 WBCs. The chemistry profile
no increase in the number of blasts. Further studies failed to was normal except for an increased lactate dehydrogenase
identify an underlying etiology for the patient’s pancytopenia. value and an increased indirect bilirubin. The bone marrow
The patient was followed for the next 3 months with no reso- analysis revealed a hypercellular marrow with relative ery-
lution of her symptoms and no improvement in her blood throid hyperplasia. There were adequate numbers of myeloid
count. She received two transfusions of red blood cells as a and platelet precursors.
result of worsening anemia. Further studies were performed after initial test results
The patient’s family was tested, and she was found to were evaluated. The urinary hemosiderin, sugar water test,
have a sister who was HLA compatible. The sister had and the Ham’s test were all positive. Flow cytometry
a collection of peripheral stem cells for an allogeneic trans- showed a population of red cells with decreased expression
plantation. The stem cells were infused into the patient of CD55 and CD59.
continued continued
172 Chapter 8 Aplastic Anemia

Case Study 2-conva ™ J | .

|

QUESTIONS
te What is the most likely diagnosis?
2 . What type of hemolysis is evident in this patient?
3: If the granulocytes in this patient were scored for LAP
activity, what would you expect the result to be?
4. What is the clinical outcome in this patient?

ANSWERS
. Paroxysmal nocturnal hemoglobinuria.
. Intravascular hemolysis.
. LAP would be expected to be decreased.
. Clinical outcome is variable but patients often have sig-
BRWN

nificant anemia and are at increased risk for venous

thrombosis, bleeding, or infection. Often this is a
chronic disorder treated with supportive care, with sur-
vival of more than 10 years.

Questions
k How isaplastic anemia best defined? ae Always causes fatal, severe aplastic anemia
a. A condition in which bone marrow production of = d. May cause a spectrum of disease that may not manifest
cells, white blood cells, and platelets has failed until several years following the benzene exposure
b. A condition in which severe anemia is seen
6. Which of the following statements about chloramphenicol-
c. A condition in which platelets are decreased
induced aplastic anemia is true?
d. A condition in which there is pancytopenia with a
a. The onset of the bone marrow aplasia is not pre-
hypercellular bone marrow
dictable by the dose or the duration of drug exposure
. Which is the most common cause of aplastic anemia? b. Low doses of chloramphenicol never lead to aplastic
a. Drug ingestion anemias
b. Toxin exposure c. People who receive chloramphenicol are 500 times
c. Idiopathic or unknown more likely to develop aplastic anemia than the gen-
d. lonizing radiation eral population
. Which of the following represents the most complete list d. The longer a patient receives chloramphenicol, the more
of etiologies causing aplastic anemia? likely it is that he or she will develop aplastic anemia
a. Secondary and congenital . Which of the following drugs is not associated with the
b. Idiopathic and congenital development of aplastic anemia?
c. Secondary and idiopathic a. Chloramphenicol
d. Secondary, idiopathic, and congenital b. Phenylbutazone
. Which of the following has not been associated with an c. Aspirin
acquired type of aplastic anemia? d. Chemotherapeutic agents
a. lonizing radiation . lonizing radiation causes aplastic anemia by which of
b. Increased chromosomal breakage the following mechanisms?
c. Chemical agents a. A dose-dependent destruction of bone marrow stem
d. Drugs cells
. The aplastic anemia associated with benzene exposure is b. An idiosyncratic delayed development of bone
characterized by which of the following statements? marrow aplasia
a. Always occurs while the exposure to benzene is c. Disruption of chemical bonds to form free radicals
occurring that damage bone marrow cells
b. Is always irreversible d. All of the above
 Chapter 8 Aplastic Anemia 173

. What is the most common congenital disorder associated 16. Which statement best describes paroxysmal nocturnal
with aplastic anemia? hemoglobinuria?
a. Fanconi’s anemia a. Acquired hemolytic anemia associated with cellular
b. Thrombocytopenia-absent radius (TAR) syndrome membrane abnormalities
c. Congenital dyserythropoietic anemia, type | b. Congenital hemolytic anemia associated with the
d. Diamond—Blackfan anemia inflammatory response
10. Which of the following is not seen in the peripheral c. A premalignant condition that almost always results in
blood of patients with aplastic anemia? development of acute leukemia.
a. Normochromic, normocytic anemia d. A common disorder that frequently resolves with time.
b. Increased reticulocyte count . What causes the red cell defect of PNH?
c. Relative lymphocytosis a. Rare red cell antigens
d. Decreased neutrophils b. Lack of GPI-anchored proteins on the erythrocyte
ke What is the appearance of the bone marrow in aplastic membrane
anemia? c. Excessive amounts of complement components
a. Hypercellular Ca 10. C9
b. Normocellular d. Glucose-6-phosphate dehydrogenase enzyme
c. Hypocellular deficiency
d. Fibrotic - 18. Which of the following is a correct description of the
|W, The differential diagnostic considerations for bone mar- sugar water test (sucrose hemolysis test)?
row hypoplasia do not include which of the following a. PNH cells are lysed by complement after exposure to
disorders? low-ionic-strength sugar water.
a. Severe malnutrition b. PNH cells are lysed by antibody and complement
b. Myeloproliferative disorder after heating to 56°C in sugar water solution (5%).
c. Aplastic anemia c. Patient’s serum is acidified to enhance complement
d. Recent chemotherapy binding and lysis of patient cells.
d. Patient’s serum is heat-inactivated and treated with
is What is the treatment of choice for severe aplastic ane- HCl; complement is added; patient cell lysis occurs.
mia in patients who are younger than age 50?
a. Multiple transfusions 19) What is a correct description of Ham’s test (acidified
b. Androgens serum lysis test)?
c. Bone marrow transplantation a. PNH cells are lysed by complement after exposure to
d. Erythropoietin therapy low-ionic-strength sugar water.
b. PNH cells are lysed by antibody and complement
14. What is the definition of pure red cell aplasia? after heating to 56°C in sugar water solution (5%).
a. Lack of hematopoietic precursors in the bone marrow c. Patient’s serum is acidified to enhance complement
b. Abnormal, giant normoblasts in the bone marrow binding and lysis of patient cells.
c. Lack of erythroid precursors with normal white blood d. Patient’s serum is heat inactivated and treated with
cell and megakaryocytic precursors HCI; complement is added; patient cell lysis occurs.
d. Dysplastic red cell precursors with normal white cell
. The basis of the flow cytometric test for diagnosis of
and megakaryocytic precursors
PNH is which of the following?
LS: What features do the congenital dyserythropoietic ane- a. PNH will have increased amounts of complement
mias (CDAs) have in common? detected on the cell surface.
a. Anemia, microcytosis, erythroid hyperplasia, and b. PNH cells are easily lysed and will show decreased
abnormal erythroblasts number when analyzed.
b. Anemia, erythroid hyperplasia with abnormal c. An affected patient will show decreased levels of
erythroblasts, and indirect hyperbilirubinemia CD55 and CD59 binding in a subset of cells.
c. Anemia, lysis in acidified serum, and indirect hyper- d. All of the patient cells will show decreased levels of
bilirubinemia GPI-anchored proteins on the erythrocyte membrane.
d. Anemia, macrocytosis, erythroid hyperplasia with
abnormal erythroblasts, and indirect hyperbilirubinemia See answers at the back of this book.
174. Chapter 8 Aplastic Anemia

m= Bone marrow examination is required for diagnosis of

aplastic anemia to document bone marrow hypocellularity
and exclude other causes (i.e., metastatic tumor) for pan-
w Aplastic anemia is defined as a failure of bone marrow and cytopenia.
loss of bone marrow cellularity leading to decreased pro-
w Aplastic anemia in patients younger than 50 years of age
duction of erythrocytes, leukocytes, and platelets, and
is usually treated by bone marrow transplantation.
development of peripheral blood cytopenias.
Patients unsuitable for transplantation therapy may be
w Clinical criteria defining aplastic anemia include a bone treated with supportive transfusion therapy, immunomod-
marrow cellularity of less than 25% and at least two ulatory therapy, or androgens.
peripheral blood cytopenias.
w Pure red cell aplasia is defined as a selective loss of bone
w Aplastic anemia usually arises as a result of acquired dam- marrow red cell precursors and presents clinically as an
age, although rare cases are hereditary. The underlying isolated hypoproliferative anemia. Causes of pure red cell
cause of aplastic anemia is idiopathic, or unknown, in the aplasia include infections (parvovirus B19), reactions to
majority of cases. drugs, and immune abnormalities. A congenital form is
mw Of those cases of aplastic anemia in which an etiologic recognized, called Diamond—Blackfan anemia.
agent can be identified, drugs, chemicals (especially m= Congenital dyserythropoietic anemias (CDAs) are
benzene-type compounds), irradiation, and some infec- familial or inherited anemias that are characterized by
tions have been implicated as causes of aplastic anemia. ineffective erythropoiesis, bone marrow erythroid hyper-
w Drug-induced aplastic anemia is usually an idiosyncratic plasia, and bizarre erythroid precursors with multiple
(not dose-related) reaction; the most commonly impli- nuclei and intranuclear chromatin bridges.
cated drugs are chloramphenicol and phenylbutazone. w Type 2 CDA is associated with an abnormal lysis in acid-
w lonizing radiation usually causes bone marrow aplasia in ified serum (Ham’s test). It is also known as HEMPAS
an immediate dose-dependent manner, although late (hereditary erythroblast multinuclearity with positive acid
effects may also be seen. serum test) and is inherited in an autosomal recessive
w Infections and altered or autoimmune states may also fashion.
cause bone marrow aplasia, probably as a result of m Paroxysmal nocturnal hemoglobinuria is an acquired
immune destruction of the bone marrow hematopoietic hemolytic anemia resulting from a hematopoietic stem
precursor or stem cell. cell abnormality causing decreased levels of GPI-
w The congenital form of aplastic anemia, Fanconi’s anemia, anchored proteins on the cells surface. The lack of some
is characterized by an autosomal recessive inheritance of these proteins leads to increased sensitivity to comple-
pattern and progressive development of pancytopenia and ment lysis of red cells.
bone marrow hypoplasia as well as an increased incidence mw Diagnosis of PNH is made utilizing the sugar water test,
of acute leukemia and other malignancies. Ham’s test and flow cytometric analysis for CD55 and
mwFanconi’s anemia is characterized at a molecular and CD59.
cytogenetic level by increased chromosomal breakage and m There appears to be an interrelationship between aplastic
defective DNA repair. anemia, PNH, and other bone marrow failure syndromes.
# Blood findings in aplastic anemia include normochromic, Many patients with aplastic anemia show hematopoietic
normocytic anemia with low numbers of reticulocytes clones that have a PNH phenotype, suggesting the possi-
(hypoproliferative anemia), leukopenia (especially of bility of a pathophysiologic relationship between these
myeloid and monocytic cells) and thrombocytopenia. disorders.
Lymphocyte counts may be normal or decreased.

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review of literature. Hematology 7:238, induced pure red cell aplasia. Pharma- uria and aplastic anaemia: Increasing
2002. cotherapy 16:1002, 1996. evidence for overlap of haemopoietic
28. Sanders, JE: Chronic graft-versus-host 46. Masuda, M, et al: Pure red cell aplasia defect. Transfus Med 13:377, 2003.
disease and late effects after hematopoi- with thymona: evidence of T-cell clonal 62. Krauss, JS: Laboratory diagnosis of
etic stem cell transplantation. Int J disorder. Am J Hematol 54:324, 1997. paroxysmal nocturnal hemoglobinuria.
Hematol 76(Suppl 2):15, 2002. 47. Ward, JH: Autoimmunity in chronic lym- Ann Clin Lab Sci 33:401, 2003.
. Comerci, GD: Medical complications of phocytic leukemia. Curr Treat Options 63. Parker, C, et al: Diagnosis and manage-
anorexia nervosa and bulimia nervosa. Oncol 2:253, 2001. ment of paroxysmal nocturnal hemoglo-
Med Clin North Am 74:1293, 1990. A8. Go, RS, et al: Aplastic anemia and pure binuria. Blood 106:3699, 2005.
. Smith, OP: Shwachman-Diamond syn- red cell aplasia associated with large 64. Meyers, G, and Parker, CJ: Management
drome. Semin Hematol 39:95, 2002. granular lymphocyte leukemia. Semin issues in paroxysmal nocturnal hemoglo-
. Tischkowitz, MD, and Hodgson, SV: Hematol 40:196, 2003. binuria. Int JHematol 77:125, 2003.
Fanconi anaemia. J Med Genet 40:1, 2003. 49. Willig, TN, et al: Diamond-Blackfan 65. Leach, A, and Gaumer, H: Diagnosis and
. Collins, N, and Kupfer, GM: Molecular anemia. Curr Opin Hematol 7:85, 2000. detection of PNH using GPI anchored
pathogenesis of Fanconi anemia. Int J . Wickramasinghe, SN, and Wood, WG: proteins. Clin Lab Sci 9:91, 1996.
Hematol 82:176, 2005. Advances in the understanding of the
. Kennedy, RD, and D’ Andrea, AD: The congenital dyserythropoietic anaemias.
Fanconi anemia/BRCA pathway: New Br JHaematol 131:431, 2005.
 Chapter ‘
Se

Introduction to
Hemolytic Anemias
Intracorpuscular Defects:
I. Hereditary Defects of the Red Cell
Membrane
Théresa L. Coetzer, PhD
Stan Zail, MBBCh, MD, FRCPath

Classification of Hemolytic OBJECTIVES

Anemias
At the end of this chapter, the learner should be able to:
Approach to Diagnosis of a
Hemolytic State 1. Define intracorpuscular and extracorpuscular red cell defects as related to hemolytic
Establishing the Presence of processes.
Hemolysis . List laboratory tests that reflect increased red cell destruction.
Establishing the Cause of
Hemolysis . Calculate a reticulocyte production index.

Hereditary Defects of the . Name laboratory tests that help to classify the cause of red cell hemolysis.
Red Cell Membrane
. Identify the red cell membrane abnormalities associated with hereditary spherocytosis.
Red Cell Membrane
Structure . Recognize abnormal laboratory results associated with hereditary spherocytosis.
Classification
. Name the functional abnormalities affecting membrane skeleton proteins in
Aa
oy
6b
Go
—
Hereditary Spherocytosis
hereditary elliptocytosis.
Case Study 1
Hereditary Elliptocytosis 8. Recall laboratory findings associated with hereditary elliptocytosis.
Clinical Phenotypes 9. Identify the abnormalities that cause the severe fragmentation and microspherocytosis
Case Study 2 characteristic of hereditary pyropoikilocytosis.

Disorders of Membrane 10. List disorders of membrane cation permeability.

Cation Permeability
Hereditary Stomatocytosis
and Hereditary
Xerocytosis
Case Study 3

176
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: I. Hereditary Defects of the Red Cell Membrane —-177

Classification of Hemolytic Anemias Approach to Diagnosis

A hemolytic state exists when the in vivo survival of red cells of a Hemolytic State
is shortened. The presence of anemia in an individual patient The approach to diagnosis of a hemolytic state initially
is, however, dependent on the degree of hemolysis and the involves establishing the fact that the rate of red cell destruc-
compensatory response of the erythroid elements of the bone tion 1s increased and then focuses on determining the cause of
marrow. Normal bone marrow is able to increase its output hemolysis.
about six- to eightfold, so that anemia is not manifest until
this capacity is exceeded, corresponding to a red cell life span
of about 15 to 20 days or less. Anemia may, however, occur Establishing the Presence of Hemolysis
with more moderate shortening of the red cell life span if
Diagnostic tests used to establish the presence of hemolysis
there is an associated depression of bone marrow function,
rely on the fact that hemolysis is characterized by both in-
which may occur with certain systemic diseases or exposure
creased cell destruction and increased production.
to chemicals or drugs.
A useful classification of the hemolytic anemias
TESTS REFLECTING INCREASED RED CELL
entails their subdivision into those disorders associated
DESTRUCTION
with an intrinsic (intracorpuscular) defect of the red cell
The most frequently used tests in this category are the serum un-
and those associated with an extrinsic (extracorpuscular)
conjugated (indirect) bilirubin and serum haptoglobin determi-
abnormality. Red cells from a patient with an intracorpus-
nations. The serum unconjugated bilirubin level seldom exceeds
cular defect have a shortened survival in both the patient
3 to 4 mg/dL in uncomplicated hemolytic states and reflects the
and a normal recipient, whereas normal donor red cells sur-
catabolism of heme derived from red cells phagocytosed by the
vive normally in the patient. In contrast, normal red cells
reticuloendothelial system (Fig. 9-1). The test is, however, rel-
are destroyed more rapidly when transfused into a patient
atively insensitive, as is the measurement of fecal stercobilino-
with an extracorpuscular abnormality. The patient’s red
gen and urine urobilinogen that represents further stages in
cells, when transfused into a healthy recipient, have normal
the degradation of unconjugated bilirubin by the liver (see
survival, provided that they have not been irreversibly
Fig. 9-1). Because the unconjugated bilirubin is bound to albu-
damaged. Hemolyti¢ states have also traditionally been
min jf cannot pass the glomerular filter, and the jaundice is said
regarded as intravascular or extravascular; that is, seques-
to be “acholuric.” On the other hand, a decreased serum hapto-
tration occurs in reticuloendothelial tissue. However, vigor-
globin level is a very sensitive test of both intravascular and
ous extravascular hemolysis may often be associated with
extravascular hemolysis, and reflects the rapid clearance by the
signs of hemoglobin release into the plasma such as hemo-
reticuloendothelial system of a complex formed between liber-
globinemia and decreased haptoglobin levels. The distinc-
ated hemoglobin and circulatory haptoglobin. Drawbacks to
tion still is useful from a clinical standpoint because certain
the use of serum haptoglobin levels are that low levels may
hemolytic states are associated with predominantly intravas-
occur in hepatocellular disease, reflecting decreased synthesis
cular hemolysis (e.g., paroxysmal nocturnal hemoglobinuria
by the liver, and that some individuals, particularly in black
and infections caused by Clostridium or Plasmodium
populations, may have a genetically determined deficiency of
falciparum).
haptoglobin. Increased synthesis of haptoglobin in acute inflam-
Hemolytic anemias may be classified as follows:
matory states or malignancy may also mask depletion of serum
1. Intracorpuscular defects haptoglobin caused by hemolysis.
a. Hereditary defects Other tests that reflect increased red cell destruction, par-
(1) Defects in the red cell membrane ticularly if it is primarily intravascular, are those that test for
(2) Enzyme defects the presence of hemoglobinemia, hemoglobinuria, and hemo-
(3) Hemoglobinopathies siderinuria. The assessment of hemoglobinemia requires
(4) Thalassemia syndromes stringent precautions in the prevention of hemolysis during
b. Acquired defects blood collection. Once the hemoglobin-binding capacity of
(1) Paroxysmal nocturnal hemoglobinuria serum haptoglobin is exceeded, hemoglobin passes through
2. Extracorpuscular defects the glomerulus as alpha—beta (a®8) chain dimers and reassoci-
a. Immune hemolytic anemias ates to a,f, tetramers in the tubule, where the hemoglobin is
b. Infections reabsorbed and degraded. The liberated iron is conserved as
c. Exposure to chemicals and toxins ferritin and hemosiderin. When the tubular reabsorptive
d. Exposure to physical agents capacity for hemoglobin is exceeded, hemoglobinuria ensues
e. Microangiopathic and macroangiopathic hemolytic and is detectable by either spectroscopic examination or com-
anemias mercially available dipsticks that detect heme. Staining of the
f. Splenic sequestration (hypersplenism) urine sediment for iron (e.g., with Prussian blue) will detect
g. General systemic disorders (in which hemolysis is not the hemosiderin- and ferritin-containing renal tubular cells
the dominant feature of the anemia) that are sloughed several days after a hemolytic episode.
178 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

R.E. CELL
Serum
Fe transferrin
Hb Globin Amino acid
Bilirubin pool

Hb

Bilirubin-albumin
Hb-dimer Met-Hb Hb-haptoglobin (unconjugated)

Ferri-heme + globin

Heme-
>
hemopexin
Methem-albumin ——~>

|
|
Hemoglobinuria
Hemosiderinuria
Urobilinogenuria

Figure 9-1 Diagrammatic representation of the degradation of hemoglobin after intravascular or extravascular destruction of red cells.
Fe = iron; Hb = hemoglobin; RBC = red blood cell; R.E. = reticuloendothelial cell.

Some of the free plasma hemoglobin may be oxidized to normal maturation time of | day, again leading to a falsely
methemoglobin with subsequent dissociation of ferriheme, elevated reticulocyte count. These cells (so-called shift retic-
which combines with albumin to form methemalbumin. ulocytes) are recognizable as large bluish-gray erythrocytes
Methemalbumin can be detected spectroscopically by the on Romanowsky (Wright’s, Giemsa) stains.
Schumm’s test. This test is relatively insensitive and is seldom The reticulocyte production index (RPI) corrects the
positive in mild hemolytic states. In routine practice, determi- hematocrit to a normal value of 45% and takes into account
nation of red cell survival using Cr°'-labeled red cells is seldom the maturation time of the reticulocyte at a particular hemat-
required to document an increased rate of red cell destruction. ocrit (approximately 1.0 day at a hematocrit of 45%, 1.5 days
The fate of hemoglobin when processed intravascularly or at 35%, 2.0 days at 25%, and 2.5 days at 15%).!
extravascularly is shown diagrammatically in Figure 9-1.
RPI = % Reticulocytes xX Hematocrit
Reticulocyte maturation time 45
TESTS REFLECTING INCREASED RED CELL
PRODUCTION For example, an RPI of 5.3 is calculated for a patient
The compensatory bone marrow response to hemolysis suspected of having a hemolytic state with the following
results in the delivery of young red cells in the form of retic- indices: hemoglobin, 12.0 g/dL; hematocrit, 36%; reticulo-
ulocytes into the circulation. These young cells contain RNA, cyte count, 10%; shift cells present.
which stains supravitally with dyes such as new methylene An RPI of greater than 2.5 to 3.0 is generally regarded as
blue or brilliant cresyl blue. The normal reticulocyte count indicative of a hemolytic state, but it is very important to
has a range of 0.5% to 2.0%, reflecting the fact that each day exclude the presence of hemorrhage in a particular patient, as
approximately 1% of the red cell mass is destroyed and this too may lead to an elevated RPI. Although the RPI is
replaced by young red cells from the bone marrow, because probably the single most useful test to detect a hemolytic
red cell survival is approximately 120 days. The reticulocyte state, a cautionary note is in order, as the test may not be sen-
count is always elevated in a hemolytic state in which there is sitive enough to detect mild hemolytic states (see Chap. 31).
a normal compensatory bone marrow response. However, a
more accurate assessment of red cell production is required,
Establishing the Cause of Hemolysis
because the percentage of reticulocytes may be “spuriously”
elevated as the reticulocytes may be diluted into a lesser Once having documented the presence of hemolysis, it is our
number of total circulating red cells. In addition, in response experience that the approach followed by Lux and Glader? in
to the anemia, reticulocytes may leave the bone marrow pre- establishing the cause of hemolysis is pragmatic and logical,
maturely and mature in the circulation for longer than the and this is the technique that is followed in this chapter and is
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane yas

_—. Cell surface area-to-volume ratio

ESTABLISH PRESENCE OF HEMOLYSIS
ated g=t-ty-fo Ml5}={Ome (-ty tal leitlola 2. The viscoelastic properties of the membrane, which depend
ated g=t-KJ-10 Hla1310M ofKololtle t(o)]
on the structural and functional integrity of the membrane
skeleton
3. The cytoplasmic viscosity, which is determined primarily
by hemoglobin.

POSITIVE The structural organization of the protein and lipid compo-

nents of the red cell membrane has been reviewed in Chap-
49
hemolytic ter 3, and only some aspects of the membrane proteins
implicated in the pathogenesis of hemolytic anemia are
emphasized here.
The red cell membrane skeleton underlies the lipid bilayer
and is a loosely knit two-dimensional protein network consist-
ing mainly of the structural proteins a- and B-spectrin, actin,
and protein 4.1.* Negatively stained stretched skeletons viewed
via high-resolution electron microscopy* reveal a hexagonal
LABORATORY INVESTIGATIONS lattice of predominantly spectrin tetramers with some hexam-
Appropriate for RBC morphology ers joined together by junctional protein complexes (Fig. 9-3).
These junctional complexes are composed of protein 4.1, short
F-actin filaments, as well as the minor actin-binding proteins
DEFINIT!VE DIAGNOSIS adducin, dematin (protein 4.9), tropomyosin, and tropomod-
ulin.’ The skeleton is linked to the lipid bilayer by interactions
Figure 9-2 ™ Diagnostic approach to hemolytic anemias. with the integral membrane proteins band 3 and glycophorin C.
The primary attachment occurs via a high-affinity interaction
with ankyrin, which binds to both band 3 and B-spectrin.® Pro-
shown diagrammatically in Figure 9-2. The initial step con- tein 4.2 stabilizes this complex. Secondary attachment sites of
sists of separating patients into Coombs test—positive (i.e., lower affinity are provided by interactions between band 3 and
immunohemolytic anemias) and Coombs _ test—-negative protein 4.1,’ as well as among glycophorin C, protein 4.1, and
groups. The latter group is then further divided into “smear- protein p55.° Spectrin and protein 4.1 also interact directly with
positive” and “‘smear-negative/nonspecific” subgroups. It is the lipid bilayer.
fundamentally important to assess morphology in peripheral Red cell spectrin, the major skeletal protein, is an elon-
smears that are free of artifact. On the basis of the classifica- gated flexible heterodimer composed of stoichiometric amounts
tion according to the predominant morphology associated of two structurally related, but functionally distinct, a and
with a particular disease state (Box 9-1), it is possible to con- 8 polypeptides that are encoded by separate genes."'' Both a-
siderably narrow the differential diagnosis and then institute and B-spectrin can be subdivided into structural domains (al to
further appropriate tests to make a definitive diagnosis. aV and BI to BIV, respectively) that are resistant to mild prote-
It is also worth emphasizing that many hemolytic states olysis by trypsin’? (Fig. 9-4A). Each spectrin molecule consists
are associated with an underlying disease, as will become ap- of several 106-amino-acid repeats that fold into coiled triple
parent in the ensuing chapters, and this should be considered helices.'*'* Repeat 10 of a-spectrin is atypical and represents
in the assessment of the individual patient. an SH3 ___domain, whereas the C-terminal has an EF hand
motif.'° The N-terminal of B-spectrin contains binding sites
for protein 4.1, actin, and adducin, and repeat 15 binds
Hereditary Defects of the Red Cell ankyrin”"'! (see Fig. 9-4A). The a and B monomers interact
Membrane along their length in an antiparallel fashion starting at
nucleation sites at the tail end of the molecules.'° The head
Red Cell Membrane Structure
regions of the aB heterodimers self-associate into tetramers
An understanding of the etiology and pathophysiology of via an interaction between the two helices from repeat 17 of
hemolytic states caused by defects of the red cell membrane the BI ___domain at the phosphorylated C-terminal, and a third
requires some knowledge of its structural organization. The helix from the N-terminal repeat | of the al ___domain to
membrane consists of a relatively fluid lipid bilayer stabilized form a complete triple helix.'° An important aspect in
by interactions with integral membrane proteins within the the biogenesis of the membrane skeleton is that a-spectrin
bilayer and with the underlying membrane protein skeleton. is synthesized in approximately threefold excess over
The membrane provides the red cell with the necessary strength B-spectrin'’ and undergoes slower degradation,'* indicating
and flexibility to survive the circulatory shear stress and numer- that 6-spectrin is the rate-limiting component in spectrin
ous passages through the spleen during its 4-month life span. assembly.
The ability of the red cell to deform and to subsequently regain Red cell band 3, the major integral membrane protein, is
its original biconcave disc shape is determined by three factors: the anion exchanger.'’ It performs an important transport
180 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

" Box 9-1 Predominant Red Cell Morphology Commonly Associated with Nonimmune
Hemolytic Disorders
j

Spherocytes Prominent Basophilic Stippling

Hereditary spherocytosis Thalassemias
Acute oxidant injury (HMP shunt defects during hemolytic Unstable hemoglobins
crisis, oxidant drugs and chemicals) Lead poisoning
|
Clostridium welchii septicemia Pyrimidine-5 -nucleotidase deficiency
Severe burns, other red cell thermal injuries Spiculated or Crenated Red Cells
| Spider, bee, and snake venoms Acute hepatic necrosis (spur cell anemia)
Severe hypophosphatemia Uremia
|

Elliptocytes Infantile pyknocytosis

|

Hereditary elliptocytosis Abetalipoproteinemia

Thalassemias McLeod blood group
Iron deficiency Target Cells
Megaloblastic anemia Hemoglobins S, C, D, and E
|
Bizarre Poikilocytes Thalassemias
Red cell fragmentation syndrome (microangiopathic and Hereditary xerocytosis
macroangiopathic hemolytic anemias) Nonspecific or Normal Morphology
Hereditary elliptocytosis in neonates Embden—Meyerhof pathway defects
Hereditary pyropoikilocytosis HMP shunt defects
Stomatocytes Adenosine deaminase hyperactivity with low red cell ATP
Hereditary stomatocytosis and related disorders Unstable hemoglobins
Stomatocytic elliptocytosis Paroxysmal nocturnal hemoglobinuria
Irreversibly Sickled Cells Dyserythropoietic anemias
Sickle cell anemia Copper toxicity (Wilson’s disease)
Symptomatic sickle syndromes Cation permeability defects
Intraeryihrocytic Parasites Erythropoietic porphyria
Malaria Vitamin E deficiency
Babesiosis Hypersplenism
Bartonellosis
|ATP = adenosine triphosphate; HMP = hexose monophosphate.

function by regulating HCO;/Cl exchange and facilitating proteins, as well as to myosin, and the N-terminal 30-kD
the transfer of carbon dioxide from tissues to lungs. It is divided ___domain interacts with glycophorin C, p55, and band 3. The pro-
into two structurally and functionally distinct domains (see tein 4.1 mRNA is subject to extensive alternative splicing, yield-
Fig. 9-4B). The 43-kD N-terminal cytoplasmic ___domain contains ing tissue- and development-specific isoforms.”' Posttranslational
binding sites for ankyrin and proteins 4.1 and 4.2, and also binds deamidation of asparagine 502 occurs during development
glycolytic enzymes and hemoglobin. The 52-kD C-terminal of reticulocytes into mature red cells, and these two forms are
region has 14 transmembrane segments intercalated in the lipid designated protein 4.1a and b.
bilayer that form the anion-exchange channel.'” The genes coding for the major membrane proteins
Ankyrin is the major connecting protein that links the have been cloned and sequenced and their chromosomal
membrane skeleton to the bilayer.*’ It is a pyramid-shaped localization identified (Table 9-1). Mutations in any of
protein with an N-terminal 89 kD band 3-binding ___domain, these genes that alter the amount or function of the
formed by 24 tandem repeats containing 33 amino acids each expressed proteins compromise the integrity of the mem-
(see Fig. 9-4C). Spectrin binds to the central 62-kD ___domain brane and manifest in altered red cell morphology. These
of ankyrin, whereas the 55-kD C-terminal portion is a regula- cells are unable to survive passage through the spleen,
tory ___domain that is alternatively spliced at the mRNA level, resulting in hemolytic anemia.
yielding ankyrin isoforms, which modulate the interaction
with spectrin and band 3.
Classification
Protein 4.1 is a globular protein with four structural
domains as depicted in Figure 94D.' A 10-kD ___domain The hereditary hemolytic anemias due to red cell mem-
enhances the spectrin—actin interaction by binding to both brane protein defects may be classified according to the
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 18]

Figure 9-5 @ Transmission electron micrographs of negatively stained red blood cell membrane skeletons. A. An area of spread skeleton net-
work. B, C. The hexagonal lattice made up of spectrin tetramers (Sp4), hexamers (Sp6), or double tetramers (2Sp4). Cross-linking
junctional complexes contain short F-actin filaments and protein 4.1. Globular ankyrin structures are bound to spectrin filaments about
80 nm from their distal ends. (From Liu, SC, et al: Visualization of the hexagonal lattice in the erythrocyte membrane skeleton. | Cell Biol
104:527, 1987, with permission.)

Molecular
Mass* Chromosomal
Protein Localization Diseases

Skeletal Proteins
a-Spectrin 1q21 > q23 HS, HE, HPP
6-Spectrin 14q23 > q24.2 jay, JENE, Nae
Protein 4.1 ; 1p33 > p34.2 HE

Integral Proteins
Band 3 17q21 > q22 HS, SAO
Glycophorin C 2q14 > q21 HE

Linker Proteins
Ankyrin : 206 8p11.2
Protein 4.2 : 77 15q15 > q21

*Proteins were separated via SDS-PAGE and stained with Coomassie blue or periodic acid—Schiff reagent (PAS).
‘Molecular weight is calculated from the amino acid sequence and expressed as kilodaltons (kD).
HS = hereditary spherocytosis; HE = hereditary elliptocytosis; HPP = hereditary pyropoikilocytosis;
SAO = Southeast Asian ovalocytosis.
182 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

!
a Spectrin
21

NH> all 46 kD | alll 52 kD aV 41 kD | COOH

| EF hand
Spectrin self-association

B Spectrin
17

[
pi2eKo|— BILGSkD pils3kD |
Spectrin Ankyrin Protein 4.1
self-association Actin
Adducin
A

Band 3
14 Transmembrane
Cytoplasmic ___domain segments Cytoplasm

NH» 43 kD 52 kD COOH

Ankyrin, 4.1, 4.2

Glycolytic enzymes
B Hemoglobin

Ankyrin

NH> 89 kD 62 kD 55 kD COOH

Cc Band 3 Spectrin

Protein 4.1

NH> 30 kD

Glycophorin C Spectrin
Band 3 Actin
D p55 Myosin

Figure 9-4 @ Schematic representation of the structure of four of the red blood cell membrane proteins involved in hereditary hemolytic
anemias. The molecular masses of the various domains are given in kilodaltons (kD). A. Spectrin. The subunit structure of a- and B-spectrin,
showing the antiparallel arrangement of the N- and C-terminals, the homologous triple helical spectrin repeat units (indicated above the a
and chains), and the trypsin-resistant domains (open squares). a-Spectrin has five domains (al to aV) and 21 repeats. B-Spectrin has four
domains (8! to BIV) and 17 repeats. Proteins interacting with spectrin are indicated below the relevant domains. B. Band 3. The 52-kD mem-
brane ___domain contains 14 transmembrane segments and is responsible for anion exchange. The 43-kD N-terminal cytoplasmic ___domain and
the proteins binding to this ___domain are shown. C. Ankyrin. The N-terminal 89-kD ___domain consists of 24 ankyrin repeat units, which bind
to band 3. The 62-kD ___domain interacts with spectrin and the C-terminal 55-kD regulatory ___domain modulates the activity of the protein.
D. Protein 4.1. The four structural domains of the protein are depicted as open rectangles and the proteins binding to each ___domain are
shown below.

understanding of the molecular basis of these disorders. To gain

morphological abnormality of the red cells. Four main groups
insight into the pathogenesis and to enable a correlation of the
are delineated:
genotype with the observed morphological phenotype, it is
|. Hereditary spherocytosis (HS) useful to divide the interactions between the red cell membrane
2. Hereditary elliptocytosis (HE) and morphologically related components into two categories (Fig. 9-5), as follows:
disorders, including hereditary pyropoikilocytosis (HPP)
1. Vertical interactions occur between the membrane skeleton
and Southeast Asian ovalocytosis (SAO)
3. Hereditary stomatocytosis and the bilayer and mainly involve spectrin—ankyrin—band
Ai . Hereditary xerocytosis
3 associations, as well as weak contacts between spectrin
and the negatively charged lipids of the inner half of the
By far the most common and well-characterized groups of disor- membrane bilayer.
ders are HS and HE, and this chapter focuses on these important i). Horizontal interactions occur between components of
entities. In recent years there have been major advances in our the membrane skeleton and include spectrin dimer
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: I. Hereditary Defects of the Red Cell Membrane 183

Sp-lipid Sp-2.1-3 Sp-lipid Sp-4.1-GP

interaction interaction interaction interaction
sane Wires
- oad

interactions
Vertical
in
SpD-D interaction

HYPOTHESIS:

Vertical defect Horizonal defect

Lipid bilayer destabilization Skeletal destabilization

Mild ¥ \ Severe
Loss of membrane material Defective shape Fragmentation
recovery
y y

e a = pn as =
HPP
Figure 9-5 ® Diagrammatic illustration of the vertical and horizontal interactions between the red cell membrane components (top). The bot-
tom section illustrates the pathophysiology of the red cell lesion in hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary py-
ropoikilocytosis (HPP). A defect in a vertical interaction resulting in spherocytes and HS is illustrated at the bottom left. A defect in a horizontal in-
teraction resulting in elliptocytes and poikilocytes (HE and HPP) is illustrated at the bottom right. (From Palek, J: Disorders of red cell membrane
skeleton: An overview. In Kruckeberg, WL, et al (Eds): Erythrocyte Membranes 3: Recent Clinical and Experimental Advances. Alan R. Liss,
New York, 1984, p 177, with permission.)

majority of cases, the resulting protein abnormalities are

self-association and spectrin—actin—protein 4.1 complex quantitative with decreased amounts of the membrane pro-
formation. teins involved in vertical interactions between the bilayer and
the skeleton (Box 9-2). The causative gene mutations are
As first predicted by Palek in 1984°* and subsequently veri-
fied by mutation analyses, defects in vertical interactions generally unique to each kindred.
manifest as spherocytosis, whereas defects in horizontal inter-
actions lead to elliptocytosis (see Fig. 9-5).

Hereditary Spherocytosis
MODE OF INHERITANCE
HS is characterized by osmotically fragile, spherical red cells,
and is the most common hereditary hemolytic anemia in people
of northern European origin (Fig. 9-6). It has been documented
in other race groups, such as the Japanese and South African
blacks, but with a lower prevalence. In at least 75% of cases it
follows a classic autosomal dominant pattern of inheritance, but
in the remaining families both parents are clinically normal,
suggesting autosomal recessive inheritance or variable pene-
trance of a dominant gene or a de novo mutation.
Figure 9-6 @ Photomicrograph of peripheral blood smear from a
patient with hereditary spherocytosis (HS). Note the microsphero-
MOLECULAR DEFECTS cytes (small condensed spherocytes with no central pallor), indi-
The underlying molecular defects in HS are heterogeneous cated by the black arrow and the pincered (mushroom-shaped)
and several genetic loci have been implicated.”*** In the vast cell, indicated by the white arrow.
184 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

these often result in substitution of highly conserved amino

Box 9—2 Defects of Red Cell Membrane acids that play a crucial role in the stabilization of the protein
Proteins in Hereditary and its insertion into the membrane. The abnormal amino acids
Spherocytosis may also influence the anion transport function of band 3 if they
are located in the transmembrane ___domain, or alter the binding of
protein 4.2 if they occur in the cytoplasmic ___domain. Arginine
Protein Deficiencies
¢ Spectrin
|
i substitutions are common®! and arginine 760 (encoded by CGG)
¢ Spectrin and ankyrin appears to be a mutation “hotspot,” because it is frequently
¢ Band 3 mutated to either CAG (glutamine) or TGG (tryptophan).
+ Protein 4.2 Protein 4.2 deficiency is rare in Europeans but relatively
}}} common in the Japanese population, mainly because of pro-
tein 4.2 Nippon, which is caused by a point mutation that
Spectrin deficiency is a common underlying cause of HS alters mRNA processing.**
in population groups originating from northern Europe and is
also found in the majority of South African blacks. It was first PATHOPHYSIOLOGY
documented by Agre and coworkers,* who noted that the The fundamental expression of the membrane defect in HS is
number of spherocytes, severity of the disease, and response to a loss of surface area of the red cell, resulting in a decreased
splenectomy correlated closely with the reduced spectrin con- surface-to-volume ratio. This is manifested morphologically
tent. Spectrin deficiency is often secondary to a decreased as spherocytosis, although it should be noted that the majority
amount of ankyrin (see later discussion) because the loss of of HS cells are spherostomatocytic rather than truly sphero-
ankyrin attachment sites prevents binding of spectrin to band 3. cytic. Such cells tolerate less swelling than normal red cells
Mutations in the a-spectrin gene have no effect in the het- and are osmotically fragile. The decrease in surface-to-volume
erozygous state, because a-spectrin is synthesized in excess. ratio also makes the cells less deformable than normal. This
However, frameshift errors caused by splice site mutations in has a particularly deleterious effect on their survival in the
both a-spectrin alleles have been implicated in severe reces- spleen and explains one of the hallmarks of HS, which is the
sive HS.”° B-Spectrin defects are associated with dominant HS excellent clinical response to splenectomy in most, but not all
because -spectrin synthesis is rate limiting in the assembly of cases (see later discussion). The exact mechanism of sphero-
spectrin in the membrane. Several different mutations have cyte formation and subsequent hemolysis is not yet fully elu-
been described in individual families.** These include mis- cidated, but a postulated pathway’ of the pathophysiology of
sense mutations, splice-site mutations, as well as deletions and HS is summarized in Figure 9-7. The primary red cell mem-
insertions, which ultimately result in a shift in the reading brane protein defects in HS are thought to weaken the vertical
frame of the gene, creating abnormal stop codons that termi- interactions between the skeleton and the bilayer. Spectrin- or
nate translation prematurely. These, together with nonsense band 3-deficient areas destabilize the membrane and allow the
mutations, as well as a point mutation in the translation initia- skeleton to be uncoupled from the bilayer. This results in loss
tion codon that prevents translation of the protein, silence the of membrane in the form of microvesicles, which reduces the
expression of the mutant allele and manifest as null mutations. membrane surface area and decreases the surface-to-volume
Ankyrin deficiency, resulting in a concomitant decrease ratio of the cell. These cells are selectively trapped and “condi-
in spectrin, was first described by Coetzer and coworkers,” tioned” in the spleen, where they are exposed to an acidic,
and subsequently shown to be caused by decreased ankyrin oxidant-rich environment, which damages the cells further,
mRNA production and synthesis of an unstable molecule.” causing them to lose progressively more surface area until they
A combined ankyrin and spectrin deficiency is the most are ultimately destroyed by phagocytosis.*~**
common abnormality underlying HS in Europe and North
America.**™* Interstitial deletions of chromosome 8 involving CLINICAL MANIFESTATIONS
the ankyrin gene (8p11.2), as well as balanced translocations The classic presenting features of patients with HS are the triad
involving chromosome 8p11, have been reported.”’*° Nonsense of jaundice, anemia, and enlarged spleen, but many patients do
and frameshift mutations are prevalent in dominant HS, result- not show all these signs. The age at presentation can vary from
ing in either unstable mRNA transcripts or truncated proteins within a day or two after birth to old age, and sometimes the
with functional abnormalities or decreased stability. In recessive condition is diagnosed only during family studies or investiga-
HS, the defects are usually missense or promoter mutations.”* tion for other reasons. Approximately two-thirds of HS patients
Band 3 deficiency is found in approximately 20% of present with the classic signs and a mild, uncompensated
American and European HS patients but is more common hemolytic anemia. Characteristically the jaundice is said to be
among Japanese and South African whites, comprising nearly “acholuric,” because unconjugated bilirubin cannot pass the
half of the cases in the latter group. The decreased band 3 con- glomerular filter. Many of these patients have pigment gall-
tent often results in a secondary deficiency of protein 4.2. Null stones, presumably caused by increased concentrations of
mutations are common and may be caused by single nucleotide bilirubin in the bile. About one-quarter of HS patients have a
insertions or deletions that alter the reading frame of the mutant mild hemolytic state that is compensated for, and such patients
band 3 allele or by nonsense mutations or splicing defects.”° are not anemic and are usually asymptomatic. A minority of
Small in-frame deletions or insertions have been documented in patients (about 10%) have a severe hemolytic anemia that
a few cases. Band 3 missense mutations are very common, and may require blood transfusion. Aplastic crises, in which
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 185

QUANTITATIVE RED BLOOD CELL both before and after splenectomy but can be low, normal, or
MEMBRANE PROTEIN DEFECT high. The mean corpuscular hemoglobin (MCH) tends to paral-
lel the MCV. Although the MCV is usually normal, because of
the red cell’s spherical shape, the diameter of some cells is sub-
stantially decreased and these appear as dark, rounded micros-
pherocytes on the peripheral smear (see Fig. 9-6). The mean
corpuscular hemoglobin concentration (MCHC) is elevated
(higher than 36%) in about 50% of cases and probably reflects
Din eete sth,
mild cellular dehydration, particularly of cells that have under-
gone splenic conditioning and that have low levels of cell water
and potassium (see the earlier section on Pathophysiology).
Automated blood counters using laser light scattering or
profiles obtained by aperture impedance analysers are very
useful and have a high degree of accuracy in diagnosing HS.
These detect the right shift in the distribution of red cell he-
moglobin concentration and the presence of a subpopulation
Splenic sequestration
Erythrostasis of microspherocytes. An example is shown in Figure 9-8.

MORPHOLOGY OF PERIPHERAL SMEAR The morphologi-

Conditioning cal hallmark of HS is the spherocyte (see Fig. 9-6). Although in
many instances the detection of these cells may present no dif-
ficulty, in some patients their detection may provoke argument
Microspherocytes Hemolysis even among experienced hematologists. It is particularly impor-
tant to examine well prepared smears free of any artifact. In typ-
ical cases, before splenectomy there may be varying degrees of
Figure 9-7 ™ Schematic representation of postulated mechanisms
of “conditioning” and destruction of HS red cells in the spleen. polychromasia, poikilocytosis, and anisocytosis with many
(From Lux, SE, and Glader, BE: Disorders of the red cell membrane. normal discoid cells, but the overriding impression is one of
In: Nathan, DG, and Oski, FA (Eds): Hematology of Infancy and increased numbers of uniformly round cells (see Fig. 9-6).
Childhood, ed 2. WB Saunders, Philadelphia, 1981.) Some of the cells appear as microspherocytes and are dark,
round, and lack central pallor. Pincered or mushroom-shaped

erythropoiesis is suppressed leading to more pronounced ane-

mia, occurs particularly in this group but may supervene in
patients with milder forms of the disease. The usual cause of
such crises is infection of erythropoietic precursors by
parvovirus B19. An uncommon complication of prolonged
hemolysis, which is not limited to HS, is chronic leg ulceration.
MCV
An interesting observation is that the clinical severity may
vary in patients with the same gene mutation, implying that
other factors influence the clinical presentation. These factors
may include a second mutation or polymorphism inherited
either in cis or in trans to the affected allele, which influences
either its level of expression” or its stability or function.

CLINICAL LABORATORY FINDINGS

EVIDENCE OF HEMOLYTIC PROCESS The laboratory
features of extravascular hemolysis, outlined earlier, are usu-
MCHC
ally apparent. Hyperbilirubinemia is found in about half the
patients, and haptoglobins are variably reduced. Classic
features of intravascular hemolysis such as hemoglobinemia,
hemoglobinuria, or hemosiderinuria do not occur. The reticu- B AS rN %
28 41
locyte production index is elevated above 2.5 in most cases
(presplenectomy). Figure 9-8 @ Laboratory diagnosis of HS using a Technicon H1 laser
scattering automated blood counter. A. The histogram indicates a
subpopulation of microcytes with a low mean cell volume (MCV) in a
RED CELL INDICES Anemia is usually mild. The mean
patient with HS. B. The histogram depicts a subpopulation of
level of hemoglobin in several series is about 12 to 13 g/dL, dehydrated HS red cells with a high mean cell hemoglobin concen-
but individual cases may vary widely depending on the sever- tration (MCHC). (From Pati AR, Patton WN, Harris RI. The use of the
ity of hemolysis and the degree of compensation. The mean Technicon H1 in the diagnosis of hereditary spherocytosis. Clin Lab
corpuscular volume (MCV) is usually within normal range Haematol 11:27, 1989, with permission).
186 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

cells are often, but not exclusively, found in band 3-deficient have abnormal osmotic fragility of red cells that have been
patients. An interesting observation is the presence of acantho- stressed by prior sterile incubation for 24 hours. During
cytes on the peripheral blood smears of HS patients with the 24-hour incubation, HS cells have greater loss of mem-
8-spectrin defects. brane surface because of the relative membrane instability (see
Fig. 9-9). A corollary of the use of the incubated osmotic
SPECIAL LABORATORY TESTS
fragility test is that if the test result is normal, it is highly
OSMOTIC FRAGILITY TEST This test is essentially a mea- unlikely that one is dealing with a patient with HS. Increased
sure of the surface-to-volume ratio of the red cell. If the test osmotic fragility is however independent of the cause of spher-
is performed on fresh red cells, it is then also a measure of the ical cells; for example, it may also be found in HPP (see section
proportion of cells that have undergone splenic conditioning. later in this chapter), autoimmune hemolytic anemia, and burns.
When red cells are placed in a series of graded hypotonic salt
solutions, water rapidly enters the cells and osmotic equilib- AUTOHEMOLYSIS TEST This relatively sensitive test in the
rium is achieved. The cells swell and become spherical, and diagnosis of HS measures the structural and metabolic integrity
eventually a critical volume is reached, at which point the cel- of the HS red cell membrane under conditions of erythrostasis
lular contents (hemoglobin) leak out and ultimately the cell and relative glucose depletion, that is, sterile incubation of red
may burst. Red cells of patients with HS, because of their cells in their own plasma for 48 hours at 37°C. The HS red cell
decreased surface area-to-volume ratio, can tolerate less is leaky to sodium. To “keep its head above water,” the cell uti-
swelling than normal cells and lyse at higher concentrations lizes adenosine triphosphate (ATP) and glucose to drive the
of salt than do normal cells (Fig. 9-9). About 25% of HS cation pump to a greater extent than normal. Associated with
patients have normal osmotic fragility of fresh red cells, par- the increased activity of the pump, there is a greater turnover of
ticularly in the very group that is mildly affected and is diffi- membrane phospholipids and associated membrane fragmenta-
cult to diagnose on morphological grounds. Generally, patients tion with a decrease in surface-to-volume ratio until the critical
in the latter group, as well as patients with more typical cases, hemolytic volume is reached and autohemolysis occurs. The
usual range of autohemolysis in HS cells is variable and is
about 10% to 50%, compared with control values of 0.2% to
100 2.0%. However, a minority of patients show only minimally
elevated autohemolysis or may even be within the normal
range. In most HS patients, addition of glucose markedly
diminishes autohemolysis but not usually to within the normal
range of samples incubated with glucose (0% to 1.0%). A
oljo) minority of patients show no correction of autohemolysis with
glucose, a finding also obtained with many patients with sphe-
Haemolysis
% rocytosis associated with autoimmune hemolytic anemia. It
should be noted that many laboratories do not use this test rou-
tinely (see Chap. 31 for a description of the procedure).

ACIDIFIED GLYCEROL LYSIS TEST A modification of this

0.0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
test described by Eber and colleagues* is a useful rapid test
% NaCl showing sensitivity and specificity similar to the incubated
osmotic fragility test. The time taken for 20 uL of EDTA blood
to hemolyze when diluted in 9.3 mM sodium phosphate-
buffered 2 M glycerol-saline at pH 6.9 is determined by a
decrease in absorbance at 625 nm. HS samples lyse with a f¢,,
of less than 5 minutes compared to greater than 30 minutes for
normal samples.

RED CELL MEMBRANE STUDIES

Haemolysis
% Densitometric quantitation of red cell membrane proteins sep-
arated by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) reveals the underlying quantitative
defect in the majority of HS patients. The abnormality in the
remaining cases may be either a subtle deficiency in one of
00 02 03 04 05 06 07 08 09
the proteins, which is not detected by SDS-PAGE, or an
% NaCl altered functional interaction between membrane proteins.
Figure 9-9 ™ Osmotic fragility curves of fresh blood (top) and Once the defective protein has been identified, DNA and
incubated blood (bottom) obtained from a patient with HS. The RNA analyses are required to elucidate the causative muta-
normal range is shown by blue areas. Note the increased fragility of tions. These molecular tests are usually carried out in research
the HS red cells to osmotic lysis. laboratories because most kindred have unique mutations and
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 187

molecular screening is thus not a feasible option for a routine

hematology laboratory. were found to have mild, compensated hemolytic states
associated with spherocytosis. In view of the risk of recur-
TREATMENT rence of common bile duct calculi, an elective splenectomy
was performed 6 months later, curing the hemolytic state.
From the foregoing discussion of the pathophysiology of the HS
red cell, and the central role of the spleen in “conditioning” such
cells which ultimately leads to their destruction, it should not be
QUESTIONS
surprising that splenectomy is functionally curative in the . Why was a Coombs test performed on this patient?
majority of patients with this disease. In the relatively rare . What type of hemolysis is occurring in this patient?
patient with severe spectrin deficiency (less than 40% spectrin), —
WN. What is the surface-to-volume ratio of these red cells as
clinical improvement occurs after splenectomy but ongoing indicated by the osmotic fragility test?
hemolysis and anemia may continue. In the usual case, although 4. In what type of hemolytic anemia is the osmotic fragility
uncorrected by glucose?
spherocytosis persists, “conditioned” microspherocytes are no
5. Which of the red blood cell (RBC) indices is typically
longer seen and red cell life span is normal or very near normal.
increased in HS?
At one time, many authorities recommended splenectomy uni-
formly in all patients with HS because of the risks of biliary tract
disease and the development of aplastic crises, but this view has
been considerably tempered in recent years. Patients with mild, Hereditary Elliptocytosis
compensated cases of HS are usually not offered splenectomy
MODE OF INHERITANCE
unless the previously mentioned complications intervene. An
Hereditary elliptocytosis (HE) is a group of disorders found
important consideration in infants and young children is the risk
in all race groups and characterized by the presence of ellip-
of postsplenectomy sepsis, particularly with Streptococcus
tical red cells in the peripheral blood (Fig. 9-10). The
pneumoniae, so that most authorities recommend deferment of
HE syndrome 1s heterogeneous in terms of inheritance, clin-
splenectomy until about 6 years of age. In severe cases, how-
ical manifestations, and underlying molecular defects. It is
ever, splenectomy may have to be performed earlier; but in
usually inherited in an autosomal dominant fashion, except
either event, treatment with pneumococcal vaccine is recom-
for hereditary pyropoikilocytosis (HPP), which is a reces-
mended, preferably starting before splenectomy. Younger chil-
sive and very severe HE variant.
dren may also require prophylactic penicillin or other
antibiotics post-splenectomy, but the latter course is controver-
sial. Failure of splenectomy is almost always associated with an Clinical Phenotypes
accessory spleen not removed at surgery, or more rarely is
caused by autotransplantation of splenic tissue in the peritoneal Three major clinical and morphological phenotypes have
cavity, leading to splenosis. been delineated by Palek and Lux: common HE, including
HPP; spherocytic HE; and Southeast Asian ovalocytosis
(SAO) (Table 9-2).**°? Common HE is the most prevalent,
especially in African populations. There is marked clinical
Case Study 1 and morphological heterogeneity, ranging from an asympto-
matic carrier state with normal morphology to homozygous
A 40-year-old woman presented to her physician with an HE and HPP with severe hemolysis and bizarre poikilocytic
attack of acute cholecystitis. Physical examination revealed
a palpable spleen in addition to the signs of acute cholecys-
titis. On investigation she was found to have numerous
gallstones, and a routine blood count showed a mild, com-
pensated hemolytic state: Hb, 13.8 g/dL; Het, 38%; MCV,
80 fL; MCHC, 36%; reticulocyte count, 7%; polymorphs
present; RPI, 3.9. The peripheral smear showed moderate
numbers of spherocytes and a few microspherocytes.
The Coombs test was negative. Unconjugated bilirubin was
2.5 mg/dL, and the conjugated bilirubin was 0.5 mg/dL.
Haptoglobin concentration was less than 10 mg/dL (normal
range is 25 to 180 mg/dL). Further investigation revealed
that osmotic fragility of both fresh and incubated venous
blood was increased. Autohemolysis was 25% after
48 hours’ incubation, corrected to 3% in the presence of
glucose. After the acute episode had settled, an elective
cholecystectomy was performed. A diagnosis of hereditary
spherocytosis was made and confirmed in a subsequent
study of the patient’s family when two of her three children Figure 9-10 @ Photomicrograph of peripheral blood smear from a
continued patient with mild hereditary elliptocytosis (HE). Note the high
percentage of elliptocytes.
188 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

Box 9-3 Defects of Red Cell Membrane

Proteins in Hereditary
Elliptocytosis
Protein Dysfunction
¢ Spectrin dimer self-association
* a-Spectrin mutations
¢ B-Spectrin mutations
¢ Truncated a- or B-spectrin
¢ Spectrin-ankyrin interaction
¢ Protein 4.1-spectrin interaction
* Band 3 (SAO)
Protein Deficiencies
Fan le,
|¢ Protein 4.1
Figure 9-1 1 @ Photomicrograph of peripheral blood smear from a ¢ Glycophorin C
patient with hereditary pyropoikilocytosis (HPP). Note the bizarre ¢ Spectrin (HPP)
micropoikilocytosis, red cell budding, and very few elliptocytes.
| HPP = hereditary pyropoikilocytosis; SAO = Southeast Asian ovalo-
cytosis.
and microspherocytic morphology (Fig. 9-11). The most fre-
quently occurring clinical form is mild HE with no or mini-
mal hemolysis. Spherocytic HE is a rare phenotypic hybrid of
HE and HS, in which the clinical course resembles HS and affected ___domain, as well as the size of the abnormal tryptic pep-
responds well to splenectomy. Southeast Asian ovalocytosis is tide, is used in the nomenclature of these defects, and Spal/74,
very common in Melanesian and other Southeast Asian pop- Spal/65, and Spal/46 are the most common. The extent of
ulation groups because of the protective effect toward impairment of spectrin dimer self-association and the amount
malaria; it also occurs in the Cape Coloured population of and type of abnormal tryptic peptide correlate with the clinical
South Africa.** severity. Several mutations have been described in both
a- and B-spectrin genes.
COMMON HEREDITARY ELLIPTOCYTOSIS Point mutations in the a-spectrin gene, resulting in single
MOLECULAR DEFECTS The underlying abnormality in HE amino acid substitutions close to the tryptic cleavage site, are
resides in the red cell membrane skeleton and _ usually most common. Codon 28 of the a-spectrin gene, CGT, is a
involves spectrin or protein 4.1 (Box 9-3). “hotspot” for mutations, and the normal arginine is replaced
Spectrin mutations that impair the self-association of by one of four different amino acids, causing Spal/74.*° Inter-
spectrin dimers into tetramers are the most common. This func- estingly, a histidine (CAT) substitution predominates in Amer-
tional defect is caused by an alteration in the structure of the ican blacks who originated from West Africa, whereas South
spectrin self-association site, usually involving the N-terminal African blacks have a cysteine (TG7) mutation. Spal/65 is
___domain of a-spectrin, or less frequently, the C-terminal ___domain invariably caused by a duplication of codon 154, which inserts
of B spectrin. This disrupts the 106-amino acid spectrin repeats an additional leucine into the protein, and is common in
and alters the partial tryptic cleavage pattern of spectrin. The Central and North Africa.*’"* In-frame deletions have been

Phenotype Hemolysis Morphology Most Common Defects

Common HE Asymptomatic Elliptocytes Impaired spectrin tetramer

formation or
Protein 4.1 deficiency
HE with infantile poikilocytosis | Moderately severe up Elliptocytes Impaired spectrin tetramer
to age two years Poikilocytes formation or
Protein 4.1 deficiency
Severe Microspherocytes Impaired spectrin tetramer
Poikilocytes formation and spectrin
Few elliptocytes deficiency
Large ovalocytes 9-amino acid deletion in band 3
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 189

documented in e-spectrin; for example, in Spectrin Dayton the

insertion of a novel mobile genetic element into the a-spectrin
gene causes skipping of exon 5.*° Spectrin St. Claude has a
single point mutation in intron 19 of «-spectrin, which creates
two different RNA splice forms. At the protein level this ulti-
mately has long-range negative effects on tetramer formation
and on the binding of B-spectrin to ankyrin.*°
Mutations in B-spectrin include missense point muta-
tions, as well as frameshift errors caused by small deletions,
insertions, or splice-site mutations that result in mutant
B-spectrin molecules with truncated C-terminal domains.?*4
A decreased amount of protein 4.1 has also been impli-
cated in HE. This is usually the result of mutations influenc-
ing the translation initiation site, or more rarely, of deletions
that shorten the protein or abolish spectrin binding.*!??4
Figure 9-12 = Photomicrograph of peripheral blood smear from a
patient with Southeast Asian ovalocytosis (SAO). Note the charac-
HEREDITARY PYROPOIKILOCYTOSIS
teristic spoon-shaped oval cells with a band across the central area.
HPP is an interesting, relatively rare, severe hemolytic
disease that is part of the HE group of disorders. The periph-
eral blood smear is characterized by microspherocytosis, with hemolysis.** SAO is characterized by rigid, spoon-shaped
micropoikilocytosis, fragments, and relatively few, if ovalocytes (Fig. 9-12) that are resistant to invasion by malaria
any, elliptocytes (see Fig. 9-11). Patients with HPP are com- parasites and provide an example of the selective pressure of
pound heterozygotes, and all cases thus far investigated malaria.* The underlying molecular defect is a deletion of
exhibit two genetic defects: 27 base pairs in exon 11 of the band 3 gene, resulting in the
absence of nine amino acids at the junction of the cytoplasmic
1. A mutant a- or B-spectrin that shows severe impairment of
and transmembrane domains of the band 3 protein.**“° This
spectrin dimer self-association.»
alters the structure and function of the mutant band 3, causing
2.A partial spectrin deficiency resulting from decreased
increased binding to ankyrin,*’ an inability to transport anions,**
synthesis of a-spectrin, which causes the characteristic
and markedly restricted lateral and rotational mobility in the
microspherocytosis.1!*
membrane.”
PATHOPHYSIOLOGY OF COMMON HE AND HPP
CLINICAL LABORATORY FINDINGS
The molecular defects in common HE involve horizontal
EVIDENCE OF HEMOLYTIC PROCESS The usual picture
interactions between proteins of the membrane skeleton (see
in the most common variant (mild HE) is that of a very mild,
Fig. 9-5). Defective spectrin dimer self-association or protein
compensated hemolytic anemia in which the only features
4.1 deficiency weakens the skeleton and, under the influence
may be a slight reticulocytosis and decreased haptoglobin
of shear stress in the circulation, the cells become distorted
levels. Many patients show no biochemical evidence of a
and progressively lose their ability to regain their original disc
hemolytic process. In the more severe cases, such as in
shape, resulting in elliptocytes and poikilocytes. The clinical
spherocytic HE, HE with infantile poikilocytosis, homozy-
expression of HE is variable and can differ between individu-
gous HE, and HPP, the usual features of extravascular
als of the same family with the identical mutation, implying
hemolysis outlined earlier are found.
that other mutations or polymorphisms influence the expres-
sion of the mutant allele. A low expression a-spectrin allele,
MORPHOLOGY OF PERIPHERAL SMEAR The morphol-
SpLELY, is an example of a modifying polymorphism that
ogy of the peripheral smear varies with the clinical phenotypes
exacerbates the clinical phenotype when inherited in trans to
of HE. In the usual variant of mild HE with no hemolysis or a
a mutant a-spectrin allele.*°
compensated hemolytic state, the red cells show prominent
In the case of HPP, the clinical severity and morphology
uniform elliptocytosis, the cells being elliptical rather than
are thought to result from a combination of horizontal and
oval or egg shaped (see Fig. 9-10). Usually, more than 30%
vertical defects. Spectrin self-association (horizontal defect) of the red cells are elliptocytic, but many patients have a
is severely impaired in HPP, which markedly decreases the higher proportion of elliptocytes, for example, more than
strength and stability of the skeleton, resulting in poikilocytes 75%. Very elongated or rod-shaped cells are characteristic
and fragmentation. Spectrin deficiency impairs the vertical and often constitute more than 10% of the red cells. In
interaction of the skeleton with the lipid bilayer and gives rise patients with uncompensated hemolysis (mild HE with spo-
to microspherocytes as described for HS (see Fig. 9-5). radic hemolysis), the red cells show more prominent poikilo-
SOUTHEAST ASIAN OVALOCYTOSIS cytosis, and a small proportion of elliptocytes may have
SAO is a very common, asymptomatic condition in ethnic budlike projections.
Southeast Asian populations. It also occurs in the Cape Infants with mild HE and poikilocytosis of infancy
Coloured population of South Africa, where it can be associated exhibit prominent poikilocytosis, microspherocytosis,
190 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: I. Hereditary Defects of the Red Cell Membrane

2 “eGy
x 28
a
CDSS
Ine osmotic fragility are markedly increased, and autohemolysis is
increased and unaffected by glucose. Pre- and postincubation
osmotic fragility are uniformly increased in spherocytic HE, and
autohemolysis is characteristically increased but corrected by
glucose. Children with HE and infantile poikilocytosis have
increased osmotic fragility and autohemolysis in the early
neonatal period that revert to normal with the development of
more prominent elliptocytosis.

coy RED CELL MEMBRANE STUDIES

Cag @ =e Red cell membrane protein deficiencies or size abnormali-

: @ © atbG0° og ties may be detected via SDS-PAGE and quantitation, as

described earlier for HS. Spectrin dimer self-association is
nO Are * Oe “ab analyzed by extracting spectrin from the membrane at 4°C
followed by nondenaturing PAGE and quantitation of spec-
Figure 9-13 ™ Photomicrograph of peripheral blood smear from a
patient with mild HE and poikilocytosis of infancy. Note the poikilo- trin dimers and tetramers. A suspected diagnosis of SAO
cytosis and fragmentation. is easily confirmed by detecting the presence of the charac-
teristic 27-base-pair deletion in the band 3 gene using the
polymerase chain reaction (PCR). The underlying gene
mutations in HE and HPP are identified by DNA and RNA
fragmentation, budding of red cells, and a variable degree of analyses. These specialized tests are usually performed in
elliptocytosis (Fig. 9-13). By the time the infant reaches the research laboratories.
age of | to 2 years, the morphology has changed to that
characteristic of mild HE. In the neonatal period, the red TREATMENT
cells show increased thermal sensitivity (which is also a Patients with compensated mild HE and no splenomegaly
characteristic of HPP), but the diagnosis is suggested by have a benign disorder and require no therapeutic interven-
finding evidence of mild HE in one parent. tion. Those with HE and uncompensated hemolysis usually
The rare patients with homozygous HE or HPP (see benefit from splenectomy, which is also uniformly beneficial
Fig. 9-11) present with marked microspherocytosis, microp- to patients with HPP and spherocytic HE. Patients with HE
oikilocytosis, fragments, and very few, if any, elliptocytes. and infantile poikilocytosis should be recognized and treated
Red cell morphology in spherocytic HE is very variable, symptomatically, because they will improve spontaneously
but the hallmarks are less prominent elliptocytosis with sphe- with the development of a clinical picture indistinguishable
rocytes and microspherocytes. The proportion of spherocytes from mild HE.
and elliptocytes varies in different kindred and even within
the same kindred.
Patients with stomatocytic HE or SAO have very distinc-
tive red cell morphology. The elliptocytes are more rounded
and oval and are often quite large with a transverse bar that
divides the central pale space (see Fig. 9-12).
“Case Study? 4.
RED CELL INDICES In the common variants of mild HE
with compensated and uncompensated hemolysis, the MCV A 45-year-old woman presented to her physician complain-
is usually normal or slightly elevated, the latter finding ing of malaise and tiredness on mild exertion. On physical
probably reflecting an associated reticulocytosis. MCH and examination, she was found to have slight scleral icterus and
a two-finger splenomegaly. A blood count revealed the fol-
MCHC are also usually within the normal range. In infants
lowing: Hb, 11.0 g/dL; Het, 32%; MCHC, 34.3%; MCV,
with HE and poikilocytosis, the MCV may be decreased
100 fL; reticulocyte count, 12.0%; shift cells on peripheral
and the MCHC is either normal or slightly elevated. In
smear; and RPI, 5.7. The peripheral smear showed about
HPP, the MCV is always decreased and the MCHC is usu- 80% elliptocytes with some poikilocytosis consisting of a
ally elevated. few fragmented cells and budding elliptocytes. Unconju-
gated bilirubin was 3.5 mg/dL, conjugated bilirubin,
SPECIAL LABORATORY TESTS 0.6 mg/dL, and haptoglobin, 15 mg/dL (normal range is 25 to
OSMOTIC FRAGILITY AND AUTOHEMOLYSIS The osmotic 180 mg/dL). Pre- and post-incubation osmotic fragility and
fragility and autohemolysis tests are useful additional tests in autohemolysis were within the normal range. Examination
delineating some of the HE phenotypes. In patients with mild of red cells from the patient’s family showed striking
HE (compensated and uncompensated) both the preincubation elliptocytes with normal hemoglobin and reticulocyte count
and the postincubation osmotic fragility and autohemolysis in her father and in one of her three children. A diagnosis of
mild HE with sporadic hemolysis was made, and a good
results are normal. Rarely, patients with mild HE and uncom-
response to splenectomy was obtained.
pensated hemolysis may have increased autohemolysis cor-
continued
rected by glucose. However, in HPP, pre- and postincubation
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 19]

QUESTIONS
1. How can the anemia presented here be classified?
2. What parameter(s) is (are) suggestive of effective ery-
thropoiesis?
3. Do the laboratory values presented here indicate hemo-
lysis in this patient?
4. What is the significance of the RPI value?

Disorders of Membrane Cation

Permeability
Hereditary Stomatocytosis and Hereditary
Xerocytosis Figure 9-15 m Photomicrograph of peripheral blood smear from a
patient with hereditary xerocytosis. Note the characteristic target
This is a heterogeneous group of rare disorders characterized cells and cells with hemoglobin concentrated on one side of the cell.
by alterations in the permeability of the red cell membrane to
cations.**** Two main clinical and morphological syndromes
sodium and potassium. To maintain osmotic equilibrium,
have been described: hereditary stomatocytosis (hydrocyto-
water enters cells in which the total cation content is
sis), in which the red cells are swollen (Fig. 9-14); and hered-
increased, leading to swelling and hydrocyte formation. In
itary xerocytosis, in which the cells are markedly dehydrated
contrast, a net loss of cations results in a movement of water
(Fig. 9-15). Several intermediate syndromes have been iden-
out of the cell with formation of dehydrated cells (xerocytes).
tified, but these are not considered here.
The basic abnormality of sfomatocytic red cells is a
marked increase in the passive permeability of sodium into
MODE OF INHERIFANCE
the cell and of potassium out of the cell. The defect in sodium
Hereditary stomatocytosis is inherited in an autosomal domi-
permeability is greater than that for potassium. Although the
nant fashion, but some patients with severe hemolysis show
sodium—potassium pump is stimulated by the influx of
autosomal recessive inheritance. Hereditary xerocytosis is
sodium, it cannot cope with the influx, and the total cation
inherited by autosomal dominant transmission.
content of the cell increases, with resultant water influx and
formation of hydrocytes. A deficiency of one component of
ETIOLOGY AND PATHOPHYSIOLOGY
band 7, designated stomatin, has been reported in some of
An important determinant of the water content of red cells is
these patients, but the coding sequence of the gene is normal
the total intracellular concentration of the monovalent cations
and mice lacking stomatin are clinically and hematologically
normal.** These findings indicate that stomatin deficiency is
not the primary molecular defect in hereditary stomatocyto-
sis. The causative abnormality may reside in another, as yet
uncharacterized protein, that associates with stomatin and sta-
bilizes it in the membrane.*** Because of the influx of water,
stomatocytes have an increased volume with a decreased
surface-to-volume ratio and the attendant consequences of
decreased red cell deformability and susceptibility to splenic
sequestration. Although splenectomy is predictably beneficial
in most patients with hereditary stomatocytosis, paradoxically
some patients with severe permeability defects do not have
significant hemolysis, suggesting that other, still unknown
factors may be important in the destruction of these cells.
Stomatocytes are also found in Rh null disease, a hemolytic
anemia characterized by an absence of the Rh antigens. This dis-
order is clinically and biochemically heterogeneous, and the
molecular pathophysiology is not completely understood.
Red cells from patients with hereditary xerocytosis have
an increased efflux of potassium that approximates sodium
influx. Although the sodium—potassium pump is stimulated by
Figure 9-14 m Photomicrograph of peripheral blood smear from a the influx of sodium, it is insufficient to correct the loss of potas-
patient with hereditary stomatocytosis. Note the high percentage of sium. Irreversible potassium and total cation loss occurs with
red cells with a central slit of pallor. (From Bell, A: Hematology. In:
Listen, Look and Learn. Health and Education Resources, Inc.,
resultant dehydration and formation of xerocytes. The underly-
Bethesda, MD, with permission.) ing molecular pathology is not understood, but the disease locus
192. Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

has been mapped to chromosome |6q23-qter.” Xerocytes have with hereditary xerocytosis do not benefit from splenec-
an increased surface-to-volume ratio, an increased MCHC, and tomy, presumably because of more generalized sequestra-
presumably increased cell viscosity, which make them less tion of these cells.
deformable and liable to sequestration in the reticuloendothelial
system. The red cells are not specifically sequestered in the
spleen, so that splenectomy does not have a beneficial effect.

CLINICAL LABORATORY FINDINGS Case Study 3

MORPHOLOGY OF PERIPHERAL SMEAR The characteristic
morphological features of hereditary stomatocytosis are a ten- A 6-year-old boy was noted by his mother to have slight
dency toward macrocytosis and the presence of stomatocytes on scleral icterus and was referred for further investigation. He
complained of some tiredness on exertion but was otherwise
the peripheral smear. These are red cells with a central slit or
symptom-free. Physical examination showed only a one-
stoma (see Fig. 9-14). On phase contrast or scanning electron
finger splenomegaly. A blood count showed the following:
microscopy, the cells have a bowl-like appearance. In hereditary Hb, 10.8 g/dL; Het, 29%; MCHC, 37%; MCV, 100 fL; retic-
xerocytosis, target cells are present, reflecting the greater sur- ulocyte count, 10%. Numerous target cells, some spiculated
face-to-volume ratio of these cells. Small spiculated echinocytes cells, and a few cells showing eccentric concentration of he-
and cells with hemoglobin concentrated in one part of the cell moglobin at one pole of the red cell were seen on
are also features of hereditary xerocytosis (see Fig. 9-15). the peripheral smear. The unconjugated bilirubin level was
mildly elevated, and serum haptoglobin levels were decreased.
RED CELL INDICES The MCV in both hereditary stomato- There was no hemoglobinemia or hemosiderinuria. The
cytosis and hereditary xerocytosis is elevated, despite the cel- osmotic fragility curve was strikingly decreased. Determina-
lular dehydration in the latter condition. The MCHC is de- tion of red cell cation concentrations revealed a markedly
creased in hereditary stomatocytosis and increased in decreased red cell potassium level of 65 mEq/L of RBCs (nor-
hereditary xerocytosis. mal is 90 to 104 mEq/L) and a slightly elevated red cell
sodium level of 15 mEq/L of RBCs (normal is 5 to 12 mEq/L).
Similar findings were obtained in the child’s father, who had
SPECIAL LABORATORY TESTS
previously been diagnosed at another center as having an
Osmotic fragility is increased in hereditary stomatocytosis
“unusual” form of anemia. A diagnosis of hereditary xerocyto-
and reflects the decreased surface-to-volume ratio. Red cell sis was made. Splenectomy was not advised, and the child has
sodium concentration is markedly elevated, and potassium maintained a hemoglobin Jevel varying between 9.5 and
concentration is decreased. Total monovalent cation content is 11.0 g/dL over the past 2 years.
increased. In contrast, red cells in hereditary xerocytosis have
strikingly decreased osmotic fragility, reflecting the increased QUESTIONS
surface-to-volume ratio. Red cell potassium concentration is
markedly decreased, sodium concentration may be normal or 1. How can this anemia be classified, as indicated by the
RBC indices?
increased, and total cation concentration is decreased.
2. What does the decreased osmotic fragility represent?
3. Why would a splenectomy not be beneficial in this case?
TREATMENT
4. Why are these red cells (xerocytes) said to be dehy-
Most patients with hemolysis caused by hereditary stoma- drated with regard to osmotic equilibrium?
tocytosis respond well to splenectomy. However, patients

Ou axtions

1. What happens when normal donor red cells are transfused hematocrit, 23%: reticulocyte count, 8%; polymorphs on
into a patient with an intracorpuscular red cell defect? peripheral smear. What is the RPI?
a. Donor cells are destroyed. a. 8
@) Donor cells have normal survival. b. 4
c. Depends on the severity of the defect.
d. Depends on the severity of the anemia.
2. Which of the following tests is not used to determine 4. What tests are useful in the classification of the cause of
increased red cell destruction? ted cell hemolysis?
a. Unconjugated (indirect) bilirubin ( aCoombs test
b. Serum haptoglobin b. Hemoglobin level and electrophoresis
c. Schumm’s test c. Reticulocyte count
Fa .
fd) Reticulocyte count d. Red cell enzyme studies and iron-binding
3. An anemic patient investigated for a hemolytic state has capacity
the following laboratory findings: hemoglobin, 8 g/dL;
 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane 15

5. Which of the following red cell membrane protein defi- a. Susceptibility of spectrin to thermal denaturation
ciencies does not cause hereditary spherocytosis? .)Defective membrane spectrin tetramer assembly and
a. Ankyrin spectrin deficiency
“b.\Protein 4.1 c. Unstable membrane lipids
c. Protein 4.2 d. Membrane ankyrin deficiency
d. Band 3
9. Which of the following findings are not typical of South-
6. Which of the following laboratory tests would not be east Asian ovalocytosis?
typical of hereditary spherocytosis? a. A deletion of nine amino acids in band 3
a. Increased osmotic fragility b. Resistance to infection by malaria parasite
b,Spherocytes on peripheral smear €)a deficiency of band 3
c. Decreased MCHC d. Rigid spoon-shaped ovalocytes
d. Increased RPI
10. Which disorders are classified as disorders of membrane
7. Which is the most frequent functional abnormality cation permeability?
affecting membrane skeleton proteins in common A. Hereditary stomatocytosis and hereditary xerocytosis
hereditary elliptocytosis? b. Sideroblastic anemia and myelofibrosis
a. Defective binding of spectrin to ankyrin c. Autoimmune hemolytic anemia and microangiopathic
@ Defective spectrin tetramer assembly hemolytic anemia
c. Defective binding of ankyrin to protein 3 d. Ehlers—Danlos syndrome and Bernard—Soulier syn-
d. Deficiency of protein 4.1 drome
8. Which of the following abnormalities is (are) thought to
cause the severe fragmentation and microspherocytes See answers at the back of this book.
_ characteristic of hereditary pyropoikilocytosis? %

~) SUMMARY m The most frequently used test reflecting increased red cell
destruction is the serum unconjugated (indirect) bilirubin
and serum haptoglobin determinations. Other tests, if
mw Anemia may occur with a moderate shortening of the red destruction is primarily intravascular, are those that test for
cell life span if there is an associated depression of bone the presence of hemoglobinemia, hemoglobinuria, and
marrow function, which may occur with certain systemic hemosiderinuria.
diseases or exposure to chemicals.
g The reticulocyte production index (RPI) is calculated by
ms Hemolytic anemias may be classified as follows: the following equation:
Intracorpuscular RPI = % Reticulocytes y Hematocrit
¢ Hereditary defects Reticulocyte maturation time 45
* Defects in the red cell membrane mg The red blood cell membrane skeleton consists of struc-
¢ Enzyme defects tural proteins, «- and ®-spectrin, actin, and protein 4.1.

¢ Hemoglobinopathies w The major integral red blood cell membrane proteins are
band 3 and the glycophorins.
¢ Thalassemia syndromes
w Hereditary hemolytic anemias due to red blood cell mem-
e Acquired defects
brane protein defects are classified into four main groups:
¢ Paroxysmal nocturnal hemoglobinuria
¢ Hereditary spherocytosis (HS)
Extracorpuscular
¢ Hereditary elliptocytosis (HE) including hereditary
¢ Immune hemolytic anemias pyropoikilocytosis (HPP) and Southeast Asian Ovalocy-
¢ Infections tosis (SAO)

¢ Exposure to chemicals or toxins ¢ Hereditary stomatocytosis

¢ Exposure to physical agents ¢ Hereditary xerocytosis

¢ Microangiopathic or macroangiopathic a HS is caused by a deficiency of one of the membrane pro-

teins involved in vertical interactions between the skele-
* Splenic sequestration (hypersplenism) ton and the lipid bilayer.
¢ General systemic disorders
continued
 194 Chapter 9 Hemolytic Anemias: Intracorpuscular Defects: |. Hereditary Defects of the Red Cell Membrane

~') SUMMARY CH m Inherited hemolytic anemias characteristically

mean corpuscular volume (MCV) that is normal (but can
have a

be low or high); a mean corpuscular hemoglobin (MCH)

we HE and HPP are mainly due to spectrin self-association that parallels the MCV; and a mean corpuscular hemoglo-
defects that compromise horizontal interactions in the bin concentration (MCHC) that is usually elevated.
membrane.
= Osmotic fragility measures the surface-to-volume ratio of
mw SAO is caused by a 27 base pair deletion in the band the red blood cell. Red blood cells of patients with HS tol-
3 gene. erate less swelling than normal cells and lyse at a higher
m Hereditary stomatocytosis and xerocytosis are disorders concentration of salt.
of membrane cation permeability.

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Sik Sahr, KE, et al: Sequence and exon-in- Al. Coetzer, TL, and Palek, J: Partial spec- 47. Liu, S-C, et al: Molecular defect of the
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 Chapter

Hemolytic Anemias
Intracorpuscular Defects:
Il. Hereditary Enzyme Deficiencies
Armand B. Glassman, MD
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)

History OBJECTIVES
Enzyme Deficiencies: Hexose At the end of this chapter, the learner should be able to:
Monophosphate Pathway
Glucose-6-Phosphate 1. Name the most common glycolytic enzyme deficiency associated with the hexose
monophosphate shunt or pentose phosphate pathway.
Dehydrogenase
Deficiency 2 _Name the most common glycolytic enzyme deficiency associated with the
Mode of Inheritance Embden—Meyerhof pathway.
Pathogenesis
Clinical Manifestations 3. Identify the red blood cell cytoplasmic inclusions associated with oxidative
Laboratory Testing denaturation of hemoglobin.

Enzyme Deficiencies: 4. List laboratory test results that would suggest a deficiency of glucose-6-phosphate
Glycolytic Pathway dehydrogenase (G6PD).
Pyruvate Kinase Deficiency
5. Identify a laboratory test result that would indicate a pyruvate kinase (PK) deficiency.
Other Enzyme Deficiencies
of the Glycolytic Pathway 6. Name the deficiency that causes hemoglobin to be oxidized from the ferrous to the
awa bs ferric state.
Enzyme Deficiencies: :
Methemoglobin Reductase
Pathway
Methemoglobin Reductase
Deficiency
Case Study

History group, which was later found to be associated with red cell
enzyme abnormalities. Biochemical and molecular studies
In 1926, 72 plantation workers in Panama suffered acute hemol- rapidly advanced the further characterization of these anemias.
ysis after receiving the antimalarial drug 8-aminoquinoline. The most common anemia in this group 1s caused by the
Subsequent reports from widely scattered geographic locations deficiency of glucose-6-phosphate dehydrogenase (G6PD),
added credence to the relationship of hemolysis, cyanosis, and an enzyme in the hexose monophosphate, shunt, or phospho-
methemoglobinemia with the ingestion of certain antimalarial gluconate pathway. The second most frequent enzyme defi-
drugs. In 1953 Dacie and associates! evaluated apparently het- ciency is that of pyruvate kinase (PK), an essential enzyme in
erogeneous cases of congenital hemolytic anemias that had sev- the Embden—Meyerhof pathway. Many other enzyme defi-
eral common characteristics. There was no detectable abnormal ciencies of these pathways have also been identified. They are
hemoglobin, the antiglobulin test result was negative, the variably associated with HNSHA. Laboratory testing is
osmotic fragility test was normal, and there were no spherocytes directed toward identification of the specific enzyme defi-
seen on the peripheral smear. The term hereditary nonsphero- ciency. Treatment is generally supportive, although experi-
cytic hemolytic anemia (HNSHA) was used to describe the mental gene therapy may have a future role.

196
 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies 197

Enzyme Deficiencies: Hexose gene, there is a reduced activity variety designated Gd A—,
Monophosphate Pathway which can be demonstrated in 10% to 15% of the men. Approx-
imately 20% of African American women are heterozygous for
Glucose-6-Phosphate Dehydrogenase the Gd A— gene. Gd A— is the prototype of the mild form of
Deficiency G6PD deficiency.
Among whites, G6PD Mediterranean (G6PD Med) is the
G6PD deficiency, one of the most common genetic abnormali- most common abnormal variant, although the overall preva-
ties known, is thought to affect more than 400 million people lence is low. Among Kurdish Jews, however, the incidence of
worldwide. Carson and associates? identified the enzyme G6PD G6PD Med may be as high as 50% to 60%. G6PD Med
deficiency in 1956 in an individual who developed hemolytic (also known as G6PD B-—) is the prototype of a more severe
anemia after the administration of the antimalarial drug pri- enzyme deficiency associated with acute hemolytic anemia,
maquine (8-aminoquinoline). Yoshida’ first purified the enzyme including favism. The variant Gd Canton is more commonly
from human red cells in 1966. Further progress characterized found in native people of Southeast Asia and China. There is a
the diverse variants of G6PD by sequencing of amino acids, high frequency of G6PD deficiency in Taiwan and southern
cloning of cDNA, and sequencing of nucleotides. China. Approximately 20% to 40% of neonatal jaundice in these
More than 400 variants of G6PD enzyme have been areas is related to G6PD deficiency, whereas neonatal jaundice
described on the basis of biochemical and genetic analyses.*° is rarely attributed to G6PD deficiency in the United States.'”
Recent advances in molecular biology have enabled classifi- The type of G6PD variant found in selected populations 1s listed
cation of the variants into approximately 50 gene mutation in Table 10-1.
groups.*’ Nearly all of these variants are the results of point The variants have been generally designated by geo-
mutations* that produce structurally abnormal or functionally graphic names. With the use of modern techniques of molec-
defective enzymes, or both. ular biology, these variants have been reclassified in terms of
the exact sites of nucleotide substitutions. Using this nomen-
Mode of Inheritance clature, Gd A+ would be designated as G6PD A?’°S to indi-
cate the presence of guanine at nucleotide 376."
G6PD deficiency is transmitted by a mutant gene located on
the X chromosome.? The gene encoding G6PD has been
Pathogenesis
mapped to the Xq28 region in humans.'° The disorder is fully
expressed in men who are hemizygous when they inherit the G6PD catalyzes the first step in the hexose monophosphate
mutant gene from their mothers, who are usually silent carri- shunt (or pentose phosphate) aerobic glycolytic pathway.
ers. In women, full expression of the disorder occurs when Oxidative catabolism of glucose is accompanied by reduction
two mutant genes (homozygous) are inherited. The heterozy- of nicotinamide adenine dinucleotide phosphate (NADP) to
gous woman has a mosaic of one red blood cell population NADPH (Fig. 10-1), which is subsequently required to reduce
with normal enzyme activity and another with deficient glutathione. Reduced glutathione (GSH) is an important source
enzyme activity.'' The expression of G6PD deficiency varies of reducing potential that protects hemoglobin from oxidative
markedly among heterozygotes, which is explained in part by denaturation. The sequence of biochemical reactions shown in
the X-inactivation hypothesis.'? In females, one of the two X Figure 10-2 occur within the normal RBC with adequate lev-
chromosomes (maternally or paternally derived) becomes els of appropriate enzymes and substrate to prevent the accu-
randomly inactivated in each cell of the early embryo. Thus, mulation of intracellular oxidants.
each somatic cell in a heterozygote expresses either one or the The activity of G6PD is highest in young erythrocytes and
other Gd allele. The ratio of the two cell types may vary decreases with cell aging. Under normal conditions, the individ-
widely, not only in different individuals but also among dif- ual with G6PD deficiency compensates for the shortened life
ferent tissues, even within the same individual." span of the erythrocytes by producing more early red cells
Distribution of the mutant gene for G6PD deficiency is
worldwide; however, the highest incidence occurs in the darkly
pigmented racial and ethnic groups. In fact, most of the studies
of G6PD variants have been performed on samples from Table 10-.
African Americans, people of Mediterranean ancestry, and
Asians. The prevalence of G6PD deficiency varies remarkably,
Enzyme Type Population Usually Associated
dependent upon the racial and ethnic group. Normally active
G6PD, type Gd B, is the most common form of the enzyme in Gd B (normal variant) All
all populations and exists in 99% of whites in the United States. Gd Med (also known Whites (Mediterranean
as Gd B—) area)
Another variety of the G6PD enzyme, Gd A+, is commonly
Gd A+ (normal variant) Blacks (~ 16% of African
found in Africans, has normal activity, but differs from Gd B by Americans)
a single amino acid substitution that alters its electrophoretic Gd A-— Africans
mobility.'? The Gd A+ variant is found in about 20% of African Gd Canton Asians
men.'>'6 Among African Americans who possess the Gd A+
198 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies

PHOSPHOGLUCONATE
PATHWAY
(oxidative)

EMBDEN-MEYERHOF :
PATHWAY GSH
(non-oxidative)
Glucose

aop> [we] NADP NADPH

ee
Glucose 6-P 6-P-Gluconate

[eri]!
Fructose 6-P Pentose-P

[Pre]
Fructose 1,6-diP

METHEMOGLOBIN |
REDUCTASE
Glyceraldehyde <>» DHAP
PATHWAY LUEBERING-RAPAPORT
H lobi NAD PATHWAY
Fy tlemoaionin [ll |(Nap [caro |
1,3-diP-Glycerate

arp? —[Pek]|
2,3-diP-Glycerate

3-P-Glycerate

[row] 2-P-Glycerate

[el]
P-Enolpyruvate

are» [PK]
Pyruvate
NADH
NAD [-ou]{
Lactate

Figure 10-1 @ Red cell metabolic pathways. The nucleated red cell depends almost exclusively on the breakdown of glucose for energy
requirements. The Embden—Meyerhof (nonoxidative or anaerobic) pathway is responsible for most of the glucose utilization and generation of
ATP. In addition, this pathway plays an essential role in maintaining pyridine nucleotides in a reduced state to support methemoglobin
reduction (the methemoglobin reductase pathway) and 2,3-diphosphoglycerate synthesis (the Luebering—Rapaport pathway). The phospho-
gluconate pathway couples oxidative metabolism with pyridine nucleotide and glutathione reduction. It serves to protect red cells from
environmental oxidants. (Adapted from Hillman, RS, and Finch, C: Red Cell Manual, ed 7. FA Davis, Philadelphia, 1996, p 15, with permission.)

(reticulocytosis). Oxidative stress, however, can lead to a mild fail to maintain adequate levels of GSH.*!” 21,22 The resulting oxida-
to severe hemolytic episode. A deficiency of GSH results in tion of hemoglobin leads to progressive precipitation of
oxidative destruction of certain erythrocyte components, includ- irreversibly denatured hemoglobin (Heinz bodies) (Fig. 10-3).
ing sulfhydryl groups of globin chains and the cell mem- The cells lack normal deformability when sulfhydryl groups
brane.'?*' In addition, more than 50 chemical agents may are oxidized and consequently encounter difficulties passing
induce hemolysis in G6PD-deficient erythrocytes. The drugs through the microcirculation. Premature destruction of the cells
that have more commonly been reported to induce hemolysis in results when they undergo intravascular lysis or are sequestered
individuals with G6PD deficiency are listed in Table 10-2. The and destroyed in the liver and spleen. This early destruction may
hemolytic episode results when G6PD-deficient erythrocytes sometimes be detected in the peripheral blood smear with the

Reaction A. RBC + infection or oxidant ——_—_—_———-—-_-_____> H,O,

Glutathione peroxidase
Reaction B. HO, + 2GSH (reduced glutathione) GSSG (oxidized glutathione) + 2H,O
Glutathione reductase
Reaction C. GSSG + NADPH (reduced form) + H* 2GSH + NADP* (oxidized form)
Glucose-6-phosphate
Reaction D. G-6-P (glucose-6-phosphate) + NADP* 6-PG (6-phosphogluconate) + NADPH + H*
dehydrogenase

Figure 10-2 ™ Reactions with erythrocytes to prevent accumulation of oxidants.

 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies 199

lem
ency
bee aca

Acetanilide Pamaquine
Chloramphenicol Pentaquine
Dapsone Phenylhydrazine
Daunorubicin Primaquine
Doxorubicin Sulfacetamide
Methylene blue Sulfamethoxazole
(Gantanol)
Nalidixic acid Sulfanilamide
Naphthalene Sulfapyridine
Niridazole (Ambilhar) Thiazolesulfone
Nitrofurantoin (Furadantoin) Toluidine blue
Trinitrotoluene (TNT) Figure 10-4 ® Peripheral blood smear from a patient with a G6PD
deficiency. Note the small, condensed “bite” or “helmet” cells.
Source: Modified from Beutler, E, and Yoshida, A: Genetic variation
of glucose-6-phosphate dehydrogenase: A catalog and future
prospects. Medicine 67:311, 1988. *
However, newborns with this intrinsic defect and adults who
take certain therapeutic drugs or develop infections may suf-
fer various degrees of hemolysis from these challenges to the
formation of small, condensed bite- or helmet-shaped red cells G6oPD-deficient erythrocytes.
(Fig. 10-4). Symptoms of the disorder are related to the severity of the
Certain G6PD-deficient individuals also exhibit sensitiv- hemolytic episode. Two to three days after the administration of
ity to the fava bean (favism) (Fig. 10-5). These individuals the offending drug, the erythrocyte count decreases, along with
develop severe hemolysis after ingesting the fava bean or the hemoglobin content. The anemia appears normochromic
even after inhaling the plant’s pollen. Favism is found in some and normocytic, and there is an increase in reticulocytes. The
individuals with G6PD deficiency of the Mediterranean and patient may experience back pain. Hemoglobinuria and jaun-
Canton types. Some chemicals isolated from fava beans dice may also be evidence of the hemolytic process. The clini-
destroy red cell glutathione, leading to symptomatic hemoly- cal features of the two most common variants are compared in
sis in susceptible G6PD-deficient individuals. Table 10-3.
The hemolytic episode in Gd A— is usually self-limiting.
Clinical Manifestations Young cells that are produced in response to the anemia have
levels of G6PD that are nearly normal’ and have better sur-
The majority of G6PD-deficient persons are asymptomatic vival characteristics. Hemolysis associated with G6PD Med
most of the time and go through life without ever being aware is more easily induced, usually more severe, and has been
of their genetic trait. G6PD enzymatic activity that is 20% of reported to result in death on occasion. Red blood cell trans-
normal or even slightly less is sufficient for normal red fusions may be indicated for hemolytic episodes in patients
cell function and survival under ordinary circumstances. with G6PD Med.

Figure 10-3 ™ Heinz bodies, using peripheral blood from a patient Figure 10-5 @ Fava beans.
with G6PD deficiency, stained with the supravital stain, crystal violet.
200 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies

with blood and is placed on filter paper, G6PD converts the

Table 10-3 Comparison ofClinical NADP to its reduced form, NADPH. When the filter paper is
“Features of GdA~and observed under fluorescent light, those erythrocytes that fail

Gd Med (GdiB= vais ote to convert NADP to NADPH (i.e., are deficient in G6PD) will
lack fluorescence. The quantitative assay of G6PD is based on
Clinical Feature Gd A-— Gd Med the measurement of the rate of reduction of NADP to NADPH
measured at 340 nm.2’ G6PD variants can also be identified
Cells affected Aging All erythrocytes
via electrophoretic methods.
by defect erythrocytes
Hemolysis with drugs Unusual Common The diagnosis of G6PD deficiency during an acute
Hemolysis with Common Common hemolytic episode may be difficult. The deficiency may be
infection obscured by the younger erythrocyte population (which has
Favism No Occasionally
more G6PD) as the older G6PD-deficient erythrocytes are
Degree of hemolysis | Moderate Severe
Transfusions required No Occasionally destroyed.
Chronic hemolysis No No
Hemolytic disease Rare Occasionally
of newborn Enzyme Deficiencies: Glycolytic
Pathway
A number of hereditary enzyme deficiencies have been
Laboratory Testing described in the RBC glycolytic pathway (Embden—Meyerhof
pathway). Most of these glycolytic enzyme deficiencies are
G6PD deficiency should be suspected after a clinical episode
inherited autosomal recessive and do not demonstrate any
of acute hemolysis after administration of chemical or thera-
abnormal RBC morphology. This differentiates them from RBC
peutic agénts known to cause the reaction in patients with the
membrane defects and hemoglobinopathies, which do have spe-
disorder. Laboratory changes of the nonspecific type include:
cific abnormal RBC morphology associated with the specific
a fall in hemoglobin (and hematocrit), hemoglobinuria (urine
disease (see Chaps. 9 and 11). These inherited glycolytic
can turn brown to almost black secondary to the presence of
enzyme deficiencies generally cause chronic nonspherocytic
hemoglobin), Heinz bodies in the erythrocytes, evidence of
hemolytic anemia (CNSHA), which vary in the degree of sever-
hemolysis in the serum, elevated serum bilirubin levels, and
ity. The ability to compensate for anemia caused by the hemol-
markedly decreased or absent haptoglobin levels. Generally
ysis is reflected in the increased production of RBCs in the bone
there are no significant alterations in leukocyte or platelet
marrow and reticulocytosis. Reticulocytes still have cytoplas-
counts or function.
mic organelles and are capable of protein synthesis and oxida-
Laboratory investigation of hemolytic anemia when there
tive phosphorylation leading to ATP production. Several
is evidence (family history or drug sensitivity, or both) of
enzymes including pyruvate kinase (PK), hexokinase (HK), and
G6PD deficiency may include several screening procedures.
aldolase have a much higher activity in reticulocytes and are
Oxidative denaturation of hemoglobin results in formation of
often termed the “age-related” enzymes.
Heinz bodies. These small particles of precipitated hemoglo-
bin can be visualized by supravital staining using certain basic
dyes such as crystal violet (see Fig. 10-3). Heinz bodies will Pyruvate Kinase Deficiency
appear as small (1- to 4-um) purple inclusions, usually on the
cell periphery. They are not seen with Romanowsky stains In 1960 DeGruchy and associates**”’ reported that some patients
such as Wright’s stain. Although Heinz bodies may be seen in with hereditary nonspherocytic hemolytic anemia (HNSHA)
some other enzyme deficiencies, they are not seen in PK defi- had elevated red blood concentrations of 2,3-diphosphoglycer-
ciency, which is the second most common RBC enzyme defi- ate (2,3-DPG). This elevation suggested a block in anaerobic
ciency. Some unstable hemoglobins also form Heinz bodies glycolysis further down the pathway (see Fig. 10-1). The
after incubation of erythrocytes at 37°C for 48 hours. enzyme was identified in 1961, when a severe deficiency of red
Other test procedures that may be used to screen for blood cell PK was found in three patients with HNSHA.*° PK
G6PD deficiency include the methemoglobin reduction test™* catalyzes one of the reaction steps in the Embden—Meyerhof
and the ascorbate-cyanide test.*° In the methemoglobin reduc- pathway of anaerobic glycolysis. Because mature red blood
tion test, which is a simple and sensitive screening procedure, cells lack mitochondria, they are dependent on anaerobic gly-
G6PD-deficient erythrocytes fail to reduce methemoglobin in colysis for the generation of adenosine 5’-triphosphate (ATP).
the presence of methylene blue. The ascorbate-cyanide test, The diminished capacity to generate ATP in PK-deficient red
which measures perioxidative denaturation of hemoglobin, is blood cells results in cell membrane fragility and a hemolytic
not specific for G6PD deficiency, because it will yield moder- anemia.
ately positive results if the patient has PK deficiency or cer- Since its discovery in 1961, more than 300 cases of PK
tain unstable hemoglobins. deficiency have been reported, and many of these were cases
The fluorescent spot test and the specific G6PD assay are of variant enzymes with different biochemical characteris-
positive only with G6PD deficiency.*° When a mixture of tics.*'*? The nucleotide sequence of cDNA for the human
glucose-6-phosphate, NADP, saponin, and buffer is mixed PK gene and sequence of several of the mutations that cause
 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: II. Hereditary Enzyme Deficiencies 201

HNSHA have been described.***4 PK deficiency is the most Interestingly, these patients may tolerate exercise to a
common enzymatic disease involving the anaerobic gly- greater degree than might be expected from the extent of their
colytic pathway of the red blood cell. Together, G6PD defi- anemia. Red blood cell concentrations of 2,3-DPG are increased
ciency and PK deficiency constitute most cases of HNSHA up to three times the normal levels in patients with PK defi-
arising from red blood cell enzyme deficiencies. ciency because of the enzyme block* (see Fig. 10-1). The
increase of 2,3-DPG decreases the affinity for O,, which is more
MODE OF INHERITANCE readily released to the tissues where it is needed. For this reason,
PK deficiency is inherited as an autosomal recessive trait, transfusion therapy should be based on the patient’s tolerance of
but true homozygotes are rare and are restricted to children the anemia. Removal of the spleen benefits some patients
of consanguineous parents. The most common mode of because it increases the life span of the altered red blood cells.
inheritance is that of double heterozygosity; that is, when
two mutant variants of the PK enzyme are simultaneously LABORATORY TESTING
inherited from each parent.*?*>° To date, approximately 20 The peripheral blood smears of patients with PK deficiency
different mutations of the PK gene are known to produce typically show a normochromic, normocytic anemia with
hemolytic anemia.*’ Thus, the clinical symptoms of PK varying degrees of reticulocytosis. Accelerated erythropoiesis
deficiency are observed both in true homozygotes and in may result in polychromasia, poikilocytosis, anisocytosis, and
double heterozygotes for the PK gene. Both sexes appear to nucleated red blood cells. Both the hemoglobin and the hema-
be affected equally. There is increasing evidence that PK tocrit levels are decreased from normal. The serum usually
deficiency is worldwide in distribution, with most of the has a moderate increase in unconjugated bilirubin, and the
cases reported to date in northern Europe, the United States, haptoglobin level is decreased or absent.*°-*”
and Japan. Other cases have been reported in Australia, Several screening tests may be used to distinguish the
Canada, China, Costa Rica, Hong Kong, Italy, Mexico, the nonspherocytic anemia of PK deficiency from the anemias of
Near East, New Zealand, the Philippines, Saudi Arabia, hereditary spherocytosis and the unstable hemoglobinopathies.
Spain, and Venezuela.*°*3*! These tests are nonspecific and serve only as a mechanism for
The Pennsylvania Amish have a high frequency of PK classifying the type of anemia. Diagnosis is made on the basis
deficiency, which has been traced back to a single immigrant of specific testing for the PK enzyme.
couple. In affected families, consanguinity is common. Thus Screening tests may include the osmotic fragility test and
the PK deficiency in the Amish population is the result of a the autohemolysis test (see Chap. 31), as well as the antiglobu-
true homozygote condition.” lin test and red blood cell survival tests. Erythrocytes that are
PK-deficient have osmotic fragility which is nearly normal
PATHOGENESIS when the test is performed on freshly drawn blood. If the blood
PK deficiency results in a decreased capacity to generate ATP is incubated for a few hours, some patients exhibit an increase
(see Fig. 10-1 and Chap. 3). The ATP-requiring membrane in osmotic fragility.'” Sterile defibrinated blood is used to per-
pumps that maintain the proper electrochemical gradients form the test for autohemolysis. When normal erythrocytes are
begin to fail with decreasing concentrations of ATP. This incubated in their own serum at 37°C, they gradually lyse, show-
results in cell water loss with cell shrinkage, distortion of cell ing up to 3.5% lysis after 48 hours. Erythrocytes from patients
shape, and increased membrane rigidity.**** These membrane with nonspherocytic anemias, as well as those with hereditary
abnormalities lead to premature destruction of the red blood spherocytosis, demonstrate an increased amount of autohemol-
cells in the spleen and liver with consequent anemia. It has ysis. When glucose is added before incubation, erythrocytes
been shown that PK-deficient reticulocytes consume six to from the patient with hereditary spherocytosis demonstrate a
seven times more oxygen than normal reticulocytes.** In most decreased amount of autohemolysis. The addition of glucose
cells, the drop in ATP regeneration because of a block in the does not correct the increased autohemolysis of PK-deficient
glycolytic pathway would be compensated for by oxidative erythrocytes. The antiglobulin test in PK deficiency is negative,
phosphorylation, but that capacity is lost in red blood cells as and the red blood cell survival is decreased.
they mature and they lose mitochondria. There is a fluorescence screening test, which is relatively
simple and sensitive. It is used for the presumptive diagnosis
CLINICAL MANIFESTATIONS of PK deficiency. The test is based on the following coupled
The severity of the hemolytic disease associated with PK defi- enzyme assay:
ciency varies from mild to severe, depending on the properties
PEP +2 ADP + Ms** PK enzyme
Pyruvate + ATP
of the mutant enzymes.***>“° True homozygotes are anemic
and jaundiced at birth and may require repeated transfusions
Pyruvate + NADH + Ht —##“» Lactate + NAD*
during life. Less severely affected patients may come to clini-
cal attention later in childhood or early adulthood because of (UV fluorescence) (No fluorescence)
anemia, jaundice, or an enlarged spleen. The hemolytic ane-
mia is often more pronounced during periods of infection or This assay takes advantage of the fact that NADH fluo-
other stresses. There is an increased incidence of pigmented resces when it is illuminated with long-wave ultraviolet (UV)
gallstone formation in these patients, as is true with all chronic light, whereas NAD does not fluoresce. Phosphoenolpyruvate
hemolytic disorders. (PEP), NADH, adenosine diphosphate (ADP), Mg’*, and
202 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies

lactate dehydrogenase (LDH) are added to a patient sample of phosphate isomerase (GPI) is the third most commonly
blood, which is spotted on filter paper and examined with a identified enzyme deficiency in the glycolytic pathway.
UV light. If the blood lacks PK enzyme, NADH will not be Although there have been reports of other enzyme deficien-
oxidized and the fluorescence will persist for 45 minutes to cies (glycolytic and nonglycolytic), not all such deficiencies
an hour. If the blood is normal and has the PK enzyme, the flu- have been associated with hemolytic anemia. The other
orescence will disappear in 15 minutes, because NAD* does reported glycolytic enzyme deficiencies are summarized in
not fluoresce.***’ It should be noted that leukocytes contain a Table 10-4, and the prevalence and characteristics listed.”
PK isoenzyme that will also catalyze the same reaction. There- Laboratory tests are available to assay many of the spe-
fore, blood must be centrifuged and plasma and buffy coat cific enzymes. Some of these tests may be available only
removed prior to testing the erythrocytes. In addition, patients through reference laboratories. Most laboratories, however,
who have recently been transfused may have enough donor will be able to screen patients with a suspected hemolytic ane-
cells remaining in circulation to give erroneous test results. mia caused by enzyme deficiency. The antiglobulin, erythro-
Any abnormal fluorescence spot test should be followed cyte survival, autohemolysis, osmotic fragility, and Heinz
with a confirmatory quantitative PK enzyme assay. This body tests can all be useful in distinguishing the enzyme
involves the same coupled reaction mechanisms as previously deficiencies from hereditary spherocytosis and the unstable
described, but the conversion of NADH to NAD* is measured hemoglobinopathies.
spectrophotometrically at 340 nm under standard conditions.
Most PK-deficient individuals have 5% to 25% of normal
activity.°°47 Enzyme Deficiencies: Methemoglobin
Reductase Pathway
Other Enzyme Deficiencies Methemoglobin Reductase Deficiency
of the Glycolytic Pathway
Hemoglobin that is oxidized from the ferrous to the ferric state is
Except for the deficiencies of PK, reports of hereditary called methemoglobin. Normally, about 1% of the circulating
enzyme deficiencies are relatively rare. The prevalence of PK hemoglobin is in the form of methemoglobin. The balance
deficiencies has been reported to be 51 cases per million between methemoglobin formation and reduction is maintained
white population based on gene frequency studies.** Glucose by the NADH-methemoglobin reductase (also called diaphorase)

Enzyme Prevalence Characteristics

Hexokinase deficiency 17 families described Wide range of severity described from

a compensated chronic hemolytic
anemia to severe neonatal hemolysis
and death. Splenectomy is generally
beneficial to the patient.
Glucose-6-phosphate 50 families described Chronic hemolytic anemia of variable
isomerase deficiency (GPI) Second in frequency severity. Viral or bacterial infections
to PK deficiency can precipitate hemolytic crises.
Hydrops fetalis is more common in
GPI deficiency than in any other
enzyme deficiency.
Phosphofructokinase deficiency 30 families described. Patients may exhibit a mild hemolytic
Approximately one third of the patients anemia with or without myopathy
are of Jewish descent. depending upon the RBC PFK
isoenzyme deficiency.
Aldolase deficiency Very rare, 6 deficiencies in Moderate chronic hemolytic anemia,
5 families described. mental retardation, and myopathy.
Triosephosphate isomerase 14 cases described RBC TPI activity is not maturation
deficiency (TPI)
dependent. Neonatal hemolytic anemia
with hyperbilirubinemia (usually
requiring an exchange transfusion),
progressive neurological dysfunction,
cardiomyopathy, and infection. Most
individuals die in childhood
before 6 years of age.
Phosphoglycerate 14 cases described Chronic hemolytic anemia of varying
kinase deficiency severity, myopathy, and CNS dysfunction
 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies N 03

pathway. Methemoglobinemia may occur either when there is

decreased enzyme activity or when production of methemoglobin
exceeds the reducing capacity of the enzyme system. Hereditary Case Study
deficiency of NADH-methemoglobin reductase results in
A 26-year-old African American man was referred to the clin-
increased levels of methemoglobin. This congenital deficiency is
ical laboratory for investigation of reported hemoglobinuria.
inherited as an autosomal recessive trait.°° Heterozygotes do not
The patient had recently been diagnosed as having infectious
usually show signs of methemoglobinemia unless challenged
mononucleosis. The following laboratory data were obtained:
with certain drugs.
The major clinical feature of methemoglobinemia is RBC 8 LOMA
cyanosis. Because methemoglobin cannot carry oxygen, some Hgb 11.0 g/dL
patients exhibit symptoms similar to those of anemia, and Het 32%
some patients develop a compensatory mild polycythemia (see MCV 86.0 fL
MCHC 34.0%
Chap. 19). The course of this disorder is generally benign; how-
WBC 9.5 X 10°/L
ever, Some patients are treated only because they find their life- Differential
long cyanosis to be a cosmetic hardship. In cases of severe Segmented neutrophils 40%
cyanosis, methylene blue is administered intravenously to acti- Bands 3%
vate the NADH-methemoglobin reductase system. Lymphocytes 48% (many atypical)
Monocytes 1%
Acquired methemoglobinemia can also occur in healthy
Eosinophils 2%
individuals when drugs or other toxic substances oxidize Platelets Adequate
hemoglobin in circulation. Patients who appear to be sympto- Reticulocytes 14.5% (uncorrected)
matic from methemoglobinemia should be treated promptly
with intravenous methylene blue.
The red blood cell (RBC) morphology was normochromic
In addition to the hereditary deficiency of NADH-
and normocytic. Polychromasia was noted. A slight poikilo-
methemoglobin reductase, methemoglobinemia may be cytosis was also noted, with some red cells showing irregu-
caused by the hemoglobin M diseases (see Chap. 11). lar protrusions. On further investigation, the antiglobulin
The laboratory differentiation of the types of methe- test result was found to be negative. On the basis of the
moglobinemia is shéwn in Table 10-5. Methemoglobin has antiglobulin test, the hemolytic process was considered not
a maximum absorbance band at 630 nm. The addition of to be the result of an immune reaction. A normal hemoglo-
cyanide causes the band to disappear, and the change in bin electrophoresis was reported.
absorbance is directly proportional to the concentration of The hematologist suggested that the patient return in
methemoglobin.*?' Methemoglobin is increased to varying 30 days for testing for erythrocyte enzyme deficiency. At
that time the patient was found to have an adequate erythro-
degrees in all three disorders; enzyme activity is decreased
cyte G6PD content. Spuriously elevated G6PD levels may
only in hereditary NADH-methemoglobin reductase defi-
be found during or immediately after the hemolytic episode
ciency. Hemoglobin electrophoresis produces normal-
because of the presence of younger red blood cells.
appearing results in patients with methemoglobinemia
except in the hemoglobin M diseases. QUESTIONS TO CONSIDER
. What does the reticulocyte count in this patient represent?
Acknowledgment . What is the corrected reticulocyte count?
WN. What type of G6PD correlates to this patient’s familial
This work was supported in part by the Olla Stribling Fund. background?
4. What RBC inclusion might also be found on the periph-
eral blood smear with supravital stains?
5. What further testing can be performed in diagnosing
G6PD deficiency?
6. What type of hemolysis is present here?

Lari anit itaandainitanivicateeinaehmenmenteenpeiatriameaiarcoemeaTeeemmmemaerceeneteeemeenetmeemeememetemeseiemmemenaseerammmememanenamemenemmemmeaene

‘apie 10-5Laboratory Differentiation of Methemoglobinemia

Methemoglobinemia Resulting From Methemoglobin Level Enzyme Activity Hemoglobin Electrophoresis

Hereditary enzyme deficiency Increased Decreased Normal

Toxic substance exposure Increased Normal Normal
Hemoglobin M disease Increased Normal Abnormal
204 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: II. Hereditary Enzyme Deficiencies

Qu esevens
1. What is the most common glycolytic enzyme deficiency a. Increased formation of Heinz bodies
associated with the pentose phosphate pathway (aerobic b. Lack of fluorescence in the fluorescent spot test
pathway)? c. Failure to reduce methemoglobin in the presence of
a. PK deficiency methylene blue
(Dy G6PD deficiency ()AII of the above
c. Hexokinase deficiency 5. Which laboratory test result would indicate a patient who
d. Glutathione reductase deficiency is PK-deficient?
2. What is the most common glycolytic enzyme deficiency @) Abnormal rate of hemolysis that is independent of the
associated with the Embden—Meyerhof pathway (anaero- presence or absence of glucose in the incubation
bic pathway)? medium of the autohemolysis test
(@)PK deficiency b. Lack of fluorescence in the fluorescent spot test
b. G6PD deficiency c. A change in the indicator from red to yellow in the
c. Hexokinase deficiency orthocresol red test
d. Glutathione reductase deficiency d. Increase in osmotic fragility
3. Oxidative denaturation of hemoglobin results in formation 6. What deficiency causes hemoglobin to be oxidized from
of small particles that are visualized with supravital stain- the ferrous to the ferric state’?
ing. What is the term for these particles? a. G6PD deficiency
a. Basophilic stippling b. PK deficiency
b. Howell—Jolly bodies NADH-methemoglobin reductase deficiency
c. Pappenheimer bodies d. Lactate dehydrogenase deficiency
@ Heinz bodies
See answers at the back of this book.
4. In the evaluation of a patient for G6PD deficiency, which
of the following test results would indicate a deficiency of
the enzyme?

nny

“-') SUMMARY CHAF w Patients with G6PD abnormalities are usually asympto-
matic until exposed to conditions of lowered oxygen ten-
sion, certain chemicals or substances (including fava
w Hereditary nonspherocytic hemolytic anemia (HNSHA)
beans), and some medications.
encompasses a group of disorders associated with red
blood cell (RBC) abnormalities. mw GOPD is diagnosed by clinical symptoms and laboratory
determinations. Laboratory testing includes finding the
m Glucose-6-phosphate dehydrogenase (G6PD) enzyme
presence of Heinz bodies in erythrocytes (not specific),
abnormalities are the most common cause of HNSHA.
electrophoresis of G6PD, and, where indicated, molecular
m= GOPD is an enzyme in the hexose monophosphate (or genetic studies.
pentose phosphate) shunt pathway.
@ PK deficiency, which was first identified in 1961, is asso-
mw GOPD enzyme variants are noted for their association ciated with a diminished capacity to generate ATP, result-
with particular racial and ethnic backgrounds. ing in fragile red blood cells and a hemolytic anemia.
m Pyruvate kinase, an essential enzyme of the Embden— @ Laboratory diagnosis of PK deficiency requires specific
Meyerhof pathway, is the second most common enzyme testing of PK activity. A fluorescent screening test is used.
abnormality associated with HNSHA.
@ Other red cell enzyme deficiencies include methemoglo-
m There are more than 400 G6PD variants. The gene for bin reductase and glucose phosphate isomerase deficien-
G6PD is mapped to chromosome Xq28. cies. Almost any enzyme of the aerobic and anaerobic
m Clinical expression of G6PD deficiency is more often metabolic pathways has been implicated in HNSHA,
evident in men because it is a sex-linked (X chromosome) although the levels of hemolysis are quite variable and
abnormality. hemolysis may be entirely absent.
m Deficient G6PD activity results in reduced glutathione
levels, which cause increased oxidative denaturation of
hemoglobin and subsequent hemolysis.
 Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: Il. Hereditary Enzyme Deficiencies 205

REFERENCES 16. Beutler, E: The molecular biology of . Miwa, S, and Fujii, H: Pyruvate kinase
G6PD variant and other red cell enzyme deficiency. Clin Biochem 23:155,
— . Dacie, JR, et al: Atypical congenital
defects. Annu Rev Med 43:47, 1992. 1990.
haemolytic anemia. Q J Med 22:79,
We Chiu, DTY, et al: Molecular characteri- . Miwa, S, et al: Concise review:
I)sy3}.
i). Carson, PE, et al: Enzymatic deficiency
zation of glucose-6-phosphate dehydro- Pyruvate kinase deficiency: Historical
genase (G6PD) deficiency in patients perspective and recent progress of mol-
in primaquine sensitive erythrocytes.
of Chinese descent and identification ecular genetics. Am J Hematol 42:31,
Science 124:484, 1956.
of new base substitution in the IPDS.
. Yoshida, A: Glucose-6-phosphate dehy-
human G6PD gene. Blood 81:2150, is)ies). Tani, K, et al: Human liver type pyru-
drogenase of human erythrocytes.
OB. vate kinase: Complete amino acid
I. Purification and characterization of
. Beutler, E: Glucose-6-phosphate sequence and the expression in mam-
normal (B+) enzyme. J Biol Chem
dehydrogenase: New perspectives. Blood malian cells. Proc Natl Acad Sci USA
241:4966, 1966.
PENSE, WIS). SolOZ OS ss
Beutler, E, and Yoshida, A: Genetic vari-
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~— . Vulliamy, TJ, et al: Diverse point muta-
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Inherited Disease. McGraw-Hill, New 38. Feng, CS, et al: Prevalence of pyruvate
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base transition is the basis of the com- haemolytic anaemia. Br J Haematol defective enzymes and concentrations of
mon human glucose-6-phosphate dehy- W327, Uo. high-energy phosphates with the severity
drogenase variant A(+). Genomics . Valentine, WN, et al: A specific gly- of clinical manifestations. Eur J Haema-
15228, 1987. colytic enzyme defect (pyruvate kinase) tol 49:82, 1992.
. Beutler, E: Glucose-6-phosphate dehy- in three subjects with congenital non- 46. Rapaport, SI: Introduction to Hematol-
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324:169, 1991. Assoc Am Physicians 74:100, 1961. 1987, Chaps 6 and 7.
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47. Perkins, S: Hereditary erythrocyte disor- 48. Beutler, E, and Gelbart, T: Estimating the 50. Jaffe, ER: Hereditary methemoglobine-
ders due to deficiencies of the glycolytic prevalence of pyruvate kinase deficiency mias associated with abnormalities in the
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 Chapter

Hemolytic Anemias
Intracorpuscular Defects:
Ill. The Hemoglobinopathies
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
David Yang, MD
Hallye Zeringer, MT(ASCP)SH

Introduction and Review of OBJECTIVES

Normal Hemoglobin
Structure At the end of this chapter, the learner should be able to:

Classification 1. Characterize hemoglobinopathies.

Hemoglobinopathies . Define qualitative and quantitative hemoglobin defects.
Nomenclature
. Explain the nomenclature for abnormal hemoglobins.
Sickle Cell Anemia
Sickle Cell Trait ° . Name the amino acid substitution found in sickle cell anemia.
Hemoglobin C Disease and
. List factors contributing to the sickling process.
Trait
Hemoglobin D Disease and . Name and describe the three types of sickle cell anemia.
Trait
. List tests useful in the laboratory diagnosis of sickle cell disease.
Hemoglobin E Disease and
Trait . Describe the effects on hemoglobin S cells when parasitized by Plasmodium falciparum.
Hemoglobin O,,., Disease
CO
ed
Oy. List characteristics for sickle cell trait.
ENS)
ONG
ae
CO
\Oo
and Trait
Hemoglobin S with Other . Describe the goals of treatment for sickle cell anemia.
Abnormal Hemoglobins . Name the amino acid substitution found in hemoglobin C disease.
Hemoglobin Variants with
Altered Oxygen Affinity . List findings for hemoglobin C disease.
Unstable Hemoglobins . Identify the laboratory findings that provide a diagnosis of hemoglobin SC disease.
Methemoglobinemia
. List characteristics for hemoglobin D, hemoglobin E, and other variants and combi-
General Summary of nations such as hemoglobin O,,.,, and hemoglobin SD.
Laboratory Diagnosis
13: Identify causes of methemoglobinemia.
Case Study
16. Recognize useful techniques for studying hemoglobin variants with altered oxygen
affinity.

207
208 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies

Introduction and Review of Normal Chromosome 16

Hemoglobin Structure
C a2 al
Hemoglobinopathies are defined in the broadest sense as con-
_ aL OC
ditions in which there are either qualitative or quantitative
abnormalities in the synthesis of hemoglobin. More than
900 hemoglobin variants have been described.'* However, the
majority of these variants are not clinically significant, because
Chromosome 11
the abnormality does not result in a defect in the structural
integrity or function of the hemoglobin molecule. The hemoglo-
binopathies are either inherited according to classic mendelian
genetics or arise from new genetic mutations. ee
More than 90% of the hemoglobin variants are single
Figure | 1-2 ® Location of the globin genes on chromosomes
amino acid substitutions in the alpha (a), beta (8), delta (6), or
16 and 11.
gamma (y) globin chains as a result of a single-point mutation
in one of the globin genes. Hemoglobinopathies are inherited
as codominant traits. When both parents carry one gene that
codes for an abnormal hemoglobin (such as HbS), there is a
25% chance with each pregnancy that the infant will be
homozygous (inheriting two genes for HbS), resulting in
sickle cell anemia* (Fig. 11-1C). A total of eight genes are Globin
Chains Hemoglobin Normal (%) Development
inherited on two homologous chromosomes that code for
polypeptide globin chains. The a and zeta (C) globin genes are QE, Gower 2 Embryo
located on chromosome 16, with two a and one ¢ globin gene Oe Gower | Embryo
per chromosome. The B, 5, y, and epsilon (€) globin genes are OY> Portland Embryo
Q,Y> Fetus
located on chromosome 11, with one B, 5, €, and two y globin
Qpey> Fetus
genes per chromosome (Fig. 11-2). a8, Adult
A brief review of normal hemoglobin structure is provided 08> Adult
here to aid in understanding the hemoglobinopathies; however,
the reader is referred to Chapter 3 for a more detailed discussion.

Hemoglobin is a conjugated protein composed of iron, proto-

porphyrin IX type HI, and globin. The combination of iron
and protoporphyrin is referred to as the heme moiety. The glo-
bin portion of the molecule consists of four polypeptide
chains, each with an attached heme group. These heme moi-
eties are positioned so that they are suspended in the center of
the polypeptide chains. This configuration provides an envi-
ronment where iron can exist in the reduced (ferrous) state, a
feature that is critical for oxygen transport and delivery. There
are six known polypeptide chains that make up the different
globin portions of the hemoglobin molecule: a, B, y, 5, €, and
¢. The latter two represent embryonic globin chains. The com-
position of normal physiologic hemoglobins is reviewed in
Table 11-1.

Figure 1 1-1 @ Inheritance of abnormal hemoglobins. A. With one

parent heterozygous for an abnormal hemoglobin, the offspring Classification
have a one-in-two chance of carrying the trait. B. With one parent
homozygous for an abnormal hemoglobin, all offspring will carry Classification of hemoglobinopathies is somewhat arbitrary.
the trait because that parent can contribute only an abnormal gene.
Hemoglobin disorders may be divided into two very broad cat-
C. With both parents heterozygous for the abnormality, the chances
are one in four for normal, two in four for heterozygous, and one in egories: qualitative and quantitative hemoglobinopathies. In
four for homozygous. D. With both parents carrying the same the qualitative category, hemoglobins differ in the sequence
abnormal hemoglobin—one homozygous and one heterozygous— of the amino acids composing the globin chain. This category
the offspring have a 50-50 chance of being either homozygous or is the one usually referred to in the general discussion of hemo-
heterozygous. £. With parents carrying two different abnormal
hemoglobins, offspring have a one-in-four chance of not inheriting
globinopathies. Quantitative defects are those characterized by
an abnormality, a one-in-two chance of carrying the trait for one or decreased production of hemoglobin resulting from a decreased
the other abnormality, and a one-in-four chance of carrying both synthesis of one particular globin chain, which is commonly
abnormalities in codominance. known as thalassemia (see Chap. 12).
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies 209

long or short subunits, and (3) the occurrence of fusion

subunits. !

* Abnormal hemoglobins without clinical significance Nomenclature

* Aggregating hemoglobins (structural abnormalities with
amino acid substitution away from the crevice of the heme, Investigators began naming the abnormal hemoglobins with
i.e., HbS, HbC)
capital letters; but with the end of the alphabet rapidly
* Unbalanced synthesis of hemoglobin (thalassemia)
° Unstable hemoglobins approaching, they began using the names of places. A letter
¢ Hemoglobins with abnormal heme function (structural plus a place name indicates identical mobility on electrophore-
abnormalities with amino acid substitutions near the sis, but there are different substitutions. It should be noted, in
crevice of the heme) general, that there is more than one abnormal hemoglobin
with the same letter designation (1.€., HBC georgetown? HBChariem>
HbG ppitadetphias HOGgantose» HPO arabs HBO jndonesia)-/ The description
A more inclusive method of classification allows divi- of the variant can also involve identifying the chains and the
sion of the hemoglobinopathies into five major categories: substitution. For example, homozygous HbS is a,f,° or a,8.°™"
or a8.°°"™". Hemoglobin Gpritacdeipnia the most common c-chain
1. Abnormal hemoglobins without clinical significance
variant in the black population, is written a,9P""B, or a,°%sB,
2. Aggregating hemoglobins (i.e., sickle cell anemia)
or a,°"bsB,. In addition, the exact helix of the secondary
3. Unbalanced synthesis of hemoglobin (thalassemia)
structure and the position in that helix can be indicated. For
4. Unstable hemoglobins
example, the designations would be a,f,%4*? for HbS and
5. Hemoglobins with abnormal heme function
a, ®!9B, for HbG ppitadeiphias
This classification of hemoglobinopathies is summarized in
Table 11-2.
Sickle Cell Anemia

Hemoglobinopathies HISTORIC OVERVIEW

In 1910, Herrick* described a 20-year-old black student from
The majority of hemoglobinopathies (hemoglobin variants) the West Indies as suffering from a severe hemolytic anemia in
result from B-chain abnormalities. Many of these variants which peculiarly elongated, “sickled” red cells were found on
have no associated physiologic consequences. There also can his peripheral blood smear. The classic hematologic features
be a-, y-, and 6-chain abnormalities, but these conditions are included not only the presence of sickle cells on the peripheral
usually clinically benign. Some individuals with B-chain blood smear but also nucleated red blood cells indicative of a
abnormalities present with abnormal physical properties severe anemia, cardiac enlargement, icterus, and leukocytosis.
resulting in clinical disease. From the first description of a The presence of target cells as well as a normocytic, nor-
sickle cell by Herrick in 1910,* research efforts continue to mochromic anemia is also generally associated with hemoglo-
define, understand, and treat these abnormalities associated binopathies. Sickle cell anemia represents the most common
with inherited abnormal hemoglobins. type of severe hemoglobinopathy, with an estimated preva-
Most hemoglobinopathies arise from a single amino acid lence in the United States of 1 in 375 African American live
substitution. For example, when valine substitutes for glutamic births.° [t is estimated to affect more than 50,000 Americans.*°
acid in the sixth position of the B chain, hemoglobin S (HbS) is
produced rather than hemoglobin A (HbA). When lysine DEFINITION
replaces glutamic acid at position 6 of the 8 chain, hemoglobin The term sickle cell disease is used generically to describe a
C (HbC) is produced. These changes truly represent a molec- group of genetic disorders characterized by the production of
ular alteration; a feature that was initially appreciated by the abnormal HbS.*° Sickle cell anemia (HbSS disease) is the
Linus Pauling in the late 1940s when he won the Nobel prize most common type of sickle cell disease and represents the
for defining sickle cell anemia as a molecular disease.’ The homozygous form, in which the individual inherits a double
abnormality was demonstrated by electrophoresis to be dose of the abnormal gene that codes for HbS. This type of
located in the protein portion of the hemoglobin molecule. hemoglobin differs from normal hemoglobin by the single
Other substitutions can cause instability of hemoglobin, such amino acid substitution of valine for glutamic acid in the sixth
as deformation of the three-dimensional structure, oxidation position from the NH,-terminal end of the 8 chain (Fig. 11-3).
of the ferrous iron, or alteration in residues that interact with The structural formula for sickle cell anemia (HbSS) is
heme, with 2,3-diphosphoglycerate (2,3-DPG), or at subunit a,B.°C"™". The formula alternatively may be written as a,8,°
contact points.' or «8°. The gene for HbS occurs with greatest frequency in
At the molecular level, a single-base DNA substitution tropical Africa, particularly Central Africa. Many social and
in the corresponding triplet codon produces one amino acid historic factors, such as slavery and conquest, are responsible
change, which is the most common cause of a hemoglo- for the appearance of this hemoglobin in North American and
binopathy. Other molecular changes that are more rare Middle Eastern populations.’ In the United States, the birth
include (1) multiple-base substitutions, (2) the production of incidence of the homozygous state (HbSS) is approximately
210 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

of sickled cells: reversible and irreversible.* Sickling of the red

cell is reversible up to a point. However, repeated sickling
eventually damages the red blood cell (RBC) membrane per-
manently. Originally, the formation of rigid sickled cells was
thought to cause microcirculatory obstruction because of
impaired erythrocyte deformability during capillary transit.
However, the actual mechanism involves several other ele-
ments, accounting for the phenotypic heterogeneity of this
disease. These mechanisms involve: |. the adhesion of erythro-
cytes to the endothelium of the postcapillary venule; 2. the for-
mation of aggregates composed of irreversibly sickled cells and
leukocytes contributing to vascular occlusion and inflamma-
tion; 3. neutrophil transmigration through vessel walls adding
to further increased inflammation in the microvasculature;
and 4. dysregulation of vasomotor tone by disturbance in the
Figure 1 1-5 @ Amino acid substitution in hemoglobin S. function of vasodilator mediators such as nitric oxide.’ In addi-
tion, abnormal cation homeostasis caused by hemoglobin S
polymers leads to sickle red cell dehydration, producing dehy-
0.26% (1 in 375 African American babies).*’ It is estimated drated, dense, irreversibly sickled cells that not only result in
that 8% to 10% of American blacks carry the trait (one gene) hemolytic anemia, but also play a crucial role in the initiation
for HbS. of vaso-occlusion.” Vaso-occlusion then further lowers pH and
Although in the United States sickle cell disease is most oxygen tension and increases the numbers of sickled cells,
commonly found in persons of African ancestry, it has also resulting in both acute and chronic tissue damage. This injury
been found in individuals from the Caribbean, South and results in painful crises and the infarction of organs. It should
Central America, Mediterranean (Turkey, Greece), the Middle be noted that the presence of hemoglobin F (HbF) and HbA, in
East, and India. The common variants of sickle cell disease red cells with HbS modifies the degree or severity of the sick-
are listed in Table 11—3, with their estimated incidence. ling.*’!° Other factors affecting the severity of HbS are listed
in Table 11-4.
PATHOPHYSIOLOGY
HbS is soluble and usually causes no problem when properly CLINICAL FEATURES
oxygenated. However, when the oxygen tension decreases, this The hallmark features of sickle cell disease are chronic
single amino acid substitution in the B-globin chain of HbS hemolytic anemia and vaso-occlusion, resulting in ischemic tis-
polymerizes, forming tactoids or fluid polymers (Fig. 11—4).* sue injury.°” All tissues and organs within the body are at risk
As these polymers realign, they cause the red cell to deform for damage as a result of the vascular obstruction produced by
into the characteristic sickle shape (Fig. 11-5). The sickling the sickled red cells. Organs at greatest risk include the spleen,
process is dependent on the degree of oxygenation, pH, and kidney, and bone marrow because, in these organs, blood flow
dehydration of the patient.* Decreases in oxygenation and pH, is slow in the venous sinuses and there is a reduced oxygen ten-
as well as dehydration, promote sickling. There are two types sion and low pH." The eye and head of the femur are also
target sites for ischemic injury because of the limited terminal
arterial blood supply to these areas.

Incidence in African American

Disorder Live Births

Sickle cell anemia 1 in 375 (0.26%)

(HbSS)
Sickle cell C disease 1 in 835 (0.12%)
(HbSC)
Sickle 8 thalassemia 1 in 1,667 (0.06%) fins

Source: Sickle Cell Disease Guideline Panel. Sickle cell disease:

Screening, diagnosis, management, and counseling in newborns and
infants. Clinical Practice Guideline No 6. AHCPR Pub No 93-0562.
US Department of Health and Human Services, Rockville, MD, Figure 11-4 @ Scanning electron micrograph (SEM) of sickle cells.
April 1993, (From Bell, A: Hematology. In: Listen, Look and Learn. Health Edu-
cation Resources, Inc., Bethesda, MD, with permission.)
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies 211

Figure 11-5 m Sickle cell disease (peripheral blood). Note the

sickle-shaped red cells and target cells. (From Bell, A: Hematology.
In: Listen, Look and Learn. Health Education Resources, Inc.,
Bethesda, MD, with permission.)

Sickle cell anemia is usually diagnosed early in life,

when the level of HbF declines. HoSS disease typically pre-
sents as a severe chronic hemolytic anemia, with hemoglobin
levels in the range of 6 to 8 g/dL. Characteristically the Figure 1 1-6 @ Asthenic physique with mild jaundice. (Reproduced
patient demonstrates an asthenic physique and is mildly jaun- with permission from Sandoz Pharmaceuticals Corporation.)
diced (Fig. 11-6). Many complications are associated with the
disease, with the major manifestations being “‘sickle crises.” sequestration is the cause, resulting in a decrease in hemoglobin
There are three types of crises: aplastic, hemolytic, and and hematocrit, which usually occurs in infants and young chil-
painful (vaso-occlusive).°”?:!? dren between 5 months and2 years of age.°’*!* Intrasplenic
An aplastic crisis is usually associated with infections, pooling of vast amounts of blood results in enlarged spleens of
particularly to parvoviruses, which cause a temporary sup- some children with HbSS disease. The usual clinical features of
pression of erythropoiesis. The marrow is simply overworked a hemolytic crisis include sudden weakness, rapid pulse, faint-
as a result of the stress related to the continuous stimulus for ness, pallor of the lips and mucous membranes, and abdominal
production of new red cells. With an already shortened red fullness caused by the enlarged spleen.*’'* In contrast to this
cell life span, even a temporary decrease or arrest in red cell process, multiple infarctions and subsequent fibrosis lead to a
production causes a drastic anemia. During the evaluation of process termed autosplenectomy in adult patients with HbSS
the febrile patient, a fall in the reticulocyte count can also disease, which results in a small, fibrotic, and nonfunctional
indicate the onset of aplastic crisis, which requires further spleen.
monitoring of the hemoglobin level in the HbSS disease Vaso-occlusive or painful crisis is the hallmark of sickle
patient. Aplastic crises usually spontaneously resolve within cell anemia.*’” The crisis is usually associated with severe pain,
5 to 10 daysr7== caused by occlusion of small blood vessels mediated by the
A hemolytic crisis reflects an acute exacerbation of the adhesion of sickled cells to endothelium, resulting in tissue
anemia with a resulting fall in hemoglobin and hematocrit, an damage and necrosis.”'' The decreased blood flow causes
increased reticulocyte count, and jaundice.°””'* Acute splenic regional hypoxia and acidosis, further exacerbating the ischemic
injury. A painful crisis usually lasts 4 to 6 days but sometimes
persists for weeks.>° Painful crisis can be precipitated by infec-
tion, fever, acidosis, dehydration, and exposure to extreme cold.
Some patients have reported that even emotional states such as
anxiety, stress, and depression may cause their painful crises.
Generally, three principles of therapy are applied in the
Amount of HbS Vascular stasis management of painful crisis:
Other hemoglobins Temperature
Thalassemia pH |. Adequate rehydration
G6PD deficiency Viscosity 2. Pain relief using sufficient analgesics
Deoxygenation MCHC
3. Antibiotic therapy to treat any precipitating or underlying
_ Amount ofHbF ety atanom
illness such as infection.®7!7:!9
“G6PD = pe 6. roe Po ee MCHC= mean
corpuscular hemoglobin concentration. In severe cases, exchange transfusion may be necessary to
reduce the hemoglobin S content in the blood of the patients
212 ~— Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

with HbSS disease. However, the mainstay of therapy for

painful crises is hydration (administration of fluid volumes) to
Table 11-6 Clinical Features of
correct fluid and electrolyte deficits in an attempt to maintain Sickle Cell Anemia by
normal serum electrolyte concentrations. Category — 7
Symptoms and clinical manifestations of HbSS disease
Hematologic
are many and varied. The most prominent types of clinical
manifestations associated with sickle cell anemia are listed in ¢ Aplastic crisis
Table 11-5. As mentioned previously, these clinical presenta- ¢ Hemolytic crisis
¢ Vaso-occlusive crisis
tions represent the sequelae of repeated infarction. A more
comprehensive list is provided in Table 11-6, which divides Nonhematologic
the clinical features of HbSS disease into hematologic and e Abnormal growth
nonhematologic categories. ¢ Bone and joint abnormalities
¢ Pain
VASCULOPATHY Occlusion of blood vessels and tissue ° Salmonella infection
¢ Hand-foot dactylitis
ischemia can occur virtually anywhere in the body: in bones,
¢ Genitourinary
joints, lungs, liver, kidneys, eye, central nervous system, and ¢ Renal papillary necrosis
spleen.®’ In the lungs, sickling in the pulmonary microvascula- ¢ Priapism
ture produces the acute chest syndrome in HbSS disease ¢ Spleen and liver
patients. It is the second most common cause of hospital admis- e Autosplenectomy
¢ Hepatomegaly
sion and the most common complication of surgery and anes-
¢ Jaundice
thesia.'*° The acute chest syndrome represents an acute illness ¢ Cardiopulmonary
characterized by fever, chest pain, prostration, and the presence ¢ Enlarged heart
of pulmonary infiltrates on the chest x-ray exam.’*'*:'* The syn- ¢ Heart murmurs
drome in adults is characteristically a result of pulmonary ¢ Pulmonary infarction
infarction, although other causes such as bacterial or viral e Eye
¢ Retinal hemorrhage
infection have been reported. This contrasts with the acute ¢ Central nervous system
chest syndrome in children with HbSS disease, which is usu- ¢ Leg ulcers
ally caused by an infectious agent. While children have a ¢ Risky pregnancy
higher incidence of acute chest syndrome, their mortality rate is
lower than that of adults.'° Pleuritic chest pain is the dominant
symptom of acute chest syndrome in adults, whereas fever,
cough, and tachypnea are often the only complaints in infants
and young children who are affected.’
The most common cutaneous manifestation in HbSS dis-
ease is the development of ulcers or sores on the lower leg
(Fig. 11-7). Approximately 8% to 10% of patients develop
leg ulcers, which are usually manifested between 10 and
50 years of age and are very difficult to resolve.*’'? The eti-
ology of leg ulcers is unclear; however, trauma, infection,
severe anemia, and warmer temperatures may predispose to
their formation. They rarely develop in individuals with sickle
cell C disease (HbSC) or sickle B thalassemia.'?

Table 11-5 Clinical Manifestations

of Sickle Cell Anemia
Cutaneous manifestations (leg ulcers)
Cardiac enlargement
Joint and skeletal problems
Arthritis
Renal complications (renal papillary necrosis)
Bone marrow infarctions
Conjunctival vascular abnormalities
Gastrointestinal symptoms
Hepatomegaly
Autosplenectomy
Cholelithiasis Figure 11-7 @ Leg ulcers in a patient with sickle cell anemia.
Priapism (persistent, painful penile erection) (Reproduced with permission from Sandoz Pharmaceuticals
Corporation.)
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies 213

Bones and joints in HbSS disease are frequent sites of

pathology, with musculoskeletal pain being the most common
symptom. There can be bone marrow hyperplasia, infection,
or infarction. Infarction is also commonly responsible for the
symptoms of the hand-foot syndrome observed in sickle cell
anemia. Dactylitis (the painful swelling of the hands and feet)
occurs commonly in infants and young children with HbSS
disease and is observed exclusively in patients in that age
group.®’”'* In many infants it is the first manifestation of the
disease. The characteristic “hand-foot” syndrome develops
later in life as a result of microinfarction of small bones of the
hands and feet, which leads to unequal growth and bone
deformities of the fingers and toes (Fig. 11-8). In addition,
episodes of painful swollen joints and aseptic necrosis of the
femoral head and other articulating bones are caused by the
process of infarction.

INFECTIONS Serious bacterial infections remain a major

cause of morbidity and mortality in patients with HbSS dis-
ease. The organisms implicated in causing infections in these
patients are listed in Table 11-7.
The most significant cause of death during early child-
hood is the severe overwhelming septicemia and meningitis
caused by Streptococcus pneumoniae.®”'*'> In HbSS disease,
splenic dysfunction develops during infancy and predisposes
the infant to overwhelming infections from encapsulated bac-
teria, such as S. pneumoniae and Haemophila influenzae.®"\*
After the first decade of life, anaerobic and enteric organisms
become important pathogens, causing infections in adult
patients with HbSS disease. Repeated splenic infarcts result
in autosplenectomy by the adult years. These patients then
become more prone to serious infections with encapsulated B.
organisms. Infections in patients with HbSS disease cause Figure | 1-8 @ Hand-foot syndrome in a patient with sickle cell
greater morbidity, disseminate more rapidly, and are more dif- anemia. (Reproduced with permission from Sandoz Pharmaceuticals
ficult to resolve than infections in healthy individuals. In par- Corporation.)
ticular, pyelonephritis recurs regularly in these patients, is
difficult to treat, and is often associated with septicemia.°”'?
This infection results in a predisposition to sickling in the

Table 11-7 Organisms implicated iin Causing its adnie in Patients

with Sickle Cell Anemia

Bacterial Viral F ungal — LEHERLE

te eres pneumoniae Rubeola Coenen: immitis Pn. spp.

Haemophilus influenzae Cytomegalovirus Histoplasma capsulatum
Neisseria meningitidis
Mycoplasma
pneumoniae
Staphylococcus aureus
Streptococcus pyogenes
Mycobacterium tuberculosis
Escherichia coli
Salmonella spp.

spp = species.
214 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies

renal papilla, ultimately causing renal papillary necrosis, presents itself in the second decade of life. The third decade in
which frequently develops along with the pyelonephritis (see HbSS disease is characterized by interstitial nephritis, papillary
the following discussion of Sickle Cell Nephropathies). necrosis, pyelonephritis, and the nephrotic syndrome, to name
The multiple factors responsible for the increased sus- a few of the renal disorders that may develop. In the fourth
ceptibility to infection in patients with HbSS disease are out- decade and beyond, end-stage renal disease develops as one or
lined in Table 11-8. more of the sickle cell nephropathies results in chronic renal
The relationship between the incidence of the malarial falurce2
parasite and the frequency of the abnormal HbS gene requires It should be noted that hyperuricemia and gross hema-
further explanation. Malaria, caused by a parasite of the turia occur commonly in patients with HbSS disease. Hyper-
Plasmodium species, is still a serious disease in tropical areas, uricemia occurs in approximately 15% of children and 40%
with Plasmodium falciparum being responsible for the most of adults with HbSS disease because of the increased urate
life-threatening situations. The original geographic distribu- production associated with the accelerated erythropoietic rate
tion of sickle cell disease overlaps with that of malaria. As in and decreased renal clearance of urate.'* Gross hematuria
thalassemias, these hemoglobinopathies are believed to pro- occurs commonly not only in sickle cell anemia but also in
vide some kind of selective advantage for malaria. This is sickle cell trait because of the sickling, stasis, ischemia, and
applicable only to subjects carrying one abnormal HbS gene extravasation of blood in the kidney.!*!7"*
(sickle cell trait). The precise mechanism of this protective
effect from malaria is not known; however, cells carrying
STROKE Stroke occurs in up to 22% of patients with HbSS
HbS, when parasitized by P. falciparum, may sickle more
disease.”'*!*-'°7° Stroke represents an array of neurologic
quickly than will nonparasitized cells, leading to preferential
complications caused by an ischemic or hemorrhagic lesion in
destruction of the parasitized cells.'° The sickling could affect
a specific cerebral vessel. The neurologic manifestations may
the cycle of the parasite in one of two ways: directly, by
be focal, such as hemiparesis, or more generalized, such as
killing the parasite: or indirectly, by causing the parasitized
coma or seizure. Recurrent episodes of stroke cause progres-
sickle cells to be sequestered in the spleen. The fact that per-
sively greater impairment and increased mortality. The most
sons homozygous for the HbS gene often lack splenic func-
common cause of stroke in children is cerebral infarction.'*'°
tion by the time they reach adulthood (autosplenectomy or
With age, subarachnoid and intracerebral hemorrhage become
functional asplenia) may be one reason why malaria is excep-
increasingly common. A distinguishing pathologic feature of
tionally severe, and often fatal, in these cases. Thus, HbS con-
arterial vessels involved in stroke is the presence of intimal
fers a relative degree of protection from malaria only in the
and medial proliferation, which results in severe restriction of
heterozygous (trait) state. It is balanced by the decreased fit-
blood flow and is found in approximately 80% of angiograms
ness of the homozygous state and the gene frequency eventu-
of patients with sickle cell anemia and stroke.”° Most likely
ally reaches an equilibrium in the population.
this is owing to the interaction of sickled cells with endothe-
lium, which results in endothelial damage, inflammation, and
SICKLE CELL NEPHROPATHIES In the kidney, intravascu-
the local release of growth factors that stimulate proliferation
lar sickling occurs more rapidly than in any other organ owing
of subintimal and medial cells. In the later stages of this vas-
to deoxygenation of HbS in the acidic and hyperosmolar envi-
culopathy, chronic ischemia leads to the formation of a deli-
ronment of this organ.*'* The combination of hypoxia, hyper-
cate subcortical network of collateral vessels that are prone to
tonicity, and acidosis in the kidney causes sickling, stasis, and
rupture, likely accounting for the increased incidence of hem-
ischemia of the renal medulla and papillary tip, leading to pro-
orrhagic stroke with increasing age.” HbSS disease patients
gressive renal events. Eventually, over time, a number of sickle
with hemorrhagic stroke (intracerebral or subarachnoid hem-
cell nephropathies develop. Hyposthenuria, the inability of the
orrhage) have a high mortality rate during the acute stage
kidney to concentrate the urine, is the earliest and most com-
(may be as high as 50%).°7"'8
mon nephropathy in sickle cell disease, occurring usually in the
Patients with a characteristic abnormality of cerebral
first decade.'*'’ Progressive renal pathology occurs in patients
blood flow (higher than normal velocity of flow on transcra-
with HbSS disease as renal tubular dysfunction and atrophy
nial Doppler ultrasonography) have been shown to be at much
greater risk to develop stroke.'° This risk can be significantly
reduced by maintaining these patients on a regular program of
‘Table 11-8s Factors Responsible for
blood transfusion designed to suppress erythropoiesis such
the Increased that no more than 30% of the circulating red cells are the
_ Susceptibility of” = patients own.'? A follow-up study demonstrated that the
Patients with Sickle Cell high risk of stroke returned after transfusion therapy was
Anemia to Infections discontinued.?!

* Reticuloendothelial blockage caused by increased hemolysis

* Stasis of sickled RBCs in the sinusoids of the liver and spleen Sickle Cell Trait
* Secondary splenic dysfunction
* Deficiency of nonantibody serum opsonic activity In sickle cell trait (Fig. 11-9), the heterozygous form of the dis-
ease, individuals inherit both a normal {-globin gene and a
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: II]. The Hemoglobinopathies oa

sickle globin gene (8°). As a result, individuals with sickle cell falling reticulocyte count may herald the onset of such a cri-
trait produce both normal HbA and HbS, with a predominance sis. There may be a neutrophilic leukocytosis with a shift to
of HbA in an approximate ratio of 60:40.3° The structural the left and thrombocytosis. The bone marrow reflects marked
formula is a8,8,°C"". The frequency of this heterozygous erythroid hyperplasia, except during an aplastic crisis.
condition in American blacks is approximately 8%.*° In vitro In individuals with the trait, sickled cells are not typically
experiments demonstrate cells that are homozygous for hemo- present on the peripheral blood smear. On rare occasions, how-
globin S sickle when the oxygen level is decreased to 4% to 6% ever, sickled cells may be observed in the peripheral blood
whereas cells heterozygous for hemoglobin § (sickle cell trait) smear during a crisis episode.
sickle when the oxygen level is decreased to 2%.” Individuals
with HbS trait are usually asymptomatic, but occasionally LABORATORY SCREENING FOR SICKLE CELL DISEASE
episodes of hematuria and hyposthenuria occur because of sick- According to the Clinical Practice Guideline on Sickle Cell
ling in the kidney. The potential for sickling exists, therefore, Disease published by the U.S. Department of Health and
and drastic lowering of pH or reduction in oxygen tension can Human Services in April 1993, all newborns, regardless of
precipitate a crisis. Causes for these include severe respiratory race or ethnic background, should be screened for the pres-
infections, air travel in unpressurized aircraft, anesthesia, and ence of HbS (Table 11—9).°
congestive heart failure. Even excessive exercise can lead to a Newborn screening for sickle cell disease began in the
significant buildup of lactic acid, resulting in sickling and sub- United States in the early 1970s. The initial screening pro-
sequent infarction. Several deaths of American black soldiers grams grew out of the recognition that sickle cell anemia was
with sickle cell trait have been reported as a result of rigorous associated with significant morbidity and mortality. Today
basic training at altitudes greater than 4000 feet, which led to a newborn hemoglobinopathy screening is universally required
buildup of lactic acid, followed by acidosis and subsequent by Law or Rule in the 50 U.S. states, the District of Columbia.
organ infarction.** Puerto Rico, and the Virgin Islands.”
The majority of screening programs currently use iso-
LABORATORY DIAGNOSIS electric focusing (IEF) of an eluate from dried blood spots to
The following laboratory tests should be performed to diag- characterize hemoglobin and a few programs use HPLC or
nose sickle cell disease: the complete blood count, a reticulo- cellulose acetate electrophoresis as the initial screening
cyte count, evaluation of the peripheral smear, hemoglobin method.'*’° Most programs retest abnormal screening speci-
electrophoresis or isoelectric focusing (IEF), and measure- mens using a second, complementary electrophoretic tech-
ment of hemoglobins A, and F by high-performance liquid nique, HPLC, immunologic tests, orDNA-based assays.'* IEF
chromatography (HPLC).°'!** is an equilibrium process in which hemoglobin migrates in a
The chronic anemia of HbSS typically is quite severe, pH gradient to a position of 0 net charge. The formation of
with hemoglobins ranging between 6 and 8 g/dL. The RBC discrete, sharp bands via IEF allows for more precise and
indices are normochromic and normocytic. The peripheral red accurate quantification than standard electrophoresis. HPLC
blood picture can be striking, with numerous target cells, is emerging as the method of choice for the initial screening
fragmented red cells, polychromasia, nucleated red cells, and of hemoglobin variants and quantification of HbA, and HbF.
usually sickled cells (see Fig. 11-5). Siderotic granules and Although a greater number of samples can be processed daily
Howell—Jolly bodies may be seen in the red cells as a result using IEF, the advantages of HPLC are that it is generally eas-
of rapid RBC turnover and “stressed” erythropoiesis. The ier to interpret, less prone to inconsistencies related to sample
average reticulocyte count is between 5% and 20%. This preparation, application, and staining techniques; and 1s fully
count decreases, however, during an aplastic crisis; indeed, a automated, thereby reducing the chance for clerical error.*°
Mass spectrometry (MS) is emerging as a possible screening
methodology with the introduction of robust and simple-to-
use electrospray tandem MS instruments capable of greater
throughput with comparable sensitivity and specificity of IEF
and HPLC, in preliminary studies.”
The definitive test for HbS is hemoglobin electrophore-
sis on cellulose acetate at alkaline pH, followed if necessary
by electrophoresis on citrate agar at acid pH (Fig. 11-10).
Electrophoresis separates different hemoglobins by electrical
charge, and hemoglobin separation by citrate agar elec-
trophoresis depends on the combination of charge and the
ability of the hemoglobin to combine with components in the
agar gel mixture. The patient with sickle cell anemia produces
no normal B chains; therefore, there will be no HbA on elec-
trophoresis (unless the patient has been recently transfused).
HbS constitutes 80% or more of hemoglobin, with HbF rang-
ing from 1% to 20%.°*'°'* When HbF levels are higher than
Figure 1 1-9 m Sickle trait (peripheral blood). Note the normal
appearing smear. 20%, there is a decrease in the severity of the disease.'° High
216 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

Table 11-9 Screening Methods

Cellulose Acetate High-Performance Liquid

Criteria Electrophoresis Isoelectric Focusing Chromatography

Equipment cost $2500 $4000 $30,000

Cost per test (consumables) $0.15-$0.25 $0.35-$0.50 $0.10-$1.75
Samples run per hour 200 72 5-20
Advantages Semiquantitative Sharper bands Automated
Simple to operate Quantitative
Disadvantages Densitometer for Densitometer for Complex to use
quantitation

Note: Labor costs vary with number of samples per run.

Source: Sickle Cell Disease Guideline Panel: Sickle cell disease: Screening, diagnosis, management, and counseling in
newborns and infants. Clinical Practice Guideline No 6. AHCPR Pub No 93-0562. US Department of Health and Human
Services. Rockville MD, April 1993.

levels of HbF are seen transiently in newborns and in heredi- chains to form af dimers compared with normal 6 globin. For
tary persistence of fetal hemoglobin (HPFH). In sickle cell this reason, HbS comprises less than 50% of the hemoglobin in
anemia, the HbA, level may be slightly increased, with a heterozygotes, and a further reduction is seen in HbC trait.
mean of 3.4%.*» Hemoglobins with similar charges have sim- Two previously used screening tests should not be used
ilar migration patterns during electrophoresis, especially on for newborn screening. These are (1) the sickle cell prepara-
cellulose acetate. Hemoglobins D and G both migrate to the tion using sodium metabisulfite and (2) solubility tests using a
same position as HbS at alkaline pH. Hemoglobin E (HbE) concentrated phosphate buffer, a hemolyzing agent, and
and hemoglobin O,,.., (HbO,,..,) migrate in the same position sodium dithionate. These tube solubility tests either isolate
as HbC. Citrate agar electrophoresis 1s very useful, because it HbS at an interface or cause the abnormal hemoglobin to pre-
clearly separates HbS from HbG and HbD, and HbC from cipitate (Fig. 11-11). Both tests depend on the concentration of
HbE and HbO,,,,, at acid pH.*' In sickle cell trait, hemoglobin HbS in the red cell or hemolysate. These tests are inappropri-
electrophoresis at alkaline pH shows 60% HbA, 40% HbS, ate screening tests because they are not sufficiently sensitive to
and usually elevated HbA, (mean is 3.6%).° At acid pH, one detect low levels of HbS and do not distinguish between sickle
band is present in the A position (HbA + HbA,), whereas the trait and different forms of sickle cell disease.'*°
other band migrates to the S position (see Fig. 11—10).° The predominance of HbF and low level of HbS in cells
It should be noted that most hemoglobin variant traits, of the neonate is believed to explain the unreliability of these
without coexistent conditions such as iron deficiency, or tha- tests during the newborn period. Although sickle cell disease
lassemia, have alkaline electrophoretic patterns with an approx- should not be diagnosed from either a sickle cell preparation
imate 60:40 ratio of normal to abnormal hemoglobin. This is or solubility test, because neither of these tests will reliably
because of the effect of charge on assembly of globin chains: distinguish sickle cell trait from sickle cell anemia, many
relatively positively charged globins, such as S (+1) and C hospitals still perform these tests on adult patients. It is impor-
(+2) have a slight disadvantage in assembling with the a tant to note that some rare hemoglobins also sickle, giving

A2/C S ze as C S A/Ap F

Normal

Control

AS (trait)

S disease

C disease

SC disease

—apl—_—_—————————_> + +<—— apl —>—

A. Cellulose Acetate (pH 8.4) B. Citrate Agar (pH 6.0-6.5)

Figure | 1-10 @ Electrophoretic patterns of hemoglobin on (A) cellulose acetate, run at pH 8.4, and (8B) citrate agar, run at pH 6.0 to 6.5.
apl = point of application.
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies DAG

Table 11-10 Examples of Rare

emoglobins That Sigetatt
Band Gives
e Tube Solubility =

HDC yariem

Hb Cg cactows

H Ziquinchor

HDS Mempnis

HbSpravis

1D Mesaers

Dpono-Atesre

in therapy of sickle cell disease has been the introduction of

prophylactic penicillin therapy, which has virtually elimi-
nated pneumococcal sepsis, one of the major causes of death
in children with sickle cell disease.** Twice-daily administra-
tion of oral penicillin reduces both morbidity and mortality
from pneumococcal infection in HbSS disease in infants.°’**
In addition, administration of age-appropriate immunizations
should be given, including pneumococcal, conjugated
Negative Positive H. influenzae, and hepatitis B vaccines.'*
A variety of drugs are being tested for their potential in
Figure | 1-11 @ Tube solubility screening test for sickle cell anemia. ameliorating the effects of sickle cell anemia. Development of
drug therapies is based on the pathophysiology of sickle cell
disease.*!* Studies in vitro have shown that increasing HbF
positive solubility tests. Table 11-10 lists some of the rare decreases HbS polymerization; this effect is based on the
hemoglobins that sickle. Because HbS, HbG, and HbD all reduction in concentration of HbS and on a direct inhibition
migrate in the same position on cellulose acetate elec- of polymerization by HbF. A number of candidate agents for
trophoresis, it is helpful to know that HbG and HbD do not increasing HbF have been evaluated in the past 15 to 20 years.
give a positive tube solubility test. Several studies in small groups of patients with HbSS disease
Molecular diagnosis of hemoglobin disorders via auto- showed an increase in HbF levels and an increase in the frac-
mated DNA-based techniques is available, but these techniques tion of cells containing HbF (F cells) with hydroxyurea ther-
remain expensive and are most effective when targeted to a apy. These findings were dramatically confirmed in the
specific mutation. Nevertheless, they are useful when compre- randomized, double-blind multicenter study of hydroxyurea
hensive characterization is required or when prompt diagnosis in patients with sickle cell disease (MSH).” This study demon-
is needed in a patient who has had a recent transfusion.”° strated a 50% reduction in the frequency of painful crises, and
of the incidence of acute chest syndromes, transfusion of blood,
TREATMENT and hospitalization.” Hydroxyurea had much less dramatic
With advances in the diagnosis, treatment, and prevention of effects on the chronic anemia of sickle cell disease, inducing a
complications, the life expectancy of individuals with sickle significant but modest (on average, 0.7 g/dL) increase in hemo-
cell disease has improved. There is an 85% chance that globin levels. Half of the patients had no increase or only triv-
infants born in the United States with HbSS disease will sur- ial increments in HbF levels.*? The benefit induced by the
vive to age 20.°”3 Delay in diagnosis and treatment resulting hydroxyurea therapy on the polymerization tendency of HbS
from lack of appropriate health services plays an important (estimated with measurements of the delay time for HbS poly-
role in overall morbidity and mortality in developing coun- merization) is of a magnitude to transform HbSS disease into
tries. It is believed that in Africa, 50% of infants with sickle the milder but still clinically relevant disorder similar in sever-
cell disease die in the first year of life. A large number of ity to HbSC disease.*! Thus, the erythrocytes of hydroxyurea-
young African children also die in the first decade of life from treated patients maintain a very significant polymerization
pneumococcal sepsis, malaria, meningitis, acute splenic capability. In addition, the percentage of dense, dehydrated
sequestration, or aplastic Giislcme cells, which is most likely associated with organ damage and
The principal causes of death in infants with HbSS dis- vaso-occlusion, is reduced but not totally abolished by hydrox-
ease in the United States include overwhelming infections yurea therapy. Therefore, a significant proportion of cells that
with S. pneumoniae, cerebrovascular accidents, and acute can readily polymerize are still present in hydroxyurea-treated
splenic sequestration crises.”'* One of the greatest advances patients. There are also concerns regarding the short-term
218 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: II]. The Hemoglobinopathies

marrow toxicity and long-term possible carcinogenic potential

Table 11- unTreatment ofSickle Cell
of hydroxyurea. These concerns are heightened by the use of
hydroxyurea in children, in whom it has been shown to provide
significant clinical benefits.**~* It is thus still necessary to Therapeutic A uicriies
improve on the positive response induced by hydroxyurea with
Increase the production of fetal hemoglobin in the adult.
similar or different drugs.
Decrease microvascular entrapment of sickled cells.
Alternative agents that increase HgF levels include the Modify the oxygen affinity or solubility of sickle hemoglobin.
less toxic analog of 5S-azacytidine, 5-aza-2’-deoxycytidine Change the volume of the sickle erythrocyte.
(decitibine), and short-chain fatty acids.*°’° Decitibine may Alter expression of the abnormal, sickle gene.
affect HbF levels by causing hypomethylation of the y-globin Prevent endothelial damage.
Counter oxidant-induced injury.
genes, and short-chain fatty acids, such as arginine butyrate,
are thought to enhance y-globin gene expression by inhibiting
histone deacetylase.*”
Dehydration of erythrocytes is uniquely seen in HbSS
disease and results in increased cellular concentrations of HbS. clearly the risks and benefits of the therapy, and to charac-
Because polymerization of HbS is proportional to HbS con- terize the natural history of those surviving free of sickle
centration, modulating the pathways of cellular dehydration, cell disease. Encouraging results have been reported show-
particularly the Ca**-activated K*-channel (Gardos channel) ing disease-free survival of approximately 80% to 85%,
and K*—Cl- cotransport, has been the focus of several recent however, 5% to 10% of patients died of complications, with
investigations. Oral magnesium supplementation induced graft versus host disease (GVHD) as the leading cause
inhibition of K*—Cl- cotransport and clotrimazole inhibition of death.*** The results highlighted both the curative
of the Gardos channel are currently being investigated in potential of bone marrow transplantation, but also the
human subjects.**°? severe limitations, which include constrained matched
A nonionic surfactant copolymer, poloxamer 188, has
been shown to reduce the duration and speed the resolution
of painful events by decreasing the microvascular entrap-
ment of sickle cells through modifying interactions of sickle
cells or other blood cells with endothelium.*° Finally, the
dysregulation of vasomotor tone seen in sickle cell disease
may respond to treatment with apo-lipoprotein (apo) A-1 ie Ehegapentic Approach Drees pte
mimetics, which have been shown to inhibit superoxide pro-
Inhibition of HbS / Urea Nocatee
duction and lead to improved vasodilation in sickle mice.*! polymerization Ethanol hemoglobin
The goals of these therapeutic approaches to the treatment Peptides modification
of sickle cell anemia are listed in Table 11-11. Various
approaches to specific therapy with a specific drug are listed Cyanate ~ ] Covalent
in Table 11-12. Pyridoxal + hemoglobin
Glyceraldehyde | — modification
Blood transfusions may be required for acute situations
and for prevention of certain complications of HbSS disease, DDAVP Erythrocyte
such as stroke.'? Simple transfusion has little or no benefit for Cetiedil modification
treatment of acute sickle vasculopathies, unless it is associ- Hyponatremia ch (increase in
ated with removal of sickle erythrocytes. In fact, transfusion red cell
volume,
only increases blood viscosity and further compromises
decrease in
peripheral blood flow. Exchange transfusions are indicated in 2,3-DPG)
the acute setting such as stroke, severe acute chest syndrome,
or acute priapism.°”'* For HbSS patients with higher hemat- Hydroxyurea Genetic
ocrits (20% or greater in children, 25% or higher in adults) an 5-Azacytidine : modification
exchange transfusion technique may be safer than a simple (increase in
gamma gene
blood transfusion.*”!? The general considerations and indica-
globin
tions for blood transfusions in HbSS disease are listed in expression,
Table 11-13. Chronic blood transfusions have been shown to bone marrow
reduce the risk of stroke.'?! When discontinuing chronic transplanta-
transfusions becomes necessary (for iron overload concerns, tion)
presence of multiple alloantibodies, or other reasons) patients Decreased erythrocyte { Nifedipine _} Vasodilator
should be carefully followed, because they have an increased microvascular
risk for recurrent strokes.?!“? entrapment
Bone marrow transplantation is currently being inves- DDAVP= 1- denne: 81D-arginine vasopressin.
tigated in the United States and in Europe to define more
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies 219

Consideration y indicanons

To improve the oxygen-carrying transport Severely anemic as reflected byee Asst fivporension
in red cells by simple transfusions high-output cardiac failure, angina, or cerebral dysfunction
Sudden fall in hemoglobin or hematocrit levels during acute
splenic or hepatic sequestration crisis
Hemoglobin level < 5.0 g/dL or hematocrit of 15% in patients
who exhibit fatigue and dyspnea along with erythroid
hypoplasia or aplasia
To improve microvascular perfusion by decreasing the Life-threatening events, such as cerebrovascular accidents
number of red cells containing HbS by partial exchange including stroke and transient ischemic attacks (TIAs)
transfusion Arterial hypoxia syndrome (fat embolization)
Acute progressive lung disease
Unresponsive acute priapism
Eye surgery when performed under local anesthesia and in the
nonanemic * patient
Source: Chance, S, et alal (Eds):pvenr
1 Peteandhen of Sickle Cell Disease. US pene of Health fed Human
Services, Public Health Service, National Institutes of Health. NIH Pub 91:2117, August 1991.

donor availability, a narrow application to the youngest cells, and transplantation of the hematopoietic stem cells back
patients in good clinical condition, and rates of transplant- into the diseased mouse to cure immunodeficiency.** Theoreti-
related morbidity and mortality that are somewhat fixed.* cally, a similar approach can be applied to treat sickle cell ane-
Nevertheless, as the experience of transplantation for sickle mia, and successful phenotypic correction of the sickle cell
cell disease has expanded, there has been a transition from mutation by gene replacement has already been demonstrated
using this modality as an experimental intervention in mouse embryonic stem cells.*”
reserved for those most severely affected to one in which
younger children with early signs of sickle-related morbid- Hemoglobin C Disease and Trait
ity are targeted for treatment.** Umbilical cord blood units
(CBUs) are now considered an acceptable source for Hemoglobin C (HbC) disease is found almost exclusively in
hematopoietic stem cell transplantation (HSCT) when the black population. HbC differs from normal HbA by the
HLA-identical donors are unavailable. CBUs have been single amino acid substitution of lysine for glutamic acid in
used successfully in HLA-matched sibling HSCT for chil- the sixth position from the NH, terminal end of the B chain
dren with sickle cell disease. (Fig. 11-12). This represents the same substitution point as in
It is now possible to diagnose efficiently the presence or HbS but with a positively charged amino acid. The structural
absence of sickle cell disease in embryos obtained with formula for HbC, the presence of which 1s often referred to as
in-vitro fertilization, prior to implantation.*° This powerful HbC disease, is a,B.°°"’*. HbC is seen with great frequency
technique may help carrier couples by allowing them to select in West Africa, particularly northern Ghana, where the inci-
a healthy child without facing the decision of whether or not dence is 17% to 28%.'° In the United States, only 0.02% of
to abort an affected fetus. blacks have HbC disease.'°° The clinical manifestations are
Gene therapy for HbSS disease is also currently being in- mild chronic hemolytic anemia with associated splenomegaly
vestigated. The goal of gene therapy is to replace the B’-globin and abdominal discomfort. The red cell morphology is typi-
gene with a wild-type B-globin gene in order for the affected cally normocytic, and normochromic or hyperchromic, with
patient to produce healthy cells rather than sickle cells. The numerous target cells (50% to 90%) and occasionally micros-
success of gene therapy depends on the ability of researchers pherocytes, fragmented cells, and folded cells (Fig. 11-13).
to isolate, enrich, and insert genes into hematopoietic pluripo- HbC crystals (Fig. 11—14) or “bar of gold” crystals occur more
tential stem cells and generate safe, stable, erythroid-specific often in the red cells of individuals who have undergone
replacement gene expression at a level that is sufficient to have splenectomy than in those whose spleen is intact. Figure 11—15
a clinical effect.45*7 Successful gene therapy has been shown is a scanning electron micrograph (SEM) of hemoglobin C
in a mouse model of immunodeficiency where embryonic crystals. These crystals can be demonstrated in wet prepara-
stem cells from a Rag2 gene deficient mouse were obtained by tions by washing the red cells and then suspending them in a
implanting nuclei of skin cells cultured from the diseased sodium citrate solution.'’ The reticulocyte count is slightly
mouse into donor mouse oocytes, followed by insertion of the increased. Hemoglobin bands, at alkaline pH, reveal approxi-
missing Rag2 gene by homologous recombination, differenti- mately 95% HbC plus A,, less than 7% HbF, and no HbA.
ation of the embryonic stem cells into hematopoietic stem Hemoglobins E, Oa,., C, and A, all migrate to the same
220 ~~ Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

}
Figure 1 1-12 ® Amino acid substitution in hemoglobin C. Figure 1 1-14 ™ Hemoglobin C disease (peripheral blood). Note
the particular crystals: “bar of gold” and numerous target cells
(postsplenectomy). (From Bell, A: Hematology. In: Listen, Look and
Learn. Health and Education Resources, Inc., Bethesda, MD, with
permission.).
position at alkaline pH; HbC can be separated from these
other hemoglobins at acid pH (see Fig. 11-10).
HbC trait, «.8,B,5"s, is present in 2% to 3% of Hemoglobin E Disease and Trait
American blacks, and these individuals are clinically asymp-
tomatic.°° The only significant finding on the peripheral blood Hemoglobin E (HbE) disease occurs with greatest frequency in
smear is targeting. At alkaline pH, there is approximately Burma, Thailand, Cambodia, Laos, Malaysia, and Indonesia.'*°
60% HbA and 40% HbC plus A. This variant is now prevalent in the United States because of
the influx of refugees from Southeast Asia. The homozygous
state (a,B,°°C"'>s) presents with little or no anemia, target cells,
Hemoglobin D Disease and Trait and microcytic, hypochromic red cell indices. Alkaline elec-
trophoresis reveals approximately 95% to 97% HbE plus A,,
Hemoglobin D (HbD) disease has several variants. The most
and the remainder of the hemoglobin is HbF. HbE migrates
common variant in American blacks is HbDpunjay, Which is
with HbC and HbO,,,,, at alkaline pH, but migrates with HbA
synonymous with HbD,. anscies. [ts frequency is less than
at acid pH. HbE trait (a@,8,8 °C") is asymptomatic clinically.
0.02%.°° Both the homozygous (a,B,'7'S""S"") and the het-
Microcytosis, target cells, and approximately 70% HbA and
erozygous (a,8,B,'7'S""S") states (where glycine is substi-
30% HbE plus A, are noted on routine electrophoresis.'°? HbE
tuted for glutamic acid) are asymptomatic. The peripheral
is slightly unstable, and there is an associated thalassemic com-
blood smear is unremarkable, except for a few target cells.
ponent with this hemoglobin variant. This is responsible for the
HbD migrates electrophoretically to the same position as
microcytosis and the lower than expected quantified value of
HbS and HbG at alkaline pH but migrates with HbA at acid
HbE in HbAE.*!
pH. HbD is a non-sickling soluble hemoglobin.
It has been postulated that HbE may protect against
malaria, because areas such as Thailand that are highly
endemic for malaria also have a high incidence of the HbE
gene. Some authors attribute this effect to the fact that the

Figure 11-15 ™ SEM of hemoglobin C crystals. (From Bell,

Figure | 1-135 = Hemoglobin C disease (presplenectomy). Note A: Hematology. In: Listen, Look and Learn. Health and Education
the numerous target and folded cells. Resources, Inc., Bethesda, MD, with permission.)
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies 221

parasite Plasmodium falciparum multiplies more slowly in

HbE red cells than in the HbAE or HbAA red cells.°

Hemoglobin O,,,,, Disease and Trait

Hemoglobin O4,.;, (HbO.4,.,) disease is a rare hemoglobin vari-
ant that occurs infrequently in black, Arab, and Sudanese pop-
ulations. Homozygous O,,,, (58.'?'%") disease exhibits a
mild hemolytic anemia with slight splenomegaly and target
cells on the peripheral blood smear. This hemoglobin migrates
electrophoretically with HbC, HbE, and HbA, at alkaline pH but
separates at acid pH, migrating in the HbA position (see
Fig. 11-10). In the heterozygous state of HbO,,., (28, B)!7'0"™), }
the patient is asymptomatic.*°
Figure 1 1-16 ®™ Hemoglobin SC disease (peripheral blood). Note
the formation of the SC crystal (yellow arrow) and the polychroma-
Hemoglobin S with Other Abnormal sia (black arrow).
Hemoglobins
As mentioned previously, sickle cell disease is a generic term solubility test results are positive owing to the presence
for a group of genetic disorders that includes sickle cell ane- of HbS. Hemoglobin electrophoresis at alkaline pH separates
mia and hemoglobinopathies in which HbS is found in asso- HbS and HbC in approximately equal amounts (Fig. 11-18).
ciation with another abnormal hemoglobin, and the sickle HbF is usually less than 2% compared with average HbF lev-
8 thalassemia syndromes.'*!**45!°? This section focuses on els of about 6% in sickle cell anemia. Electrophoresis at
defining the disorders that have HbS and another abnormal acid pH confirms the S and C hemoglobins (see Fig. 11-10).
hemoglobin. Sickle 8 thalassemia is briefly mentioned at the Table 11-15 compares the incidence of the most common
end of this section. Fhe common and uncommon forms of hemoglobinopathies found in American blacks.
sickle cell disease are listed in Table 11-14.
HEMOGLOBIN SD DISEASE
HEMOGLOBIN SC DISEASE The combination of HbS and HbD disease, although rare, pre-
Hemoglobin SC (HbSC) disease (a,8,°"'B,°S"*) occurs sents an interesting diagnostic problem. Because these hemo-
when the gene for HbS is inherited from one parent and that globins migrate together at alkaline pH, the electrophoretic
for HbC from the other. About 0.12% of black Americans have pattern is similar to that of HbSS disease. Solubility tests are
SC disease.'!*°° Patients with HbSC disease are generaily less positive.
anemic and experience a milder course than those with HbSS The clinical severity of HbSD disease, however, falls
disease. However, because of increased blood viscosity, this between that of sickle cell anemia and that of sickle cell
condition has a greater incidence of retinal hemorrhage, renal trait.''*°° Acid electrophoresis separates these two hemoglo-
papillary necrosis, and necrosis of the femoral head.'® bins. HbS has its own migration point, whereas HbD migrates
In addition, several cases of renal medullary carcinoma with HbA in the same position (see Fig. 11-10).
have been reported in association with hemoglobin SC dis-
ease and sickle cell trait..*°° Peripheral blood smear findings
include target cells, folded red cells, and occasionally glove-
shaped intracellular crystals (Figs. 11-16 and 11-17).°' The

Common Uncommon

(HbSS) sickle (HbSD) hemoglobin SD disease

cell anemia
(HbSC) sickle-HbC (HbSO,,.») hemoglobin SOa;a,
disease
(HbSB') sickle (HbSE) hemoglobin SE disease
B* thalassemia Figure | 1-17 @ Hemoglobin SC disease (peripheral blood). Note
(HbSB°) sickle (HbS-Lepore) hemoglobin S Lepore the type of “Washington Monument” crystals and target cells.
R° thalassemia (From Bell, A: Hematology. In: Listen, Look and Learn. Health and
Education Resources, Inc., Bethesda, MD, with permission.)
222 Chapter 11. Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

HbS/B thalassemia and other hemoglobin variants that occur

in combination with thalassemia.”*

LABORATORY DIAGNOSIS OF HbS WITH OTHER

ABNORMAL HEMOGLOBINS
When HbS is found in association with another abnormal
hemoglobin, the diagnosis in many instances can be made
from hemoglobin electrophoresis alone. However, it may be
difficult to distinguish between HbSS disease and some of the
sickle B thalassemia syndromes such as HbSB° thalassemia,
HbS8* thalassemia, HbS5B thalassemia, and HbS in associa-
tion with hereditary persistence of fetal hemoglobin syn-
drome (HbS HPFH).'***? In these cases, the electrophoresis
demonstrates only HbS, HbF, and HbA,. It is important
to properly diagnose these disorders because the clinical
Figure 11-18 ® Hemoglobin electrophoretic patterns: (7) HbAC,
(2) HbAS, (3) commercial control, (4) HbSC, (5) HbA-Lepore (see manifestations and subsequent treatment are different. For
Chap.12), and (6) HbAA normal control. example, HbSf° thalassemia is similar in severity to HbSS
disease. Patients with HbS88 thalassemia have few symp-
toms; HbS/B* thalassemia is a milder form, and HbS HPFH is
Table 11-15 Incidence of Common usually asymptomatic with no anemia. Measurement of HbF
and HbA, may be helpful in distinguishing these conditions,
fas Hemoglobinopathies because patients with HbSS, HbSB° thalassemia, HbS5B tha-
* in American Blacks lassemia, and HbS HPFH all have similar electrophoretic pat-
terns.2+°? In HbSB° thalassemia, HbA, levels are greater than
Condition Genotype Incidence (All Ages)
3.5% whereas they are low in patients with HbS6dp tha-
Hemoglobin C a.B°BO 0.02% (1 in 4500) lassemia and HbS HPFH.”*? Generally, HbF levels are higher
disease in all the HbS£ thalassemias in comparison with HbSS.
Hemoglobin C trait a,BABo 3.0% (1 in 33) Assessment of HbF in the parents may be indicated when
Sickle cell anemia aBSB> 0.26% (1 in 375)
8.0% (1 in 13)
HbS HPFH is suspected.***? The clinical and hematologic
Sickle cell trait a,B*B>
Sickle C disease a.BSB° 0.12% (1 in 835) findings in the common variants of sickle cell disease are
Sickle B a,BSB° 0.06% (1 in 1667) summarized in Table 11-16.
thalassemia

Hemoglobin Variants with Altered Oxygen

Affinity
HEMOGLOBIN SO,,,., AND S-Oman DISEASE
The combination of HbS and HbO,,.,, disease can have a clin- High-affinity hemoglobins, which are inherited as an autoso-
ical presentation that is similar in severity to that of HbSS mal dominant disorder, are seen in the heterozygous state.
disease. The anemia is severe, with typical sickled cells seen These hemoglobins bind oxygen more readily and release it
on the peripheral blood smear. This condition might initially less easily to the tissues. The result is tissue hypoxia, which
be confused with HbSC on routine electrophoresis; however, stimulates increased erythropoietin production. This, in turn,
differentiation can be made with acid electrophoresis (see causes a compensatory increase in red cell mass, with
Fig. 11-10). increases in red cell count, hemoglobin, and hematocrit, pro-
Hemoglobin S-Oman (HbS-Oman) is a unique hemo- ducing erythrocytosis and congenital polycythemia. Other
globin variant that carries both the classic sickle mutation hematologic parameters are normal. There is a shift to the left
in B° and one additional mutation in B'*'S'"s, which is the in the oxygen dissociation curve, and a diagnosis is estab-
same substitution observed in HbO,,,,. Heterozygotes for lished by measuring Ps» levels (Fig. 11-19). Individuals with
this variant express between 14% and 20% of the abnormal these hemoglobin variants are asymptomatic.°? For a review
hemoglobin. The higher levels of expression are associated of Ps, refer to Chapter 3.
with a sickle cell anemia clinical syndrome of moderate Hemoglobins with decreased oxygen affinity release oxy-
intensity.°° gen quite readily to the tissues. There is a shift to the right in the
oxygen-dissociation curve (see Fig. 11-19). As more oxygen is
HEMOGLOBIN S/B THALASSEMIA COMBINATION released per gram of hemoglobin, erythropoietin concentrations
The severity of HbS combined with 8 thalassemia depends on fall. This can result in decreased hemoglobin concentration with
the degree of suppression of 8-globin chain synthesis. Hemo- the development of a mild anemia. There may also be mild
globin S/B° thalassemia is a severe condition that clinically cyanosis associated with a decreased oxygen saturation level.
resembles sickle cell anemia; on the other hand, HbS/B* tha- Hemoglobins with increased or decreased oxygen affinities are
lassemia generally has a milder clinical presentation. The listed in Table 11-17. In cases of unexplained erythrocytosis or
reader is referred to Chapter 12 for a detailed discussion of cyanosis, oxygen affinity studies may be helpful.°°
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies 223

Hemoglobin Electrophoresis Hematologic Values*

Disease Clinical Hb RBC

Group Serene S§ (%) F OF A A(%) (g/dL) Retic (%) MCV (fL) Morphology

SS Usually >90 <10 35) 0 6-10 5-20 >80 Sickle cells

marked nRBCs
Normochromic,
normocytic
Anisocytosis
Poikilocytosis
Target cells
Howell-Jolly
bodies
Sp° Thal Marked to >80 <20 235 0 6-10 5-20 <80 Sickle cells
moderate nRBCs
Hypochromic
Microcytosis
Anisocytosis
Poikilocytosis
Target cells
SB* Thal Mild to >60 <20 <i) 20 (A) 9-12 5-10 <5) No sickle cells
moderate Hypochromic
Microcytosis
Anisocytosis
Poikilocytosis
Target cells
SC Mild to 50 ES Ie) o 10-15 5-10 75-95 Occasional SC
moderate crystals
Anisocytosis
Poikilocytosis
Target cells
S HPFH Asymptomatic >70 >30 DS) 0 12-14 1-2 S75) No sickle cells
Anisocytosis
Poikilocytosis
Rare target cells

Se iicnatlonic «values are approximate. There is a tremendousreovantabiiny en diseatesgroups and betweenni individual
patients of the same group, particularly with regard to clinical severity.
SS = sickle cell anemia; SB° Thal = sickle beta zero thalassemia; SB* Thal = sickle beta plus thalassemia; S HPFH =
HbS in association with the hereditary persistence of fetal hemoglobin syndrome; SC = hemoglobin SC disease;
nRBCs = nucleated red blood cells.
Source: Charache, S, et al (eds): Management and Therapy of Sickle Cell Disease. US Department of Health and Human
Services, Public Health Service, National Institutes of Health NIH Pub 91:2117, August 1991.

Unstable Hemoglobins
More than 130 unstable hemoglobins (Fig. 11-20) associated
with hemolytic anemias have been described.*°° Unstable
hemoglobins are hemoglobin variants in which amino acid
Increased O, Affinity Decreased O, Affinity-May substitutions or deletions have weakened the binding forces
and Polycythemia Have Mild Anemia or Cyanosis _ that maintain the structure of the molecule. The instability may
if OB, ilrinos vee “og@™B, cause hemoglobin to denature and precipitate in the red cells
268, Hb 01,%4A50B,, as Heinz bodies. Most unstable hemoglobin variants are inher-
SO oped Hb pisiacite pee ited as autosomal dominant disorders.°° However, absence of a
02877" HD agenogi 2Bo" > positive family history is not always helpful, as new mutations
Dempsey aB5"*™ Abpeth sre are common. Many mutations producing unstable hemoglo-
px Crestiane) ae pana “B04 binopathies are single amino acid substitutions in either the
ara OBC cea 4: a-, B-, y-, or 6-globin chains that affect a few key areas of the
Hb pene 09!" hemoglobin structure. By far, the majority of these substitutions
are in the B-globin chain, followed by an a-chain substitution
224 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies

100 - =

80 Hb Rainier

Q } F Hb Seattle

Seay
=
Hb A. a
—_—
5 y x Hb Kansas
® 40 J
A >
O

20 eff

0 +=
0 20 40 60. 80

Op» pressure (mm Hg) Figure 1 1-20 @ Unstable hemoglobin: Hb Zurich (peripheral
blood). (From Bell, A: Hematology. In: Listen, Look and Learn.
Figure | 1-19 @ Oxygen equilibrium curve of whole blood from Health and Education Resources, Inc., Bethesda, MD, with
subjects with Hb Rainier, Hb Seattle, Hb Kansas and normal permission.)
control (HBA).

medical attention only after exacerbation of the hemolysis

caused by infection and increased temperature or exposure
and only a few in y or 6 chains. Many of the unstable hemo- to oxidative drugs.*! Reticulocytosis is variable. Hypochro-
globins have high oxygen affinity and, therefore, may not mia may be apparent on the peripheral blood smear, and the
cause anemia, making diagnosis in this group of patients mean red cell hemoglobin (MCH) content can be low in
particularly difficult. When anemia is present, the degree of some cases because the unstable hemoglobin may be
hemolysis associated with an unstable hemoglobin varies denatured and “pitted” out of the cell by the mononuclear
considerably.°° Some patients experience severe chronic phagocytic cells of the spleen.
hemolysis with jaundice and splenomegaly. However, most Hemoglobin electrophoresis is usually not a very helpful
patients have a mild compensated condition and seek laboratory method to detect unstable hemoglobins; however,

Table 11-18 Unstable Hemoglobins*

a-Chain Abnormalities 8-Chain Abnormalities

Oo Bp
HD gorina
2 Hy iden 282°” (Glu deleted)
FO rena Ones oa Hees 085!44

Pee Ab Fretbure 05B"° (Val deleted)

aeiaaAccor
EID Cup Mbt eee oR.
Cure aey, [Elle screne 013528?
Hg iopicoke
Ebner cy,!!2Ging, Eee or)al
HD pina Ope joes 0,825"
FID} ouisvitie O5Be es
HB erpersinat apace
ADeurich OnB5=
AD routouse Ou[eyeree
AD pristat Gaon men
Hb 0,8,57A!8
Hbshephera’s Bush CHS
HD seate On Ba eee
AD gras Qe Poreae
HDsania Ana a, (3,88
Boon nin 2B." (5 a.a. deleted)
Deets a,8,2!P
leloyese a1, 28M
aleve 01,31027H
bye. 0t,3!30A8P
HDoimstea Q5Bo)
“Hemoglobins that may precipitate as Heinz bodies after splenectomy: congenital Heinz body hemolytic anemia.
a.a. = amino acids.
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies 225

laboratory tests such as a reticulocyte count, peripheral smear

evaluation, hemoglobin electrophoresis, and, if necessary, mea-
surement of hemoglobins A, and F via HPLC. Selected patient
information such as age, gender, ethnic background, family his-
tory, and physical symptoms is helpful.
Most abnormal hemoglobins are associated with RBC
indices that are normocytic and normochromic. Microcytosis
and hypochromia are seen in some variants (e.g., HbE).
HM iiwaukee
Abnormal red cell morphology may or may not be noted on
HbMyiyae Park the peripheral blood smear, and reticulocyte counts are often
elevated. Screening tests include hemoglobin electrophoresis,
tEP sand HPLC
Cellulose acetate electrophoresis at alkaline pH (see
subtle indications of an abnormality may be observed.*”” Fig. 11-17) is commonly performed first. Electrophoresis on
These include an increased level of HbA,, a common finding citrate agar at acid pH can further differentiate abnormal hemo-
in unstable B-chain hemoglobins, and the presence of minor globins. HbF quantitation should be done, after the newborn
electrophoretic components such as free a-globin chains, period, when this hemoglobin is seen on cellulose acetate.
indicative of an unstable B globin (Table 11—18).5°%” IEF, a type of sensitive electrophoresis, separates hemo-
Most hospitals still perform the isopropanol precipita- globins according to their isoelectric points. Superior [EF
tion or heat denaturation test for detection of unstable hemo- resolution differentiates some hemoglobins that migrate to the
globins. Newer techniques such as IEF can resolve many same electrophoretic point at alkaline and acid pH. On the ini-
hemoglobin mutations with only a very slight alteration in tial patient visit, other laboratory tests should also be per-
their isoelectric point, and globin chain analysis can be per- formed; these include urinalysis, liver function tests, urea,
formed via reversed phase HPLC.*°*” creatinine, and electrolytes. In addition, a chest x-ray film
should be obtained.

Methemoglobinemia
Methemoglobinemia is a clinical condition with methemoglo-
bin levels greater than 1% of the total hemoglobin.°° Methe-
moglobin contains the oxidized ferric form of iron (Fe**)
rather than the ferrous form (Fe?*). In this state, the molecule Case Study
is unable to bind oxygen, which results in cyanosis. The blood
is chocolate-brown in color. In general, there are three causes A 13-year-old black girl was admitted to the hospital appear-
of methemoglobinemia: ing acutely ill with fever and abdominal pain. On physical
examination, an enlarged spleen was evident. Laboratory
1. Hemoglobin M variants (dominant inheritance) test results were as follows:
2. NADH-methemoglobin reductase deficiency (recessive
inheritance; see Chap. 10) Hgb 5.0 g/dL
3. Toxic substance (acquired; see Chap. 10) Het 15%
RBC count 1.4 * 10”/L
There are five variants of hemoglobin M (Table 11-19), WBC count PDS AD ING:
which result from a single amino acid substitution in the a- or Reticulocyte count 1%
B-globin chain that stabilizes iron in the ferric form. If the Differential
substitution occurs in the a chain, cyanosis is present at birth. Segmented neutrophils 62%
Cyanosis does not occur with a B-chain substitution until Bands 12%
approximately 6 months of age.” This correlates with the Lymphocytes 19%
Monocytes 4%
switch from y to B chains. The presumptive diagnosis of
Eosinophils 2%
HbM is made from the absorption spectra of hemolysates, and Basophils 1%
hemoglobin electrophoresis on agar gel at pH 7.1.°° Patients RBC indices Normal
have obvious cyanosis but otherwise are generally asympto- Platelet count 400 X 10°/L
Peripheral blood smear (see Fig. 11-16)
matic. No specific treatment is indicated or possible.

Hemoglobin electrophoresis, alkaline pH, showed one

General Summary of Laboratory band in the HbS position and one band in the HbC position.
Diagnosis The hemoglobins were quantified as 55% HbS and 45%
HbC plus A. Hemoglobins S and C were confirmed by elec-
Hemoglobinopathies usually cannot be diagnosed correctly with trophoresis at acid pH.
a single laboratory procedure. It is usually necessary to correlate continued
the results of a complete blood count and indices with additional
226 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

ANSWERS
Case Seti yout #
1. Numerous target cells are present on the peripheral
blood smear along with some cells that appear to have
QUESTIONS shadows of precipitating intraerythrocytic crystals.
1. Describe the morphological features of this peripheral 2. The data suggest a diagnosis of HbSC disease.
blood smear. 3. The crystals in HbSC disease appear to be only partially
2. In reviewing the electrophoretic data, what diagnosis is formed, or there may be more than one formation. The
suggested? crystals in the red cells often are described as having a
3. Comment on crystal formation in this condition. glove-shaped appearance with several “fingers” protruding.
4. Discuss the clinical presentation of the patient. Is 1t con- 4. Generally HbSC disease has a milder presentation than
sistent with HbSC disease? HbSS disease; however, this patient appears to be expe-
5. This girl’s parents have no hematologic problems; there- riencing a severe episode of SC crisis. This is indicated
fore, for her to have HbSC disease, what would be their by her acute illness, abdominal pain, and decrease in
most likely genotypes? hemoglobin and hematocrit without an increase in the
6. The inheritance of structurally abnormal hemoglobins reticulocyte count.
follows simple mendelian laws. With parents having the 5. With no hematologic problems, one parent would be
trait form of HbS and HbC, what would be the expected expected to have HbS trait (HbAS), whereas the other
genotypes in any of four children? would most likely have HbC trait (HDAC).
continued 6. The probability for each birth would be 25% for HbAA,
25% for HbAS, 25% for HbAC, and 25% for HbSC.

Jicervom’
1. Which of the following is not a characteristic of hemo- ae 5. Which of the following are crises associated with sickle
globinopathies? cell anemia?
a. Abnormal hemoglobins are synthesized. a. Aplastic crisis with low reticulocyte count, and
b. Result from inherited abnormalities or genetic mutations. infections
Cc)AU are manifested in clinically significant conditions. b. Hemolytic crisis with splenic sequestration, decreased
d. Result in a defect in structural integrity or function of emoglobin and hematocrit, increased reticulocyte
the hemoglobin molecule. count, and jaundice
2. Which of the following are used in the nomenclature c. Vaso-occlusive or painful crises with severe pain,
system for abnormal hemoglobins? tissue damage, and necrosis
a. Capital letters d. All of the above
b: Names of places 6. What are the therapeutic goals in the treatment of sickle
c. Names of chains and substitutions cell anemia?
d\All of the above a. Decrease microvascular entrapment of sickled cells or
3. What is the amino acid substitution found in sickle cell change the volume of RBCs
anemia? b. Modify oxygen affinity or solubility of sickle hemo-
ubstitution of valine for glutamic acid in the sixth globin
position from the NH,-terminal 8 chain c. Increase production of fetal hemoglobin
b. Substitution of lysine for glutamic acid in the sixth aa of the above
position from the NH,-terminal 8 chain 7. What is the amino acid substitution found in HbC
c. Substitution of lysine for glutamic acid in the 26th disease?
position from the NH,-terminal ® chain a. Substitution of valine for glutamic acid in the sixth
d. Substitution of valine for glutamic acid in the 121st osition from the NH,-terminal B chain
position from the NH,-terminal 8 chain (Ob ubstitndes of lysine for glutamic acid in the sixth
4. What factors contribute to the sickling of RBCs? position from the NH,-terminal 6 chain
a. Increase in pH and oxygenation c. Substitution of lysine for glutamic acid in the 26th
(b)Decrease in pH and oxygenation, and dehydration position from the NH,-terminal ® chain
c. Increase in pH and decrease in oxygenation d. Substitution of valine for glutamic acid in the 121st
d. Decrease in dehydration and increase in pH and position from the NH,-terminal B chain
oxygenation
 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies a i)~—

8. Which of the following is not true for HbC disease? © Hemoglobin electrophoresis at alkaline pH
a. Mild anemia d.{RBC indices
b. Numerous target cells
10. Which of the following is a cause of methemoglobinemia?
c. Crystals in red cells
a. HbM variants :
(A)HbC can be separated from other hemoglobins at an
b. NADH-diaphorase deficiency
alkaline pH
c. Toxic substances
9. Which finding would be most useful in establishing a di- d,\All of the above
agnosis of HbSC disease?
a. Target cells and sickled cells on peripheral blood smear See answers at the back of this book.
_b. Severe anemia: Increased reticulocyte count

<’} SUMMARY ives fs

g@ In sickle cell disease (HbSS) the red blood cell (RBC)
indices are normochromic and normocytic, with hemoglo-
bins ranging from 6 to 8 g/dL, target cells, sickled cells,
= More than 90% of the hemoglobin variants are single amino
nucleated red cells, and polychromasia.
acid substitutions in the alpha (a)-, beta (B)-, delta (8)-,
or gamma (y)-globin chains as a result of a single-point w The definitive test for HbS is hemoglobin electrophoresis
mutation. on cellulose acetate and citrate agar.

= Hemoglobin S (HbS) is produced when valine substitutes mHbC disease presents with a normocytic, and nor-
for glutamic acid in the sixth position of the B chain. mochromic or hyperchromic anemia, and numerous target
cells, microspherocytes, schistocytes, folded cells, and
m= Hemoglobin C (HbC) is produced when lysine replaces “bar of gold” crystals.
glutamic acid at position six of the B chain.
g Electrophoresis in HbC disease shows 95% HbC plus A),
m Sickle cell anemia (HbSS disease) is the most common
and less than 7% HbF.
type of sickle cell disease and represents the homozygous
form in which the individual inherits a double dose of the @ Hemoglobin SC (HbSC) disease occurs when an HbS gene
abnormal gene that codes for HbS. is inherited from one parent and an HbC gene is
inherited from the other; morphology includes target cells,
m=The characteristic morphology in HbSS disease is the folded red cells, and glove-shaped intracellular crystals.
sickled cell, which increases the viscosity of the blood,
leading to hypoxia, painful crises, and infarction of the # Methemoglobinemia occurs when levels exceed 1% of
spleen, kidney, and bone marrow. total hemoglobin and may be the result of hemoglobin M
(HbM) variants, NADH-diaphorase deficiency, or toxic
mw The three types of crises associated with sickle cell dis- substances.
ease are aplastic, hemolytic, and vaso-occlusive (painful).
w Unstable hemoglobins are hemoglobin variants in which
g In sickle cell disease HbS constitutes 80% or more of the amino acid substitutions or deletions have weakened the
hemoglobin content in addition to HbF plus A). binding forces that maintain the structure of the molecule.
mw Sickle cell trait represents the heterozygous form of sickle g High-affinity hemoglobins, associated with a shift to the
cell disease in which individuals inherit both a normal B- left, bind oxygen more readily and release it less easily to
globin gene and a sickle globin gene. the tissues.
w Individuals with sickle cell trait produce both HbA and w Low-affinity hemoglobins, associated with a shift to the
HbS in a ratio of 60:40. right, release oxygen quite readily to the tissues.
228 Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: Ill. The Hemoglobinopathies

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Cell Anemia Foundation, Augusta, GA, Cell Disease, ed 3. Oxford University 103:2039, 2004.
1998. Press, New York, 2001. 35). Saunthararajah, Y, et al: Effects of 5-aza-
. Pennsylvania State University: HbVar: A . Lee, MT, et al: Stroke prevention in 2°-deoxycytidine on fetal hemoglobin
database of human haemoglobin variants sickle cell anemia (STOP): Extended levels, red cell adhesion, and hematopoi-
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 Chapter

Hemolytic Anemias
Intracorpuscular Defects:
IV. Thalassemia
Russell Aaron Higgins, MD
Chantal Ricaud Harrison, MD

Introduction OBJECTIVES
Genetics of Hemoglobin
At the end of this chapter, the learner should be able to:
Synthesis
Pathophysiology of 1. Name the hemoglobin defect of thalassemia.
Thalassemia 2. Describe the different types of mutation involved in alpha («) and beta (8)
Thalassemia Syndromes thalassemia.
Beta Thalassemia
3. Describe the clinical expression of different gene combinations of a and B
Alpha Thalassemia
thalassemia.
Delta—Beta Thalassemia
and Hemoglobin Lepore 4. Describe the condition known as hereditary persistence of fetal hemoglobin (HPFH).
Syndrome
5. List the complications of a regular blood transfusion program for patients with
Hereditary Persistence of
thalassemia major.
Fetal Hemoglobin
Pancellular and Heterocellular 6. Name the most characteristic laboratory findings for the diagnosis of thalassemia.
HPFH
Thalassemia Associated with 7. List red cell indices that help to distinguish thalassemia from iron deficiency.
Hemoglobin Variants 8. Describe the appearance of the peripheral smear in thalassemia.
A Broad Clinical Classification
of Thalassemia Syndromes 9. Name the test used as a population screening test for thalassemia carriers.
Laboratory Diagnosis of 10. List conditions in which quantitation of hemoglobin F is important.
Thalassemia
11. Describe the use of routine chemistries for differentiation of thalassemia from
Routine Hematology
iron-deficiency anemia.
Procedures
Flow Cytometry
Hemoglobin Electrophoresis
Hemoglobin Quantitation
Routine Chemistry
Differential Diagnosis of
Microcytic, Hypochromic
Anemia
Treatment of Thalassemia
Blood Transfusion in
Thalassemia
Curative Treatment of
Thalassemia
Prevention of Thalassemia
Case Study 1
Case Study 2
Case Study 3

230
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 231

Introduction thalassemia, caused by a defect in the synthesis of B chains.

The original cases described by Cooley were cases of
The thalassemia syndromes consist of a diverse group of genetic homozygous £8 thalassemia.
disorders, which clinically manifest themselves as anemia of The world distribution of thalassemia is summarized in
varying degrees. These disorders are the result of a decrease in Figure 12-1. In North and South America and northern Europe,
production of the globin portion of the hemoglobin. thalassemia has been imported and is present in immigrant pop-
In 1925 Thomas B. Cooley and Pearl Lee described the ulations originating mostly from Italy, Greece, West Africa, and
first cases of severe thalassemia in several North American Southeast Asia. For all practical purposes, thalassemia is absent
children of Mediterranean origin.' “Cooley’s anemia” is still from American Indian populations. The marked similarity in
a commonly used term for this form of severe thalassemia, worldwide distribution of thalassemia with malignant malaria
which is also termed thalassemia major. The name tha- caused by Plasmodium falciparum has been attributed to the
lassemia was actually applied to these clinical syndromes a process of gene selection secondary to a protective effect against
few years later. The term is derived from the Greek word tha- P. falciparum malaria brought about by the heterozygous state
lassa, which means sea, because at that time, all of the cases of thalassemia.’
described were from the Mediterranean coastal region. It Because the hemoglobin structural variants (such as hemo-
is now well known that the distribution of thalassemia is globin S and hemoglobin C in West Africans and African
worldwide and not restricted to the Mediterranean Sea area. Americans or hemoglobin E in Southeast Asians) occur in the
It was later realized that the original severe clinical disease same population in which a or £8 thalassemia is frequent, the
described by Cooley was the result of a homozygous defect two types of genetic defects may be found in the same person,
in hemoglobin production, whereas many milder cases resulting in variability of clinical expression of the two defects.
described as “thalassemia minima” or “thalassemia minor”
were manifestations of a heterozygous defect.
Genetics of Hemoglobin Synthesis
In contrast with hemoglobinopathies such as sickle cell
disease, hemoglobin C, or hemoglobin E, in which the genetic All normal human hemoglobins have a general tetrameric struc-
defect results in a structurally abnormal globin chain, the tha- ture consisting of two alpha-like (alpha or zeta, respectively
lassemia syndromes result from decreased production of one abbreviated as a or C) and two beta-like (beta, delta, A-gamma,
of the globin chains. With a few minor exceptions, the globin G-gamma, or epsilon, respectively abbreviated as B, 5, “y, Cy,
chains produced are structurally normal, but there is an imbal- and €) chains (see Chap. 3). In the normal adult, the majority
ance in production of the two different types of chain, result- (95% to 97%) of the hemoglobin is a,8, (hemoglobin A) and a
ing in an absolute decrease in the amount of normal hemoglo- minor fraction (about 2.5%) is a6, (hemoglobin A,). A small
bin formed, as well as an excess production of one type of amount of hemoglobin F, a,y», (always less than 2%) may also
chain that may precipitate and induce hemolysis. There are be found. The different normal hemoglobins found throughout
two major types of thalassemia: alpha (a) thalassemia, which human development, as well as the abnormal hemoglobins
is caused by a defect in the synthesis of « chains; and beta (3) found in patients with thalassemia are listed in Table 12-1.

SSS§ « Thalassemia
(57) B Thalassemia
RSS88 o + B Thalassemia

Figure 12-1 ® World distribution of alpha («) and beta (8) thalassemia.
 Paha
»
Table 12-1 Composition of Hemos ob
-and Abnormal Hemoglok
Globin Chains Hemoglobin
State

Adult a>
05>
Fetus a*y>
ay,
Embryo O4€> Gower 2
Oe Gower |
Or Portland
a Thalassemia Ba H
4 Bart’s

The ¢- and a-globin genes are found on chromosome 1. Transcriptional mutants affecting one of the promoter
16 and the genes for €-, “y-, “y-, 5-, and B-globins on chromo- sequences
some 11. All globin genes consist of three exons separated by 2. RNA processing mutants, usually affecting introns at the
two introns, with 5° promoter sequences and untranslated splice junction sites or in the consensus sites
regions in 5’ and 3” directions. Each globin gene spans approx- 3. RNA translational mutants due to nonsense or frameshift
imately 1.5 kilobases (Kb). mutations, which produce premature termination and con-
There are two closely linked genes, both active and cod- sistently result in complete lack of production of B chains
ing for identical a globin chains, although at different levels of 4. Miscellaneous mutations affecting initiation codon, 5° cap
activity in the normal adult. The a,-globin gene is expressed at site, or cleaving or polyadenylation sites at the 3° end.’
two to three times the rate of the a,-globin gene.* Detailed
In all cases the final result is a decreased or absent production
mapping of the DNA shows great similarity between the two
of the B-globin chain.
a-globin genes and between the two regions immediately
upstream (5°) of each a-globin gene.*? There are three areas of
homology in DNA blocks called x, y, and z (Fig. 12-2), includ- Pathophysiology of Thalassemia
ing or juxtaposed to the a-globin genes. These homologous
blocks render this area more susceptible to mispairing. Cross- In thalassemia, a decrease in production of one of the globin
ing over in this area of chromosome 16 may result in deletions chains causes a decrease in the amount of normal physiologic
of all or part of one a-globin gene. Occasionally a chromo- hemoglobin produced, resulting in a microcytic, hypochromic
some with three a-globin genes can be produced. This anemia. Because a- and B-globins associate in a stoichiometric
explains why the majority of a thalassemias are the result of a fashion to produce hemoglobin A (a,8;), decreased production
gene deletion. of one globin chain, as occurs in thalassemia, results in a rela-
On the other hand, the majority of 8 thalassemias are the tive excess of the other globin chain. For example, 6 tha-
result of point mutation affecting production of the B-globin lassemia results in an excess of unmatched a globin, which then
chain. A diagram of the B-globin gene fine structure, includ- accumulates in red blood cells. In both a thalassemia and 8 tha-
ing the areas involved in the regulation of expression, is lassemia, this excess globin is responsible for the damage to the
depicted in Figure 12—3. Between 5 and 20 Kb upstream of red blood cell or red blood cell precursors, manifesting as either
the e-globin gene is a B-locus control region (B-LCR), which peripheral hemolysis or ineffective erythropoiesis, respectively.
is involved mainly in the regulation of the switching on and In the case of a thalassemia, the excess y chains and B chains
off of the different B-like genes. Between 30 and 105 bases can form stable tetramers: hemoglobin Bart’s (y,) and hemoglo-
upstream of the B-globin gene lie the three classic promoter bin H (8,), respectively. However, these hemoglobins are phys-
sequences TATA, CAT, and CAC boxes. The point mutations iologically useless and will precipitate in older red cells, causing
resulting in B thalassemia can be classified into the following a shortened red cell life span. These abnormal hemoglobins
categories: may be detected in the peripheral blood as a diagnostic clue for

a By)

———————_:._vWVvO$S
5! a2

Eee] | rr eR Ee
x y Z xX y 7a

Figure | 2-2 m The areas of homology (x, y, and z) can lead to mispairing.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 233

cious 6 B 3)
nH
Chromosome 11

CAC CAT TATA

Figure 12-5 ™ Diagram of the B-globin gene expression: (top) schematic of the ___location of the B-like genes on chromosome 11 with the
locus control region (LCR) on the 5’ end; (bottom) exploded B-globin gene depicting the three exons and two introns with the three
promoter areas upstream.

a thalassemia. In the case of 8 thalassemia, the excess a chains adult hemoglobin, hemoglobin A (a,8,). The transition, or
form a, precipitates, which cause apoptosis of the red cell pre- switch, from fetal to adult hemoglobin begins in the third
cursors in the bone marrow, resulung in ineffective erythro- trimester and continues until complete at about 6 months;
poiesis.*” In severe forms of B thalassemia there is a selective therefore, infants with B thalassemia syndromes do not pre-
survival of cells producing hemoglobin F, which then becomes sent clinically until after birth. In the heterozygous form, tha-
a diagnostic clue for a homozygous form of 8 thalassemia. lassemia minor, hemoglobin A, is characteristically increased
Although both ineffective erythropoiesis and hemolysis play through an unclear mechanism. In contrast, the homozygous
roles in a and B thalassemia syndromes, hemolysis predomi- forms of B thalassemia, 8 thalassemia major and intermedia,
nates in severe a thalassemia whereas ineffective erythropoiesis characteristically have an increased hemoglobin F as
predominates in severe B thalassemia (Fig. 12-4). described in the preceding text.
The genetic background for 8 thalassemia is very hetero-
geneous. Almost 200 different mutations have been described;
Thalassemia Syndromes however, only some 20 mutations account for more than 80% of
all 8-thalassemia genes worldwide. A specific group of muta-
Beta Thalassemia
tions (four to six) is characteristically found in each geographic
8 Thalassemia does not manifest in utero because the fetus area.° Clinically, these haplotypes may be broadly subdivided
produces fetal hemoglobin, hemoglobin F (a,y,), rather than into B° and B* thalassemia.

Severe 8 Severe o thalassemia

thalassemia (hemoglobin H disease)

Globin Balanced « Decreased 3 Decreased o

production and B production production
production
Bone
marrow

Excess o
damages
precursors in
marrow causing
ineffective
erythropoiesis

Peripheral Normal hemoglobin Selective survival Hemoglobin H detectable

blood composition (97% of cells producing in blood. Hemoglobin H
hemoglobin A) | hemoglobin F precipitates and damages
RBCs, causing hemolysis

Figure 12-4 @ Diagram of globin chain imbalance. Excess globin chains precipitate and damage red blood cells and their precursors.
Destruction of red blood cells within the bone marrow (ineffective erythropoiesis) predominates in severe B thalassemia. In contrast, hemoglobin
H precipitates and damages circulating red blood cells (hemolysis) in severe a thalassemia. Notice the changes in hemoglobin constitution of
the peripheral blood with thalassemia: hemoglobin F is increased in severe B thalassemia and hemoglobin H is detectable in severe
a thalassemia.
234 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

B® THALASSEMIA HAPLOTYPES mostly of hemoglobin F and hemoglobin A;.'° B Thalassemia

These genes result in the complete absence of B chains. This intermedia is a less severe clinical expression and occurs in
particular gene expression is commonly found in the Mediter- patients homozygous for combinations of less severe B-globin
ranean area, particularly in Italy, Greece, Algeria, and Saudi mutations. These patients are not transfusion dependent at
Arabia. It is also common in Southeast Asia. baseline but may need transfusion during illnesses such as
infection.
B* THALASSEMIA HAPLOTYPES Heterozygosity for the B° or B* thalassemia gene causes
The 8*-thalassemia genes produce reduced amounts of B a mild form of chronic hypochromic, microcytic anemia that
chain. The wide variety of mutations at the molecular level has been called B thalassemia minor (Fig. 12-6). Although
produces a spectrum of 8* thalassemia genes, expressing var- the degree of anemia is variable, with hemoglobin levels from
ious levels of 8 globin. The expression of B globin is inversely 10.5 to 13.9 g/dL, it is impossible to determine whether the
proportional to the severity of clinical presentation. In other patient has the B° or B* gene on clinical grounds alone. In gen-
words, mutations causing large decreases in B-globin product eral, the levels of hemoglobin A, and hemoglobin F are mildly
will result in severe clinical syndromes, whereas mutations elevated. Some patients with very mild, B**, mutations show
causing only small decreases in B-globin product will result no clinical or laboratory evidence of anemia (normal hemo-
in mild, or even silent, clinical syndromes. The mildest of globin A,) and, for this reason, are called silent carriers.
8-globin gene mutations are sometimes referred to as B**,
although this terminology is losing favor. CLINICAL COURSE AND THERAPY OF THE 8
THALASSEMIA SYNDROMES
CLINICAL EXPRESSION OF THE DIFFERENT GENE 8 THALASSEMIA MAJOR Infants with B thalassemia major
COMBINATIONS (8 THALASSEMIA) usually present within the first year of life with failure to
B Thalassemia major is the most severe clinical expression of thrive, pallor, recurrent infections, and abdominal enlarge-
8 thalassemia and occurs in patients with homozygous B° or ment due to splenomegaly. On clinical presentation, the diag-
B* thalassemia or with compound heterozygous B° and B* tha- nosis can be confirmed with the presence of anemia (mean
lassemia. Homozygous B thalassemia refers to cases in which value of approximately 8 g/dL), abnormal blood smear (see
individuals have inherited two identical ® mutations and are Fig. 12-5), elevated hemoglobin F, and the demonstration of
encountered in geographic regions with greater consanguin- the 6 thalassemia trait in both parents.
ity. The more common compound heterozygous f£ tha- In nontransfused and inadequately transfused infants,
lassemias contain two different B thalassemia mutations. this severe chronic anemia is a strong stimulus for erythro-
When two inherited mutations are severe, they greatly poiesis. This causes marked expansion of the marrow space
decrease the production of B-globin, producing severe, trans- and characteristic skeletal changes of the skull, long bones,
fusion dependent clinical disease. In thalassemia major a and hand bones. The skull radiographs show widening of the
severe hypochromic, microcytic anemia develops during the
diploid space and characteristic radiating striations giving the
first year of life (Fig. 12-5). The hemoglobin level at the time
typical “hair-on-end” appearance (Fig. 12—7). The marrow
of presentation has a mean value of 8.3 g/dL and consists
expansion of the facial bones produces a characteristic facial
appearance with hypertrophy of the maxilla causing forward
protrusion of the upper teeth and overbite, a relativety sunken

Figure 12-5 m Peripheral smear from a patient with -thalassemia

major. Note the nucleated red cells, Howell-Jolly body in the
hypochromic microcyte (arrow), numerous target cells, and moder- Figure 12-6 m Peripheral smear from a patient with thalassemia
ate anisocytosis and poikilocytosis (Wright's stain). (From Bell, minor. Note the microcytosis and hypochromia with mild anisocy-
A: Hematology. In: Listen, Look and Learn. Health and Education tosis and poikilocytosis. A few target cells and basophilic stippling
Resources, Inc., Bethesda, MD, with permission.) are present. (Wright's stain, magnification x 400)
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia BN)

Without careful medical supervision and a therapeutic

program, including blood transfusions, iron chelation, and
early treatment of infection, these children will have numerous
complications, which include massive hepatosplenomegaly,
recurrent infections, spontaneous fractures, leg ulcers, dental
and orthodontic problems, and compression syndromes caused
by tumor masses from extramedullary hematopoiesis. If the
condition is left untreated, these children will usually die in
early childhood.
Survival of these patients is dependent on a lifelong
chronic blood transfusion program. It is now clear that a high
transfusion program (hypertransfusion) that maintains the
hemoglobin level at 11.5 g/dL on the average is better than an
intermittent program that allows the hemoglobin level to drop
to a point at which the child becomes severely symptomatic.
Hypertransfusion allows for better development, suppresses
ineffective erythropoiesis (thus preventing the serious bony
deformities), and provides for an overall better quality of life.
This, however, draws on considerable blood resources and
may not be available in the very countries where thalassemia
major is a main public health concern.
In the presence of splenomegaly, splenectomy plays a
clear role in decreasing the blood transfusion requirement.
However, it should preferably not be performed until the child
Figure 12-7 m Skull x-ray film of a 5-year-old child with homozy- has reached at least 5 years of age, to decrease the risk of over-
gous f thalassemia. Note the dilation of the diploic space and the
whelming infection, particularly of pneumococcal origin.
typical “hair-on-end” appearance caused by subperiosteal bone
growth in radiating striations. With regular blood transfusion, these children will often
be without severe symptoms of thalassemia but develop
nose, widely spaced eyes, and prominent cheek bones, result- severe iron overload owing to increased iron absorption and
ing in a Mongoloid facies (Fig. 12-8). The long bones of the to the loading of iron from the blood transfusions. This iron
hands and feet have cortical thinning with porosity of the overload results in hemochromatosis, and these patients die in
medullary space on radiographs. These changes are not a spe- their second or third decade, usually of cardiac failure. They
cific feature of B thalassemia and are found in other severe, develop multiple organ damage, with lack of pubertal devel-
chronic congenital anemias, but they are most prominent in opment probably caused by iron toxicity to the pituitary
8 thalassemia major. gland, cirrhosis of the liver (which may be the result of either
hemochromatosis or posttransfusion hepatitis), and diabetes
resulting from iron toxicity to the pancreas. This iron over-
load may be improved with the early introduction of iron
chelation therapy. Subcutaneous iron chelation therapy with
deferoxamine may result in an adequate iron balance, promot-
ing a longer survival. However, it is an expensive and difficult
regimen that is often not available or rigorously followed by
patients. New combined iron chelation therapies currently
being developed may offer a better alternative worldwide."
Hydroxyurea, a drug that increases the production of hemo-
globin F through unknown mechanisms, may also have a role
in treating severe B thalassemia.'*

B THALASSEMIA INTERMEDIA £B Thalassemia intermedia

covers a broad spectrum of clinical expression of thaiassemia,
bridging the gap between the severe B thalassemia major and
£7
the mild, often asymptomatic anemic state of B thalassemia
minor. The definition of 8 thalassemia intermedia is relative,
Figure 12-8 wm Face (A) and profile (B) of an 11-year-old child, with because the clinical state of patients in this group varies from
homozygous B thalassemia who is receiving hypertransfusion. The a mild disability to severe incapacitation without transfusion.
characteristic facial changes are not as prominent as those in an 8 Thalassemia intermedia is a form of thalassemia in which
untransfused child, but are still present. Note the bossing of the skull,
patients have variable degrees of symptomatic anemia, jaun-
hypertrophy of the maxilla with prominent malar eminences, depres-
sion of the bridge of the nose, and mongoloid slant of the eyes. dice, splenomegaly, and many of the complications of
>
236 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

8 thalassemia major, but survive into adulthood without a will be a greater variety in severity of disease, as there may be
large blood transfusion requirement. one, two, three, or four a-globin genes affected in one patient.
Patients with B thalassemia intermedia usually present at a Another characteristic of a thalassemia is the fact that
somewhat older age—generally after the age of 2—and with a the decreased or absent a chain production will result in
slightly higher level of hemoglobin (below 9 to 10 g/dL) than excess y chains during and shortly after fetal life and in
patients with 8 thalassemia major. There is great overlap excess 8 chains later on. This causes formation of stable
between the two conditions at presentation; however, it is very tetramers, such as y, (hemoglobin Bart’s) and 8, (hemoglo-
important to differentiate between them, as the only therapy for bin H), which can be detected by hemoglobin electrophore-
thalassemia major is regular blood transfusion in conjunction sis. These stable, nonfunctional tetramers precipitate in
with iron chelation, whereas the management of thalassemia older red cells, forming inclusion bodies and interfering
intermedia involves mostly supportive therapy with only occa- with membrane function, which results in decreased red
sional blood transfusion under special circumstances. cell survival and may induce a hemolytic crisis during
The serum bilirubin level is significantly more elevated infectious episodes.
in patients with 8 thalassemia intermedia than in those with B
thalassemia major. These patients may develop the physical a’ THALASSEMIA (a THALASSEMIA 1) HAPLOTYPES
and bony characteristics of B thalassemia major including a Thalassemia and « thalassemia | have been used inter-
hepatosplenomegaly. The anemia usually becomes worse changeably in the past; however, «° thalassemia is the preferred
with infections, pregnancy, or folic acid deficiency states. term for the description of the genetic determinant. The «° tha-
These patients are susceptible to frequent, sometimes severe, lassemia haplotype contains deletions of both a genes, a2 and
infections, and gallbladder problems owing to the formation al, on the same chromosome. As a consequence of this deletion
of gallstones. Children usually have an acceptable level of there is a complete absence of production of « chains from the
growth and development (although puberty may be delayed affected chromosome. Because both a-globin genes are deleted,
by a few years), and they reach adulthood if infections are the haplotype can be designated — —, and a patient who is
controlled and if they enjoy good nutrition with particular homozygous for the a° thalassemia mutation would be desig-
emphasis on prevention of folic acid deficiency. They may nated — —/— —. There are at least 21 major a°-thalassemia genes,
become transfusion dependent if severe hypersplenism which differ in the amount of DNA that has been deleted from
occurs. This usually requires splenectomy. Women with the chromosome.°® Each gene appears to be characteristic of a
8 thalassemia intermedia may become pregnant and may certain population in the world. «° Thalassemia genes are found
require blood transfusions as well as folic acid supplementa- frequently in Southeast Asia and less frequently in the Mediter-
tion throughout pregnancy. In spite of the lack of transfusion, ranean area. They also occur sporadically in other parts of the
patients with 6 thalassemia intermedia develop iron overload world. This gene may be recognized in adults through the detec-
as a result of increased gastrointestinal absorption. tion of small amounts of ¢-globin chain.'?

8 THALASSEMIA MINOR 86 Thalassemia minor, also called 8 a* THALASSEMIA (a THALASSEMIA 2) HAPLOTYPES

thalassemia trait, is a clinical entity in which the genetic defects Again, a* thalassemia and a thalassemia 2 have been used
of thalassemia are expressed as a mild microcytic, hypochromic interchangeably, but a* thalassemia is the preferred term. The
anemia, usually in the 10 to 13 g/dL range. Patients with B tha- usual a* thalassemia haplotype has a deletion of a single
lassemia minor are heterozygous for the B* thalassemia gene or a-globin gene on chromosome 16, which leaves the other
the 6° thalassemia gene. Patients are asymptomatic except dur- a-globin gene intact and able to function. Deletion of
ing periods of stress such as pregnancy, infection, or folic acid one a-globin gene is characterized by a reduction in the output
deficiency. Patients with thalassemia minor are usually diag- of a chains. This deletional mutation is also denoted as —«.
nosed incidental to a family study of an index case with Other less common types of a* thalassemia genes are caused
thalassemia major, by population screening, or by an incidental by non-deletion mutants, «'a affecting « chain synthesis. This
laboratory finding of microcytic hypochromic anemia. They is similar to the situation in the ® thalassemias in which
usually require no therapy if they maintain good nutrition. It is numerous different mutations can occur. Depending on the
important that they not be misdiagnosed as having iron- type and ___location of the mutation a number of processes can
deficiency anemia. be affected which lead to a decrease in production of the
a globin. A third type of a* thalassemia genetic background is
associated with unstable a-globin structural mutants, which pre-
Alpha Thalassemia cipitate in red blood cells. A discussion of these unstable vari-
ants is beyond the scope of this chapter. Overall, a minimum of
In contrast to B thalassemia, a thalassemia is usually mani- 40 major genetic defects resulting in the a* thalassemia gene
fested immediately at birth or in utero. This early presentation have been defined.*® These different thalassemia genes result in
reflects the switch from embryonic hemoglobin to fetal hemo- different levels of « chain output. For example, the non-deletion
globin (a,y>) around 6 to 8 weeks of gestation. Another charac- type of defects, a", are generally more severe than the single
teristic of a thalassemia is its wide range ofclinical expression. gene deletion type of defects, -~ commonly found throughout
Because each chromosome 16 carries two a-globin genes, the the Mediterranean, Middle East, Indian subcontinent, Southeast
total normal complement of a-globin genes is four. Thus, there Asia, Africa, and Malaysia.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 237

HEMOGLOBIN CONSTANT SPRING from individuals who have non-deletion mutations (— —/a'a or
The a-globin variant Constant Spring, or a, is the result of a a'a /a'a). Clinically, hemoglobin H disease is characterized by
point mutation in the «,-globin gene and may be considered an a variable degree of microcytic, hypochromic anemia.
a* thalassemia haplotype because it affects only one of Hemolytic crises may occur with infections. Because there is a
the two a-globin genes. The point mutation alters the natural switch from fetal (y) to adult (8) globins around the time of
stop codon, allowing translation to continue until the next stop birth, infants and adults have different hemoglobin composi-
codon farther downstream on the mRNA. The end result is an tions. Adults with hemoglobin H disease will have from 5% to
a-globin variant with 31 extra amino acids at its tail end. a 40% hemoglobin H; the remainder is mostly hemoglobin A with
combines with B-globin to produce hemoglobin Constant a small amount of hemoglobin A, and hemoglobin Bart’s.
Spring, Hb CS, which can be detected on hemoglobin Infants who later develop hemoglobin H disease usually have
electrophoresis at alkaline pH as a slow migrating band. between 19% and 27% hemoglobin Bart’s at birth, with the
Heterozygous patients are asymptomatic without hematologic remainder composed of hemoglobin F and hemoglobin A.
abnormalities and produce only small amounts of Hb CS (1%), Hemoglobin H and hemoglobin Bart’s can easily be identified
while homozygotes have a thalassemia minor with microcyto- by hemoglobin electrophoresis, because they migrate anodal to
sis and 5% to 8% Hb CS. Hb CS is found in Southeast hemoglobin A at alkaline pH (Fig. 12-9). These are sometimes
Asia, where it can combine with other prevalent a thalassemia referred to as fast hemoglobins. In addition, hemoglobin H
haplotypes to produce a thalassemia syndromes. shows a characteristic appearance of multiple ragged inclusions
in many red cells after incubation with brilliant cresyl blue, the
CLINICAL EXPRESSION OF THE DIFFERENT GENE so-called golf-ball appearance (Fig. 12-10).
COMBINATIONS (a THALASSEMIAS) a Thalassemia minor, also called a° thalassemia trait or
a Thalassemia can be divided into four clinical categories, a thalassemia | trait, is caused by defects of two of the four
depending on the severity of the disease. The most severe a-globin genes. This is usually the result of heterozygosity for
expression of a thalassemia 1s the hemoglobin Bart’s hydrops the a° thalassemia haplotype (— —/aa) but could also be the
fetalis syndrome, which is caused by homozygosity of the a° result of homozygosity for the a* thalassemia haplotypes
thalassemia gene haplotype (— —/— —). This is a lethal disease, (-a/-a). The condition is characterized by the presence at
and infants with hemoglobin Bart’s hydrops fetalis die either birth of 5% to 15% hemoglobin Bart’s, which disappears with
in utero or soon after birth. They produce no @ chains, and the development and is not replaced by hemoglobin H. Adults,
only hemoglobins found are hemoglobin Bart’s (y,) and therefore, will have a normal hemoglobin electrophoretic
hemoglobin Portland (C,y.). Because hemoglobin Bart’s is pattern. There is a minimal amount of anemia with slight
useless as an oxygen carrier, survival of these fetuses into the hypochromia and microcytosis present. The mean corpuscu-
third trimester or until birth is entirely due to the presence of lar volume (MCV) is usually between 70 and 75 fL. This
hemoglobin Portland. This condition is quite common in South- condition exists in 3% of African Americans and may be
east Asia in the -SEA/-SEA form but is also found sporadically confused with iron deficiency.
in the Mediterranean area in the form of -MED/—MED. The last category of a thalassemia 1s the silent carrier of
The second most severe clinical expression of a a thalassemia, also called a* thalassemia trait or a tha-
thalassemia is hemoglobin H disease. In this entity only one lassemia 2 trait. This is the result of a defect in one of the four
a-globin gene out of four is functioning. This is usually the a-globin genes and is characterized by the presence of a very
result of a compound heterozygosity of the deletional a° tha- small amount (up to 2%) of hemoglobin Bart’s at birth; after
lassemia and a*-thalassemia haplotypes (—/-c) but can also arise the disappearance of hemoglobin Bart’s during development,

+ _—

H x

Portland Bart's C,A>,

A Portland, Bart’s, F
IF Lepore,S,E,F
Lepore,A,,A,E
Lepore,S A
Constant spring
E,C,A, ~ S
Constant spring Origin
=m mense Y H
Carbonic anhydrase Portland pe Origin
Origin H — c
1
lee Cid oS py Tey CN aetoy (5) fe EhSa 8)
— + +

Cellulose acetate pH 8.4 Cellulose acetate pH 7.0 Citrate agar pH 6.0

Figure 12-9 m Diagram of the migration of the different hemoglobins at different pH: (7) normal adult (A, A2); (2) homozygous Hb Lepore
(F, Lepore); (3) HbH/Constant Spring disease a“a/(Constant Spring, A>, A, Bart’s H); (4) compound heterozygous HbE/f-thalassemia (E, F);
(5) Hb Bart’s hydrops fetalis syndrome (Portland, Bart’s); (6) HbS/C disease (S, C).
238 — Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

CLINICAL COURSE AND THERAPY OF a THALASSEMIA

SYNDROMES
HEMOGLOBIN BART’S HYDROPS FETALIS Hemoglobin
Bart’s hydrops fetalis is the result of homozygosity for the com-
plete deletion of both a-globin genes. The affected infants are
either stillborn or die within a few days after birth. At delivery,
these infants are severely anemic and edematous, and demon-
strate ascites, marked hepatomegaly, and splenomegaly. The
mothers may report a history of stillbirth or neonatal deaths. The
clinical significance of this entity is related to the obstetric prob-
lems that may arise in the affected infants’ mothers. Pregnancy
is often complicated by preeclampsia, labor complications, and
postpartum hemorrhage, all of which contribute to severe mor-
bidity and mortality. Clinical emphasis for this entity is on the
prevention of the disease through early antenatal diagnosis. In
the hands of skilled clinicians, ultrasound screening may be

ee
used to identify hemoglobin Bart’s hydrops fetalis as early as
the first trimester.'*
DAP

Figure 12-10 ® Hemoglobin H inclusions (supravital stain). (From HEMOGLOBIN H DISEASE Hemoglobin H disease is sim-
Bell, A: Hematology. In: Listen, Look and Learn. Health and Education ilar to ® thalassemia intermedia in that it covers a wide
Resources, Inc., Bethesda, MD, with permission.) range of clinical severity from the patients with very mild
anemia to patients potentially requiring regular transfusion.
The age at presentation varies from 0 to 74 years old.’
no recognizable hematologic abnormality is present, except Infants will have near normal hemoglobin levels and no
for a borderline low MCV (78 to 80 fL). This condition is splenomegaly, so the diagnosis must come from clinical
found in up to 30% of African Americans. suspicion and laboratory testing. Most cases are discovered
In the simplest terms, deletional type mutations result in in adulthood and will have episodic periods of anemia, pal-
predictable clinical syndromes such that four deletions produce lor, and weakness during which they may be discovered to
hemoglobin Bart’s hydrops fetalis, three deletions produce have a hypochromic, microcytic anemia, leading to an
hemoglobin H disease, two deletions produce a thalassemia eventual diagnosis. These episodes of anemia may be asso-
minor, and one deletion produces a silent carrier (Fig. 12-11). ciated with concurrent infections, medications, or other ill-
Because non-deletion mutations and hemoglobin variants can nesses. Other clinical features may include scleral icterus,
also combine with deletional a-globin gene mutations, the hepatosplenomagaly, gallstones, and bone marrow expan-
actual molecular basis for a thalassemia syndromes is more het- sion. The bone changes are present in about one third of
erogeneous. The different genetic backgrounds associated with cases but are milder than that seen in severe B thalassemia.
the four different clinical expressions of @ thalassemia are sum- Splenomegaly, if present, may worsen the anemia and
marized in Table 12-2. sequester platelets, so these patients may benefit from a

Mother’s haplotype

Father’s haplotype

Normal Silent carrier a thalassemia minor

6 <<
x —mmen:
Silent carrier a thalassemia minor Hb H disease

<——
ae os aS >t S<
a thalassemia minor Hb H disease Hb Bart’s

Figure 12-11 @ Simplistic look at the interactions of deletional mutations of « thalassemia. The severity of disease is predictable and
depends on the number deleted a-globin genes.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 239

Chromosome
Genes J —O asq

Q carrier Q Carrier Q carrier thal minor

q Carrier thal minor thal minor thal minor
Q carrier thal minor thal minor thal minor
Q Carrier thal minor thal minor H
thal minor H H

aa = normal haplotype; -a = deletion of one a-globin gene; a°Sa = Hb Constant Spring; a'a = nondeletion « thalassemia
gene; — = deletion of both a-globin genes (Note: the clinical phenotype resulting from the combination of these haplotypes
is found at the intersection of the corresponding column and row); N = normal clinical phenotype; « carrier = silent carrier
of a thalassemia, 5%—15% Hb Bart’s at birth, mild anemia; thal = « thalassemia minor, 0%-2% Hb Bart’s at birth, minimal
hematologic changes; H = hemoglobin H disease; Bart’s = hemoglobin Bart’s hydrops fetalis.

splenectomy. Some cases of hypercoaguability, with deep

predictive of @ thalassemia minor versus silent carrier, there
vein thrombosis and pulmonary embolism, have been
is overlap. Also, ethnicity may be helpful when estimating
reported after splenectomy. Unlike severe B thalassemia, risk. The only way to know for sure is through molecular
iron overloading is not a general teature of hemoglobin H analysis of the a genes.
disease but may occur in older patients and patients requir-
ing regular transfusion. Anemia may worsen during preg-
nancy, and when it occurs, other factors such as iron or Delta—Beta Thalassemia and Hemoglobin
folate deficiency should be investigated. Patients over Lepore Syndrome
6 years old can be considered to have hemoglobin H disease
if greater than 1% to 2% hemoglobin H is detected and Delta—beta (58) thalassemias are a diverse group of tha-
there are typical inclusion bodies in the red blood cell lassemias characterized by a combined defect in 6 and B
after incubation with brilliant cresyl blue. chain synthesis. They can be described as demonstrating a
near normal level of hemoglobin A, and an unusually high
a THALASSEMIA MINOR (HOMOZYGOUS a! level of hemoglobin F in the heterozygote, and absent hemo-
THALASSEMIA OR HETEROZYGOUS a°® THALASSEMIA) globin A and A, in the homozygote. All 58 thalassemias stud-
Patients with a thalassemia minor are generally asymptomatic ied thus far have been shown to be the result of a deletion.
with mild hypochromic microcytic anemia. They are identified They can be described at the genetic level as three different
either incidentally or by screening programs in regions with a entities, depending on the amount of DNA lost: hemoglobin
high prevalence of a thalassemia. Identification of individuals Lepore syndrome (68)* results from a partial deletion of the
with a thalassemia minor is, however, very important. Preg- 6 and B-globin genes, (58)° thalassemia from a complete
nant women with a thalassemia minor and women with o tha- deletion of the 6 and B-globin genes, and (“y6B)° thalassemia
lassemia minor who are planning pregnancies are at risk for from a deletion of the “y-globin gene in addition to the dele-
hemoglobin Bart’s hydrops fetalis and its associated morbidity tion of the 6 and B-globin genes. 68 Thalassemias are less
and mortality. Identification of thalassemia minor is also common than £ thalassemias and have been found sporadi-
important to prevent the unnecessary treatment of misdiag- cally in Greeks, Africans, Italians, and Arabs.
nosed iron deficiency. Historically 68 thalassemias were divided into two
groups according to the types of fetal hemoglobin produced.
SILENT CARRIER OF a* THALASSEMIA (HETEROZYGOUS (6B)° thalassemia produces both “y and “y chains, while
a’ THALASSEMIA) Patients who carry the a* thalassemia (“y5B)° produces only “y chains because the “y-globin gene
haplotype are asymptomatic and, again, may only be picked up has been included in the deleted segment. In order to be more
by screening programs or by incidental, abnormal laboratory consistent with nomenclature of other thalassemias, the 58
data. These individuals are not anemic and have normal blood thalassemias are now designated according to the deficiency
smears, but a slightly low MCV can be a clue to the carrier state. of globin chains: (58)*, (68)°, and (“y6B)°.
Women who are silent carriers are not at risk of hemogiobin One type of 58 thalassemia involves the production of an
Bart’s hydrops fetalis; however, they may have children with abnormal hemoglobin. This abnormal hemoglobin, called
hemoglobin H disease. hemoglobin Lepore after the name of the family in which it
There is a need to separate a thalassemia minor from was first found, has been shown to be a fusion of the 6 and B
silent carrier of a* thalassemia in women because of the risk chains, which is the product of a fusion gene formed by an
associated with hemoglobin Bart’s hydrops fetalis. Although unequal crossing over. The production of hemoglobin Lepore
the level of hemoglobin Bart’s at the time of birth may be by this unequal crossing over is indicated diagrammatically in
240 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

Compound heterozygosity, also called double heterozy-

Ay 6 B Normal
oh - chromosome II gosity, refers to the inheritance of two different abnormal
} 0 B Normal thalassemia haplotypes. For example, a patient may have a 6B
Unequal crossing over
chromosome II thalassemia haplotype on one chromosome and a B thalassemia
haplotype on the other chromosome. These compound het-
; ay
a $e} a
6 OB Chromosome coding for erozygous individuals contribute to the clinical heterogeneity
A ee m hemoglobin Lepore
of the thalassemia syndromes. Compound heterozygous 68
Y ° Bid ae Chromosome coding for
hemoglobin anti-Lepore thalassemia and 6 thalassemia produces a clinical course simi-
lar to 8 thalassemia intermedia; however, compound heterozy-
Figure 12-12 ® Hemoglobin Lepore formation. An abnormal gous hemoglobin Lepore and B thalassemia produces a clinical
crossing over between £3 and 8-globin genes gives rise to hemoglo- course similar to B thalassemia major.
bin Lepore and to hemoglobin anti-Lepore.

Hereditary Persistence of Fetal

Figure 12-12. At least three different hemoglobin Lepores Hemoglobin
have been described, varying in the exact ___location of the
unequal crossing Over. Hereditary persistence of fetal hemoglobin (HPFH) com-
Heterozygosity for 658 thalassemia and hemoglobin prises a group of conditions characterized by the persistence
Lepore results in a mild form of anemia that is clinically of fetal hemoglobin synthesis into adult life. HPFH has many
described as thalassemia minor and is similar to the condition of similarities to 68 thalassemia; however, HPFH does not pro-
patients with heterozygous B thalassemia. duce significant hematologic abnormalities. The fundamental
The y-chain synthesis in 58 thalassemia is usually more difference between the two is that HPFH produces enough
efficient than in BB thalassemia or hemoglobin Lepore, and in hemoglobin F to compensate for the loss of 5- and B-globin
general 5B thalassemia produces a milder clinical disease chains, while 58 thalassemia does not.
than the latter two. The y chain is able to combine effectively There are two major categories of genetic defects result-
with a chain to make functional hemoglobin (hemoglobin F); ing in HPFH with hemoglobin F in the 20% range. One is a
therefore, the accumulation of unmatched a chain, which can deletion of the 6 and B-globin genes somewhat like the dele-
damage cells, is reduced. The overall effect is a partial com- tion of 58 thalassemia, and the other is a non-deletion muta-
pensation by hemoglobin F for the decreased production of tion in the promoter of the “y or “y-globin gene (Fig. 12—13).
hemoglobin A and A,. Patients with homozygous 68 tha- The promoter mutations, °yB* HPFH and “yB* HPFH,
lassemia have a clinical course similar to B thalassemia inter- increase the transcription of y-globin genes by increasing
media. On the other hand, patients with the homozygous state their interaction with the locus control region, which greatly
for hemoglobin Lepore, which does not compensate as well as enhances the output of B-cluster gene with which it is associ-
dB thalassemia, have a more severe transfusion-dependent ated. The (68)° HPFH deletions are more baffling since 58
thalassemia. thalassemias also include deletions of the 6 and B-globin

Deletion of 5 and 8 genes Promoter mutation Promoter mutation

Deletion
mutation in mutation in
Sy promoter Ay promoter
region region |

|
Loss of competition Increased Increased
from deleted genes transcription of Sy transcription of Ay
allows increased y
transcription

Increased hemoglobin F

Figure | 2-15 @ Mutations seen in HPFH with hemoglobin F in 20% range. Mutations producing HPFH in this range have common themes.
One common molecular mechanism (left) is the deletion of the 8 and 6-globin genes, (88)°. This eliminates the competition of the locus
control region for the 8 and 8 promoters and allows increased y transcription. Another common theme is a point mutation in the Sy or Ay
promoters which increases the association of the locus control region with the respective y-globin gene and increases its transcription.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 241

Deletion of y silencing sequence Juxtaposition of enhancer sequence

. |y Silencing sequence Enhancer sequence

BR

Deletion Deletion

Loss of silencing sequence Juxtaposition of enhancer

allows increased y transcription sequence next to y genes
increases y transcription

s /
Increased y globin chain compensates for loss of 6 and 8 globin chains

Figure 12-14 (8p)? Deletions can be found in both HPFH and df thalassemia. A greater amount of hemoglobin F is seen in HPFH than in
5B thalassemia. Two hypotheses for increased production of y globin expression in HPFH are shown above. The 5B deletion along with an
intergenic sequence responsible for y silencing during development is shown at the /eft. A second hypothesis in which a deletion results in
the juxtaposition of a downstream enhancer with the y-globin gene, where it can have a positive effect on y transcription, is shown at the
right. The mechanisms are not exclusive of one another and may coexist. Complete compensation for loss of 8 and B globins with increased
y globin prevents a globin imbalance between a and non-a globin chains in HPFH.

genes. There are two main theoretical models to explain the This technique is discussed later in this chapter with the other
different hemoglobin F output between (68) HPFH and (6B)° laboratory methods useful in the diagnosis of thalassemia.
thalassemia (Fig. 12—14). In the first model, a (68)? HPFH
deletion brings a downstream enhancer into proximity of the PANCELLULAR HPFH
-globin gene. The enhancer then has a positive effect on Various forms of (58)° HPFH have been described, all of which
transcription of the y-globin gene. This has been supported by produce a pancellular distribution of hemoglobin F. One com-
mouse experiments in which HPFH breakpoint sequences mon form is the African (HPFH 1), in which there is a deletion
increase y transcription.'® In the second model, a region of the 6- and B-globin genes and increased synthesis of “y and
between the y and 6-globin genes, which may be responsible “vy chains, which almost completely compensate for the lack of
for y silencing around the time of birth, is deleted in HPFH.
Evidence that this intergenic region may also play a role is
supported by the demonstration of increased ‘y transcription
with its deletion.'”

Pancellular and Heterocellular HPFH

HPFH can be classified into two different categories accord-
ing to the distribution of hemoglobin F among the red cells.
Fetal hemoglobin is more resistant than adult hemoglobin to
elution at acid pH and can be demonstrated on a peripheral
smear by the acid elution test of Kleihauer and Betke. Using
this stain, the HPFH conditions can be divided into a pancel-
lular form, in which hemoglobin F is uniformly distributed
among the red cells, and a heterocellular form, in which
hemoglobin F is found in only a small percentage of the cells
(Fig. 12-15). In a normal adult, cells containing hemoglobin
Figure 12-15 @ Kleihauer—Betke stain of blood from a patient
F, also called F cells, can occasionally be found, but the
with hereditary persistence of fetal hemoglobin (HPFH). Note that all
amount is always less than 2% and is usually less than 1%. red cells stain red, owing to the varying amounts of hemoglobin F.
More recently flow cytometry has been used to separate (From Bell, A: Hematology. In Listen, Look and Learn. Health and
pancellular and heterocellular distributions of hemoglobin F. Education Resources, Inc., Bethesda, MD, with permission.)
 242 ~~ Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

production of 6 and B chains. Hemoglobin F constitutes 100% 8 THALASSEMIA WITH HEMOGLOBIN C

of the hemoglobin in the homozygous state and 15% to 30% of The £ thalassemia with hemoglobin C syndrome demon-
the hemoglobin in the heterozygous state. The hemoglobin F is strates great variability in clinical and hematologic manifesta-
homogeneously distributed among the red cells and consists of tions, which is directly related to the type of B thalassemia
a mixture of “y and “y chains. Clinically, the homozygotes will gene that interacts with the hemoglobin C gene; however, the
demonstrate features of thalassemia minor and the heterozy- great majority of patients with this syndrome are West
gotes will be hematologically normal. Africans and African-Americans. In this racial group the B*
Non-deletion HPFH, “y8* and “y®*, generally produce thalassemia gene is common, and compound heterozygosity
a pancellular distribution of hemoglobin F. An example is the for 8 thalassemia and hemoglobin C is characterized by a
Greek/Sardinian/African “yB* HPFH, in which about 15% mild degree of usually asymptomatic anemia, in which the
hemoglobin F is present in the heterozygous state. This hemo- clinical and hematologic findings are very similar to those
globin F is also found uniformly distributed among the red found in heterozygous £ thalassemia.
cells but is only of the “y type. The homozygous state for this
type of pancellular HPFH produces no significant hemato- B THALASSEMIA WITH HEMOGLOBIN E
logic abnormalities. Compound heterozygosity for 8 thalassemia and hemoglobin
E is unusual because it results in a clinical disorder that is
HETEROCELLULAR HPFH much more severe than homozygous hemoglobin E disease.
Heterocellular HPFH appears to be an inherited condition in Patients with this syndrome are distributed widely throughout
which the number of F cells is increased without concurrent the Far East. The condition follows a clinical course very sim-
alteration in 6- and B-chain production. Collectively these are ilar to that of homozygous £ thalassemia, with a very severe
called the heterocellular HPFH, or sometimes ‘Swiss HPFH’, anemia occurring in early childhood and the development of
but in reality they are a heterogeneous group, which has not the characteristic features of thalassemia major if the patient
been conipletely elucidated at the molecular level. These gen- is not started on a regular blood transfusion program.
erally produce a mild increase in hemoglobin F, which is
rarely greater than 5%. Some of these are the result of a point a THALASSEMIA WITH SICKLE CELL ANEMIA
mutation and rearrangements, while others are not even The occurrence of a thalassemia in conjunction with sickle
linked to the B-globin gene cluster. cell anemia has a positive influence on the clinical expression
of the disease. Patients with such a genetic background have
Thalassemia Associated with Hemoglobin an increased percentage of hemoglobin F, which is thought to
Variants result in a decreased severity of the sickling process. Of inter-
est is the fact that the amount of hemoglobin F present is
Although thalassemia has been described in association with a roughly proportional to the number of a-globin genes
large number of hemoglobin structural variants, the following affected. Patients with the «° thalassemia trait have an average
discussion considers only the interactions with the more com- of 16% hemoglobin F, and those with the a* thalassemia trait
mon hemoglobin variants (i.e., B thalassemia with hemoglobin have an average of 8% hemoglobin F.'*
S, hemoglobin C, and hemoglobin E, and a thalassemia with
hemoglobin S).
A Broad Clinical Classification
B THALASSEMIA WITH HEMOGLOBIN S of Thalassemia Syndromes
This condition was first recognized in individuals who had inher- Because of the heterogeneity of thalassemia at the molecu-
ited a single hemoglobin S gene and who demonstrated about lar level it may be helpful to broadly categorize the
65% hemoglobin S and 35% hemoglobin A, which is the reverse thalassemia syndromes according to clinical severity. The
of the proportions found in patients with sickle cell trait. This con- clinical course and therapy of patients with thalassemia can
dition is the result of the inheritance of a hemoglobin S gene from
be broadly subdivided into three categories: thalassemia
one parent and a f-thalassemia gene from the other. 8 Tha-
major, thalassemia intermedia, and thalassemia minor. A
lassemia with hemoglobin S (also called the 8-thalassemia sickle
fourth category, termed thalassemia minima, is applied to
cell syndrome) has been widely seen in Africa, the Mediterranean
healthy silent carriers who show no clinical symptoms and
area, the Middle East, and the West Indies. There is great variety
minimal to no hematologic abnormalities. The different
in the clinical severity of this syndrome, depending mostly on the
genetic backgrounds that result in each of these clinical out-
type of B thalassemia gene inherited. If the B thalassemia gene is
comes are summarized in Table 12-3.
the B° type, no hemoglobin A is produced and the clinical condi-
tion is indistinguishable from classic sickle cell anemia, character-
ized by severe anemia presenting in early childhood and recurrent
Laboratory Diagnosis of Thalassemia
sickling crises. If the B thalassemia gene is the B* type, some
hemoglobin A is produced, and these patients have less severe The hallmark of thalassemia is the finding of a microcytic,
sickling crises than those in the B° group. The severity of clinical hypochromic anemia. Although more sophisticated laboratory
disease reflects the degree of decreased 8-globin produced by the procedures are needed to define exactly the type of tha-
8* mutation. lassemia, the original diagnosis of thalassemia can be made or
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 243

Genetic Background Clinical Syndrome

Homozygotes
Homozygous B° Thalassemia major
Homozygous B* Thalassemia major or intermedia
Homozygous 5B Thalassemia intermedia
Homozygous Hb Lepore Thalassemia major or intermedia
Homozygous HPFH Thalassemia minima
Homozygous a°-thalassemia (—/—) Hemoglobin Bart’s hydrops fetalis
Homozyous a*-thalassemia (—o/—c) Thalassemia minor

Compound Heterozygotes
Compound heterozygous B°/severe B* Thalassemia major
Compound heterozygous B**/severe B* or B° Thalassemia major oi intermedia
Compound heterozygous 58/B° Thalassemia major or intermedia
Compound heterozygous Lepore/8B Thalassemia intermedia
Compound heterozygous Lepore/B° or B* Thalassemia major
Compound heterozygous HPFH/B’ or B* Thalassemia minor
Compound heterozygous E/f° or * Thalassemia major or intermedia
Compound heterozygous C/B° or B* Thalassemia intermedia or minor
Compound heterozygous S/B° or B* Sickle cell disease
Compound heterozygous a-thalassemia (—/—a) Thalassemia intermedia (hemoglobin H disease)

Heterozygotes
Heterozygous 8° or B* Thalassemia minor
Heterozygous B** Thalassemia minima
Heterozygous 68 Thalassemia minor
Heterozygous Lepore Thalassemia minor
Heterozygous HPFH Thalassemia minima
Heterozygous a° thalassemia Thalassemia minor
Heterozygous a* thalassemia Thalassemia minima

strongly suspected on the basis of the results of routine hema- MCH is usually below 22 pg and the MCV below 70 fL,
tology procedures. whereas the hemoglobin level is in the 9 to 11 g/dL range.
In heterozygous thalassemia, there is often a relative
Routine Hematology Procedures erythrocytosis. That is, the RBC count is high relative to the
hemoglobin level. A general rule is that, in non-thalassemic
AUTOMATED BLOOD CELL ANALYZER individuals, the hemoglobin is three times the RBC count.
Automated blood cell analyzers are commonly used in devel- Therefore a patient with a RBC count of4 million will have a
oped countries and provide hemoglobin levels, hematocrit, and hemoglobin level of approximately 12 g/dL. However,
red blood cell indices (see Chap. 31). The red bluod cell indices thalassemic patients have a relative erythrocytosis, so the
are invaluable to clinicians and laboratory personnel, who are hemoglobin level will be much less than three times the RBC
sorting through the differential diagnosis of microcytic count. For example, an individual with heterozygous B tha-
hypochromic anemia. The thalassemias in general are charac- lassemia may have a RBC count of 4 million and a hemoglo-
terized by a decrease in hemoglobin level, hematocrit, mean bin level of 9 g/dL. Demonstrating a relative erythrocytosis in
corpuscular volume (MCV), and mean corpuscular hemoglo- an individual with a low MCV should alert the interpreter of a
bin (MCH) in conjunction with a normal-to-increased red possible thalassemia, and further testing is indicated.
blood cell (RBC) count, a normal to mildly decreased mean
corpuscular hemoglobin concentration (MCHC), and a normal PERIPHERAL SMEAR EXAMINATION
red cell volume distribution width (RDW). The only exception The careful examination of a well prepared peripheral smear
is thalassemia major, in which the degree of anisocytosis is is essential to the diagnosis of thalassemia.
such that the RDW is increased. The decrease in MCV is usu-
ally striking and disproportionate to the decrease in hemoglo- WRIGHT’S STAIN In homozygous B and compound heterozy-
bin and hematocrit. This fact, in conjunction with the relatively gous non-a thalassemia, the peripheral smear demonstrates
high RBC count and the normal RDW, offers a useful discrim- extreme anisocytosis and poikilocytosis with bizarre shapes, tar-
ination index between heterozygous a or B thalassemia and get cells, ovalocytes, and large numbers of nucleated red cells
iron deficiency.'? In iron deficiency, the RDW is increased and (see Fig. 12-5). There is marked hypochromia and microcyto-
the decrease in MCV is less striking and observed only when sis. In heterozygous f thalassemia, the cells are hypochromic
the anemia is more severe. In heterozygous thalassemia, the and microcytic with a mild to moderate degree of anisocytosis
244 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

and poikilocytosis. Target cells are frequent, and basophilic stip- quality of the technique, as this technique is very sensitive to
pling is often seen (see Fig. 12-6). The peripheral smear of a many variables. Flow cytometry can also be used to assess he-
patient with the sickle cell thalassemia syndrome can be differ- moglobin F distribution (see later) and may suffer less from
entiated from that of a patient with pure sickle cell anemia by the confounding variables of the Kleihauer—Betke test.
the presence of hypochromia, microcytosis, numerous target This stain is very useful in demonstrating the distribution
cells, and only an occasional sickled cell. of hemoglobin F and can be used to differentiate between
In hemoglobin H disease, the peripheral smear demon- pancellular and heterocellular HPFH. It is also useful in dif-
strates hypochromia with microcytosis, target cells, and mild ferentiating heterozygous 58 thalassemia from heterozygous
to moderate anisopoikilocytosis. Patients with heterozygous pancellular HPFH, because the former usually has a hetero-
a’ thalassemia usually demonstrate a mild hypochromia and cellular distribution of hemoglobin F.
microcytosis, whereas those with heterozygous a* tha-
lassemia usually have a perfectly normal peripheral smear. OSMOTIC FRAGILITY
The red cells of patients with homozygous or heterozygous 8
SUPRAVITAL STAINS The reticulocyte count is usually ele- thalassemia, hemoglobin H disease, and «° thalassemia trait
vated up to 10% in hemoglobin H disease and up to 5% in have a decreased osmotic fragility. This fact is not very use-
homozygous B thalassemia but is disproportionately low in ful for diagnostic purposes in a specific patient, but it is the
relation to the degree of anemia in the latter condition. basis of a simple, inexpensive method of screening for the
In hemoglobin H disease, incubation of the red cells with thalassemia carrier state in large populations.
brilliant cresyl blue stain causes in vitro precipitation of hemo-
globin H owing to the redox action of the dye. This results in a
Flow Cytometry
characteristic appearance of the majority of the red cells, which
display multiple discrete inclusions, the appearance of which Flow cytometry of red blood cells using fluorescently labeled
has often been compared to that of a golf ball (see Fig. 12-10). anti-hemoglobin F antibodies can be used to determine if the
Occasionally, and after extensive searching, such cells contain- distribution of hemoglobin F is pancellular or heterocellular.
ing hemoglobin H inclusions can be found in the a° thalassemia Red blood cells are permeabilized and then incubated with
carrier. anti-hemoglobin F before being loaded onto the flow cytome-
In patients with homozygous § thalassemia or hemoglo- ter. As red blood cells pass through the laser in single file, they
bin H disease who have undergone splenectomy, incubation are simultaneously counted and assessed for the presence of
of the blood with methyl violet stain can demonstrate Heinz hemoglobin F. The end result is either one peak, representing
body—like inclusions, which represent in vivo precipitation of a single population of red blood cells containing hemoglobin
the abnormal hemoglobin. F, or two peaks, representing separate populations with and
without hemoglobin F.
ACID ELUTION STAIN The acid elution technique (see Flow cytometry is useful in the same situations where
Chap. 31), originally described by Kleihauer and Betke, is Kleihauer—Betke acid elution is useful. Individuals with
based on the fact that at an acid pH of about 3.3, hemoglobin hemoglobin F in the 20% range may have either heterozygous
A is eluted from an air-dried, alcohol-fixed blood smear, HPFH or heterozygous 58 thalassemia, and the flow cytome-
whereas hemoglobin F is resistant to elution. After such treat- try method can identify the pancellular and heterocellular dis-
ment and subsequent staining with eosin or erythrosin, normal tribution of hemoglobin F, respectively” (Fig. 12-16).
adult red cells appear as very faint ghosts. Red cells contain-
ing hemoglobin F demonstrate a variable amount of stain,
Hemoglobin Electrophoresis
depending on the amount of hemoglobin F present. A con-
trolled preparation containing a mixture of adult and cord cells Hemoglobin electrophoresis plays an important role in the diag-
must also be stained and examined in parallel to check the nosis of thalassemia by allowing the detection of increased

200 200
160 160
120 120

Counts 80
Counts

40

10° 101 102 10° 104 102 10° 104

Hgb.F PE Hgb F PE
Figure 12-1 6 @ Flow cytometric analysis to determine hemoglobin F distribution. The x-axis represents the
amount of hemoglobin in red cells,
and the y-axis represents the amount of red blood cells. A pancellular distribution of hemoglobin
F in an individual with HPFH is shown in the dia-
gram at Jeft. A heterocellular distribution of hemoglobin in an individual with heterozygous (58)?
thalassemia is shown in the diagram at right.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 245

HbA (%) HbA, (%) HbF (%)

Homozygous B° thal 0 2-5 95-98

Homozygous 8* or compound 5-35 2-5 60-95
heterozygous B*/®° thal
Homozygous 68 thal 100
Homozygous Hb Lepore 75 (25% Hb Lepore)
Heterozygous B thal 2-5
Heterozygous 68 thal 5-20
Heterozygous Hb Lepore 1-6 (7-15% Hb Lepore)
Homozygous HPFH 100
Heterozygous HPFH (African type) 15-35
Heterozygous HPFH (Greek type) 15-25
Normal 1-2

levels of hemoglobin A, and hemoglobin F as well as the pres- Citrate agar gel electrophoresis, which is performed at
ence of abnormal hemoglobins, such as hemoglobin H, hemo- an acid pH between 5.9 and 6.2, is helpful in identifying
globin Bart’s, hemoglobin Lepore, hemoglobin Constant hemoglobin variants that can be inherited along with tha-
Spring, or other structurally abnormal hemoglobins that can be lassemia.
found in association with thalassemia (hemoglobin S, hemoglo-
bin C, hemoglobin E). Table 124 contains a summary of the
different patterns of the hemoglobins present in the non-c tha- Hemoglobin Quantitation
lassemia syndromes. Although an experienced observer can detect an increased
Routine hemoglobin electrophoresis to confirm the diag- level of hemoglobin A, or hemoglobin F on cellulose acetate
nosis of thalassemia is performed at an alkaline pH around or starch gel electrophoresis, actual quantitation is necessary
8.4 on cellulose acetate, starch, or agarose gels. At alkaline pH, to truly establish the diagnosis of thalassemia.
the hemoglobins migrate from the most cathodal to the most
anodal in the following order: first hemoglobin Constant Spring,
then hemoglobins A,, C, and E migrate in the same band; next
hemoglobins S and Lepore, again in the same band; then hemo-
Q
globin F, followed by hemoglobin A, hemoglobin Portland, nN
“Oo
hemoglobin Bart’s, and finally, hemoglobin H. The different
patterns of migration of these hemoglobins are illustrated in
Figure 12-9. An example of alkaline electrophoresis on agarose
gel with some commonly encountered entities is shown in
€
Figure 12-17. Cellulose acetate or starch gel electrophoresis can
be performed at low to neutral pH to detect hemoglobin H and
hemoglobin Bart’s easily, as they migrate anodally (.e., in the
direction opposite to the other hemoglobins) at this pH.

CELLULOSE ACETATE
Cellulose acetate electrophoresis has replaced starch gel elec-
trophoresis in most laboratories, owing to its simple, rapid
method. It uses a smaller sample than starch gel electrophoresis, - +
and minor components such as hemoglobin Constant Spring
and small amounts of hemoglobin A, may be overlooked. Small . A2 SF AM
amounts of hemoglobin A in the presence of mostly hemoglobin CD
F also can be difficult to detect. aie
O
OTHER GELS
Starch gel electrophoresis is a little more cumbersome and Figure 12-17 m Electrophoresis at alkaline pH. Lanes 7 and 7 are
controls with hemoglobins C, S, F, and A. Lanes 3 and 4 are
time consuming. The results of the starch gel electrophoresis normal individuals (A, = 2.5%). Lane 5 is an individual with
are similar to those of the cellulose acetate procedure. Elec- 6 thalassemia minor (A, = 4.7%). Lane 6 is an individual with
trophoresis with starch gel is better at defining the presence of a thalassemia minor; a thalassemia minor has a pattern with a
hemoglobin Constant Spring and should always be used if normal A>. Lane 2 is an individual with B thalassemia minor (A; =
such a variant is suspected. 5%) and “Swiss HPFH” (F, = 7%).
246 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

HEMOGLOBIN A, QUANTITATION iron-deficiency anemia, as well as in the assessment of the iron

The elevation of hemoglobin A, is an excellent tool for the load in a patient with thalassemia major or intermedia. The
detection of a heterozygote carrier of 8 thalassemia. It is char- serum iron and serum ferritin levels are low and the TIBC
acteristic of heterozygous 8 thalassemia and has high specificity increased in patients with iron deficiency. These values are
with no overlap values between heterozygous carriers and normal in patients with thalassemia minor, unless they have
normal individuals. The level of hemoglobin A, ranges between concurrent iron deficiency. Patients with thalassemia major
3.5% and 7% in heterozygous B thalassemia, whereas normal who have been transfused have increased levels of serum iron
values are always less than 3.5%. A few rare variants of that approach 100% saturation of the TIBC. The serum ferritin
8 thalassemia with normal A,, which are called normal A, level is elevated and indicates the amount of iron deposited in
8 thalassemia, do exist, and can be distinguished in the carrier the tissues.
state from heterozygous a thalassemia only by globin-chain
synthesis. Also, in an iron-deficient patient with B thalassemia
Differential Diagnosis of Microcytic,
minor, hemoglobin A, may be reduced to normal levels.
The percentage of hemoglobin A, can be quantified by densito-
Hypochromic Anemia
metric scanning of gels after electrophoresis, microcolumn The differential diagnosis of microcytic hypochromic anemia
chromatography, or cation-exchange high-performance liquid includes iron deficiency, a thalassemia, B thalassemia, anemia
chromatography (HPLC). HPLC is gaining favor as a method of chronic disease, hemoglobin E disease, sideroblastic
for quantitating hemoglobin A, because of its higher precision anemia, and lead poisoning. Evaluation of the clinical history,
compared to the other methodologies mentioned above. hemoglobin level, and red cell indices (in particular, MCV and
Hemoglobins A, F, S, and C can also be quantitated on some MCH), and examination of the peripheral smear usually nar-
HPLC systems. Limitations of HPLC include the co-elution of row the diagnosis. The sometimes difficult differentiation of
some hemoglobin variants with hemoglobin A,, which prevents the thalassemia carrier from the iron-deficiency state can be
accurate quantitation, and overestimation of A, in carriers of achieved by evaluating the serum iron and ferritin levels and
hemoglobin S secondary to hemoglobin S adducts.*! the TIBC. A markedly elevated free erythrocyte protopor-
phyrin (FEP) identifies a child with lead poisoning. Cellulose
HEMOGLOBIN F QUANTITATION acetate electrophoresis usually allows differentiation between
The hemoglobin F levels are useful in the definition of the a B-thalassemia carrier, an a-thalassemia carrier, or the pres-
type of thalassemia involved, and a summary of the levels of ence of hemoglobin E. The differentiation between these dis-
hemoglobin F corresponding to the different types of eases is summarized in Table 12-5.
thalassemia can be found in Table 12-4. The hemoglobin F
level is normally below 2%. Approximately half of the
8-thalassemia carriers have a mildly elevated level of hemo- Treatment of Thalassemia
globin F, usually below 5%.
Blood Transfusion in Thalassemia

Routine Chemistry Three main concerns need to be addressed regarding patients

with thalassemia major who will be receiving a regular blood
The indirect bilirubin level is elevated in thalassemia major transfusion program:
and intermedia, ranging from | to 6 mg/dL. It is characteris-
1. The development of iron overload
tically more elevated in thalassemia intermedia than in tha-
2. The development of alloimmunization
lassemia major.
3. The risk of transfusion-transmitted diseases
The assessment of the iron status of the patient by the
determination of the serum iron level, total iron-binding The problem of iron overload can be approached from two
capacity (TIBC), and serum ferritin level is useful in the directions—by increasing the iron excretion or by decreas-
differentiation of a thalassemia carrier from a patient with ing the amount of iron transfused. This latter option can be

Serum Iron TIBC Serum Ferritin A, Level

Iron deficiency
a Thalassemia
8 Thalassemia
Hemoglobin E disease
Anemia of chronic disease
Sideroblastic anemia

free erythrocyte protoporphyrin; nl = normal.

 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 247

performed by increasing the length of survival of the trans-

Prevention of Thalassemia
fused red cells, which requires selection of younger red
cells (also called neocytes) for transfusion. Young red cells The current optimal therapy for thalassemia major relies on
and reticulocytes have a lower specific gravity than old red intensive use of a fairly sophisticated level of health care.
cells, and by using a differential centrifugation technique, This cannot be achieved in most of the countries where tha-
the blood unit can be separated so that the upper layer of lassemia major is a serious problem without shunting the
cells is collected. These red cells will have a longer life major thrust of the health resources in that direction. Another
expectancy and can decrease the transfusion requirement of approach to this problem at the health planning level is to
a patient by lengthening the interval of the blood transfu- decrease the number of births of infants with thalassemia
sion schedule. major. This has been successful in certain countries, particu-
Alloimmunization is a recurrent problem of all chroni- larly Cyprus, with the implementation of mass population
cally transfused patients. These patients often develop anti- screening for the detection of heterozygous carriers, genetic
bodies to white cell as well as to red cell antigens. Antibodies counselling, and antenatal diagnosis for couples at risk.
to white cell antigens cause febrile nonhemolytic transfusion Screening for heterozygous individuals may include an
reactions that are uncomfortable. These reactions can be automated blood count with RBC indices, hemoglobin elec-
avoided by routinely transfusing leukocyte-reduced red trophoresis, and estimation of A, and F levels. Cut-off values
cells. Alloimmunization to red cell antigens is a more serious of MCV< 78 fL and MCH less than 27 pg have been used to
problem, because this can cause acute or delayed hemolytic identify candidates for further study. Hemoglobin elec-
transfusion reactions and may seriously affect the availability trophoresis and estimation of A, and F can then be performed
of compatible blood. It is recommended that a complete phe- to narrow the possibilities, and molecular studies are useful to
notype of the patient’s red cells be obtained before embarking secure the diagnosis. Developing regions of the world may
on a regular transfusion program. not have access to sophisticated laboratories, so new simple
Transfusion-transmitted diseases are a common compli- methods to augment detection are being sought. An enzyme
cation in multitransfused patients. In the past, patients with linked immunosorbent assay for HbA, has been evaluated
thalassemia major often developed hepatitis. This risk has preliminarily for the detection of 8 thalassemia trait but needs
been significantly decreased in many areas of the world with to be adapted for use in screening programs.” Also a single-
the introduction of testing for hepatitis C. Some patients may tube osmotic fragility test may be useful as a screening tool
develop a chronic form of hepatitis that, in conjunction with for thalassemia in these countries.”°
the toxicity of iron overload, may result in cirrhosis of the To ensure understanding of test results and its conse-
liver. quences, proper counseling should be provided to patients
who have thalassemia trait. For example, couples who each
carry a B-globin gene mutation are at risk of having a child
Curative Treatment of Thalassemia with severe B thalassemia. Perhaps more importantly, women
who carry an «° thalassemia (— —) haplotype are at risk of hav-
Blood transfusion and iron chelation is the mainstay of treat- ing a pregnancy complicated by hemoglobin Bart’s hydrops
ment of severe thalassemia. Rigorous adherence to the trans- fetalis and the associated preeclampsia.
fusion and chelation program can allow individuals to survive Prenatal diagnosis, using DNA hybridization techniques
into the fifth decade; however, many are unable to comply on sampling from chorionic villi, enables physicians to make a
with the regime necessary to achieve maximal medical ther- diagnosis during the first trimester of pregnancy. Heterogeneity
apy. The only curative treatment is transplantation with bone of thalassemia makes molecular diagnosis difficult; therefore, it
marrow, stem cells, or cord blood. The initial experience with is essential to know the ethnicity of mother and father. In this
bone marrow transplant from 1981 to 2003 achieved a 68% way, the molecular testing can be tailored according to the
thalassemia-free survival after 20 years.*??* Stratification of mutations that are most prevalent in the region of interest.
patients into risk groups and optimization of transplant proto-
cols has led to an improvement in the high risk categories.
Even the high risk group now has an overall survival proba-
bility of 93% and a thalassemia free survival probability of
é
Case Study 1
85%.?' The risks of transplantation include acute and chronic
graft versus host disease, infections, and graft rejection with A 25-year-old man of Chinese extraction was evaluated be-
the return of thalassemia. Endocrine complications like cause he was found to be anemic when he attempted to do-
growth disturbances, infertility, and failure to enter puberty nate blood. He otherwise had no complaints. He stated that
can also occur after transplantation. Therefore, a carefu! risk he is active in sports and feels healthy. A complete blood
assessment must be made when considering medical manage- count gave the following results: RBC, 5.76 million, Hgb,
10.4 g/dL; Het, 35.9%; MCV, 62 fL; MCH, 18.1 pg; MCHC,
ment versus transplantation. New approaches such as gene
29 g/dL; RDW, 13.5%. The peripheral blood smear showed
therapy may bring cures with fewer complications. Although
hypochromic, microcytic erythrocytes with a mild anisocyto-
these strategies are investigational at this time, the B-globin sis, occasional target cells, but no basophilic stippling.
gene has been successfully inserted into a mouse model for continued
B thalassemia with good levels of expression.”
248 — Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

Case Study |eet ay Case Study 2

QUESTIONS A 21-year-old healthy Vietnamese woman is seen in a clinic
for a routine health screen. A complete blood count is per-
1. What diagnoses should be considered at this time? formed and the RBC indices are as follows: RBC, 5.16 mil-
2. What laboratory tests are most useful to diagnose the lion; hemoglobin, 11.4 g/dL; MCH, 22.0 pg; MCHC,
cause of the patient’s anemia? Further evaluation reveals 30.7 g/dL, MCV, 71.8 fL; RDW, 14.6%. A peripheral blood
the following findings: serum iron is 95 wg/dL (normal smear shows a microcytic, hypochromic red blood cell
is 60 to 150 ug/dL); TIBC, 305 ug/dL (normal is 260 to picture with frequent target cells.
360 ug/dL); and ferritin level, 175 g/dL (normal is 30
to 300 ug/dL). Cellulose acetate electrophoresis shows
QUESTIONS
an increased amount of hemoglobins F and A,, which
are quantitated to 4% and 5%, respectively. . What is your differential diagnosis?
3. What is your diagnosis now, and what is the significance . Interpret the RBC indices.
of this diagnosis for this patient? — . Iron studies were ordered and are normal. Hemoglobin
Won

electrophoresis was performed and shows a normal pat-

ANSWERS tern with 98% hemoglobin A and 2% hemoglobin A,.
What is the diagnosis?
1. The patient has a microcytic hypochromic anemia. On
clinical history alone, the possibilities of anemia of
ANSWERS
chronic disease and lead poisoning can be ruled out in a
healthy young man. This leaves the possibility of iron 1. The differential diagnosis is essentially the same as in
deficiency, a B thalassemia carrier, an a thalassemia car- the first case study.
rier, sideroblastic anemia, and hemoglobin E disease 2. First, the MCV indicates a microcytosis. Second, notice
(which should be considered in a person of Chinese that there is a relative erythrocytosis; in other words, the
extraction). The red blood cell indices are very helpful RBC count is too high compared to the hemoglobin level.
here. There is a relative erythrocytosis, in that the hemo- The RBC count (in millions) multiplied by 3 should be
globin level is less than expected for the RBC count about equal to the hemoglobin level in g/dL (5 X 3 =
(RBC count X 3 = expected hemoglobin level in g/dL). 15). In this case, the RBC count is higher than expected
Then notice that the RDW is normal here. A patient with for a hemoglobin level of 11.4 g/dL. This relative erythro-
iron deficiency will likely have an elevated RDW. The cytosis is a clue that there is a thalassemic state. Next,
degree of anemia is mild considering that the MCV is patients who are iron deficient with a low MCV are gen-
quite low; this is also a clue that the patient is likely to erally anemic; an MCV of 71.8 fL in the absence of low
have thalassemia. hemoglobin strongly argues against iron deficiency.
i). Aserum iron level, TIBC, serum ferritin level, and cellu- Finally, RDW is generally normal in heterozygous
lose acetate electrophoresis are appropriate tests that thalassemia, but is elevated in an iron deficient state.
may differentiate among these conditions. Overall, the RBC indices are suggestive of thalassemia,
3. Sideroblastic anemia and iron deficiency can be ruled and further testing should be done.
out by the normal iron level, TIBC, and ferritin level. 3. Iron studies eliminate the possibility of iron deficiency.
Although hemoglobin E migrates in the same area as The hemoglobin electrophoresis shows a normal pattern
hemoglobin A;, on cellulose acetate electrophoresis, a with a normal amount of hemoglobin A,. The RBC
patient with heterozygous or homozygous hemoglobin E indices are worrisome for heterozygous thalassemia.
would have a much larger amount of hemoglobin in that This patient most likely has a thalassemia trait
band; thus, hemoglobin E disease is ruled out. An (a° thalassemia), and family studies or molecular studies
a thalassemia carrier would have a normal hemoglobin are indicated to confirm the diagnosis. This case demon-
electrophoresis pattern; therefore, the diagnosis in this strates the importance of RBC indices. Without RBC
patient is heterozygous B thalassemia. Making the diag- indices, the patient may not have been identified. She is
nosis of B thalassemia heterozygosity in this patient is at risk of having a child with hemoglobin Bart’s or
important for two reasons. First, the patient must be hemoglobin H disease. Once thalassemia is identified,
reassured that this level of hemoglobin and hematocrit is she and her spouse have the choice of further risk
normal for him, and he should not be placed on iron evaluation for thalassemia before having a child.
therapy, which could be harmful. Second, the patient
needs to be educated regarding the possibility of his
having a child with a severe congenital anemia and its
therapeutic implications if he marries someone who is a
carrier of B thalassemia, hemoglobin E, or hemoglobin S.
His spouse should be screened for the presence of these
genes and genetic counseling such as antenatal
diagnosis offered if she is a carrier.
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 249

based on clinical presentation are iron deficiency, tha-

Case Study 3 lassemia, and hemoglobin E, all of which can cause
microcytic RBCs.
A 27-year-old African American woman is being evaluated 2. First, the MCV indicates a microcytosis. Second, notice
for fatigue. She appears pale and reports a history of heavy that there is a relative erythrocytosis; in other words, the
menstrual bleeding. RBC indices are as follows: RBC count RBC count is too high compared to the hemoglobin
4.05 million, hemoglobin level, 8.2 g/dL; MCH, 20.4 pg; level. One would expect a hemoglobin level of approxi-
MCHC, 33.2 g/dL, MCV, 61.3 fL, and RDW, 18.6%. A pe- mately 12 g/dL with a RBC count of4 million (4 X
ripheral blood smear shows a microcytic, hypochromic ane- 3 =12). This relative erythrocytosis is suggestive of a
mia with occasional target cells. thalassemic state. Next, the MCV is low and the hemo-
globin is concordantly low, as expected in iron defi-
QUESTIONS ciency. In addition, the RDW 1s elevated suggesting iron
deficiency. Overall, the RBC indices are giving a mixed
1. What is the differential diagnosis? picture of iron deficiency and thalassemia.
2. Interpret the RBC indices. 3. The iron studies indicate the patient is indeed iron defi-
3. Iron studies were performed and are as follows: serum cient. Although the hemoglobin electrophoresis pattern and
iron is 15 mg/dL (normal is 60 to 150 mg/dL); TIBC, level of hemoglobin A, appear normal, iron deficiency can
405 mg/dL (normal is 260 to 360 mg/dL); and ferritin lower the hemoglobin A,. Therefore, 8 thalassemia minor
level, 4 ug/dL (normal is 30 to 300 ug/dL). A hemoglo- cannot be ruled out in the presence of iron deficiency.
bin electrophoresis was performed and shows a normal 4. The patient should be given iron supplementation, and
pattern with 98% hemoglobin A and 2% hemoglobin A3. then hemoglobin electrophoresis with quantitation of
Interpret these finding. hemoglobin A, should be repeated.
4. What should be done next? 5. Now that the patient has been repleted with iron, the
5. Hemoglobin electrophoresis was repeated and shows the hemoglobin A, is elevated and is diagnostic of B tha-
following: 95% hemoglobin A,, 5% hemoglobin A,, and lassemia minor. This case demonstrates that iron defi-
2% hemoglobin F. What is your diagnosis now? ciency should be ruled out before interpreting the hemo-
globin A, as normal. If relative erythrocytosis was not
ANSWERS recognized, follow up studies may not have been per-
formed. The physician may have continued treating the
1. The differential diagnosis is essentially the same as in microcytosis with iron, and the patient may have
the first case study. The most important considerations, become iron overloaded.
continued

CO iastente
1. What is the hemoglobin defect found in thalassemia syn- c. B Thalassemia minor (8 */B)
dromes? d. Hemoglobin H disease (—a/— —)
a. Abnormal incorporation of iron molecule
(Defective production of the globin portion 4, What is the term for the clinical course of homozygous
(d8)° thalassemia?
c. Excessive production of porphyrins
a. Thalassemia minor
d. Amino acid substitution
b. Thalassemia major
2. What type of globin chains and hemoglobin are charac- c. Thalassemia trait
teristics of severe a thalassemia? d. {Thalassemia intermedia
a. Two a chains and two B chains (HbA)
5. Hereditary persistence of fetal hemoglobin (HPFH) is
b. Two a chains and two 6 chains (HbA,)
characterized by the persistence of fetal hemoglobin into
(¢)Four 8 chains (HbH) or four y chains (Hb Bart’s)
adult life. What are the clinical manifestations of this
d. Two @ chains and two y chains (HbF)
condition?
3. Which type of thalassemia has primarily hemoglobin a. Chronic anemia with skeletal abnormalities caused by
Bart’s and shows the following clinical expressions: excessive erythropoiesis
infants die in utero or soon after birth, severe anemia, b. Asymptomatic except during pregnancy or stressful
marked hepatomegaly and splenomegaly, and situations
ascites? c. Hydrops fetalis syndrome
(a. Homozygous «° thalassemia (— —/— —) CoN significant abnormalities for heterozygous; minor
b. Homozygous B° thalassemia (B°/B") 7 ymptoms for homozygous
250 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia

11 g/dL range; iron deficiency: increased RDW, with

6. What is the clinical manifestation of a thalassemia with
decreased MCV and MCH only in severe anemia
sickle cell anemia?
c. Heterozygous thalassemia: increased RDW, with
a. Severe, life-threatening anemia
decreased MCH and MCV and Hgb in the 5 to 9 g/dL
b. Relatively asymptomatic until placed in an oxygen-
range; iron deficiency: normal RDW, with normal
deprived environment
MCV and MCH
c.JLess severe than sickle cell anemia alone
d. Heterozygous thalassemia: normal RDW, MCH, and
d. Skeletal abnormality, but milder anemia than sickle
MCY; iron deficiency: RDW, MCH, and MCV all
cell anemia
increased
7. What is the primary risk to thalassemia major patients
10. Which of the following cells are not found in a patient
who are on a high-transfusion (hypertransfusion)
with homozygous B thalassemia?
program?
a. Target cells
a. Hyperviscosity of blood
b. Ovalocytes
b, Iron overload
c. Sickle cells
c. Citrate toxicity
d. Nucleated red cells
d. Electrolyte imbalance
11. Which test is useful in demonstrating the distribution of
8. What routine hematologic finding is indicative of tha-
hemoglobin F and in differentiating pancellular HPFH,
lassemia?
heterocellular HPFH, and heterozygous 58 thalassemia?
(a, Microcytic, hypochromic anemia
Osmotic fragility
b. Macrocytic, hypochromic anemia
leihauer—Betke acid elution test
c. Normocytic, normochromic anemia
c. Serum ferritin level
d. Macrocytic, normochromic anemia
d. Complete blood count
9. How can iron deficiency be distinguished from heterozy-
12. Which of the following findings would be indicative of
gous a or B thalassemia?
a. Heterozygous thalassemia: decreased RDW, with
heterozygous B thalassemia?
a. Hemoglobin A, level of 3.5% to 7%
increased MCH and MCV and Hgb in the 10 to
b. Hemoglobin F level less than 2%
14 g/dL range; iron deficiency: increased RDW,
c. Hemoglobin A level of 65% to 85%
MCH, and MCV
b. Heterozygous thalassemia: normal RDW, with d. Hemoglobin A, level less than 3.5%
decreased MCH and MCV and Hgb in the 9 to
See answers at the back of this book.

w @ thalassemia can be divided into three clinical categories:

a severe homozygous form with transfusion dependence,
w Thalassemia syndromes result from a decrease in synthe- 8 thalassemia major; an intermediate homozygous form,
sis of one of the globin chains. 8 thalassemia intermedia; and a mild heterozygous form
with few symptoms, B thalassemia minor.
m Two main types of thalassemia exist: alpha (a) tha-
lassemia and beta (8) thalassemia. # « Thalassemia can be divided into four clinical categories
depending on the severity of the disease: hemoglobin
m Both ineffective erythropoiesis and hemolysis occur in
Bart’s (hydrops fetalis syndrome), which is lethal; hemo-
thalassemia; however, ineffective erythropoiesis predom-
globin H disease; a thalassemia minor; and silent carrier
inates in severe B thalassemia ($6 thalassemia major and
of a thalassemia.
intermedia) srdtucietoee Selominatee in severe a tha-
lassemia (hemoglobin H disease). m Delta—beta (58) thalassemia is caused by a decrease in
5 and 6 globin chains and creates thalassemia minor in the
m In thalassemia, there is a decrease in the amount of nor-
heterozygous state and thalassemia intermedia in the
mal physiologic hemoglobin produced, resulting in a
homozygous state.
microcytic, hypochromic anemia.
= Hemoglobin Lepore is caused by a fusion of the 8 and 8
m There are two main types of 8 thalassemia mutations: B°
chains producing an abnormal globin product.
thalassemia producing no B-globin and 8+ thalassemia
producing reduced 8-globin.
continued
 Chapter 12 Hemolytic Anemias Intracorpuscular Defects: IV. Thalassemia 251

@ Hereditary persistence of fetal hemoglobin (HPFH) com- (MCV), and mean corpuscular hemoglobin (MCH), in con-
prises a group of conditions characterized by the persis- junction with a normal to increased red cell count.
tence of fetal hemoglobin synthesis into adult life without w Hemoglobin electrophoresis aids in the diagnosis of
producing significant hematologic abnormalities. thalassemia by detecting increased levels of hemoglobin
g Thalassemia has been associated with a number of hemo- A, and hemoglobin F, as well as other abnormal
globin structural variants, including hemoglobin S, hemo- hemoglobins.
globin C, and hemoglobin E.
@ The thalassemias are generally characterized by a decrease
in hemoglobin level, hematocrit, mean corpuscular volume

REFERENCES . Mourad, FH, et al: Comparison between differentiation from iron deficiency. Am
desferrioxamine and coibined therapy J Clin Pathol 80:31, 1982.
Ih. Cooley, TB, and Lee, P: A series of cases
with desferrioxamine and deferiprone in 20. Hoyer, JD, et al: Flow cytometric mea-
with splenomegaly in children with ane- surement of hemoglobin F in RBCs:
iron overloaded thalassemia. Br J
mia and peculiar bone changes. Trans Diagnostic usefulness in the distinction
Haematol 121:187, 2003.
Am Ped Soc 37:29, 1925. 12. Bradai, M, et al: Hydroxyurea can elimi- of hereditary persistence of fetal hemo-
NO. Nagel, RL, and Roth, EF: Malaria and
nate transfusion requirements in children globin (HPFH) and hemoglobin S-HPFH
red cell genetic defects. Blood 74:1213, with severe B-thalassemia. Blood from other conditions with elevated
1989. 102:1529, 2003. hemoglobin F. Am J Clin Pathol
~Liebhaber, SA, et al: Human a-globin
. Tang, WT, et al: Immunocytological test 117:857, 2002.
gene expression. The dominant role of to detect adult carriers of (S*"/) dele- . Suh, DD, et al: Influence of hemoglobin S
the a2-locus in mRNA and protein syn- tional a-thalassemia. Lancet 342:1145, adducts on hemoglobin A2 quantification
thesis. J Biol Chem 261:15327, 1986. by HPLC. Clin Chem 42:113, 1996.
1993.
. Liebhaber, SA, et al: Homology and con- . Leung TN, et al: Thalassaemia screening . Schrier, SL, and Angelucci, E: New
certed evolution at the al and «2 loci of in pregnancy. Curr Opin Obstet Gynecol strategies in the treatment of the tha-
human a-globin. Nature 290:26, 1981. EI 2Y), KOO. lassemias. Annu Rev Med 56:157, 2005.
. Higgs, DR, et al: The molecular pathol- . Gibbons, R, et al: The a thalassemias . Sevilla, J, et al: Hematopoietic transplan-
ogy of the thalassemias. In Weatherall, and their interactions with structural tation for bone marrow failure syn-
DJ, and Clegg, JB (Eds): The Tha- haemoglobin variants. In Weatherall, DJ, dromes and thalassemia. Bone Marrow
lassemia Syndromes, ed 4. Blackwell Sci- and Clegg, JB (Eds): The Thalassemia Transplant 35:S17, 2005.
entific Publications, Oxford, 2001, p 133 Syndromes, ed 4. Blackwell Scientific 24. Rivella, S, et al: A novel murine model of
ON. Huisman, THJ, et al: A syllabus of tha- Cooley anemia and its rescue by lentiviral-
Publications, Oxford, 2001, p 484.
lassemia mutations. The Sickle Cell Ane- . Katsantoni, EZ, et al: Persistent -y-globin mediated human B-globin gene transfer.
mia Foundation. Augusta, GA, 1997. expression in adult transgenic mice is Blood 101:1902, 2003.
. Ho, PJ, and Thein, SL: Gene regulation mediated by HPFH-2, HPFH-3, and DS. Ravindran, MS, et al: 8 thalassemia car-
and deregulation: A B globin perspec- HPFH-6 breakpoint sequences. Blood rier detection by ELISA: A simple
tive. Blood Rev 14:78, 2000. 102:3412, 2003. screening strategy for developing coun-
. Yuan, J, et al: Accelerated programmed . Chakalova L, et al: The Corfu 58 tha- tries. J Clin Lab Anal 19:22, 2005.
cell death (apoptosis) in erythroid precur- lassemia deletion disrupts y-globin gene . Chow, J, et al: Evaluation of single-tube
sors of patients with severe B-thalassemia silencing and reveals post-transcriptional osmotic fragility as a screening test for
(Cooley’s anemia). Blood 82:2, 1993. regulation of HbF expression. Blood thalassemia. Am J Hematol 79:198,
. Poortrakul, P, et al: A correlation of ery- 105:2154, 2005. 2005.
throkinetics, ineffective erythropoiesis, . Emburg, SH, et al: Concurrent sickle-cell
and erythroid precursor apoptosis in Thai anemia and a-thalassemia. Effect on See the Bibliography for this chapter at the
patients with thalassemia. Blood 96:7, severity of anemia. N Engl J Med back of the book.
2000. 306:270, 1982.
. Cao, A: Diagnosis of B-thalassemia . Johnson, CS, et al: Thalassemia minor:
intermedia at presentation. Birth Defects Routine erythrocyte measurements and
22a MOS8:
 Chapter

Hemolytic Anemias
Extracorpuscular Defects
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
Louann W. Lawrence, DrPH, MT(ASCP)SH, CLSpH(NCA)
Ralph Green, BAppSci(MLS), FAIMLS
Carl R. Schaub, MD

Immune Hemolytic Anemia OBJECTIVES

Definition
At the end of this chapter, the learner should be able to:
immune Hemolysis
Classification of Immune 1. List mechanisms of immune hemolysis.
Hemolytic Anemia
. Define alloimmune hemolytic anemia.
Nonimmune Hemolytic
Anemia . Characterize immediate hemolytic transfusion reactions.
Intracellular Infections . Characterize delayed hemolytic transfusion reactions.
Extracellular Infections
Mechanical Etiologies . Describe the causes of hemolytic disease of the newborn.
Chemical and Physical . Define autoimmune hemolytic anemia.
Agents
Acquired Membrane . Characterize warm autoimmune hemolytic anemia.
Disorders . List features of cold agglutinin syndrome.
Case Study 1 . Describe the principle of the Donath—Landsteiner test used for paroxysmal cold he-
ON
SS
CO
SOF
ee
2
Case Study 2 moglobinuria.
Case Study 3 10 . List three mechanisms for drug-induced immune hemolytic anemia.

11. List extracorpuscular causes of nonimmune hemolytic anemia.

Immune Hemolytic Anemia Immune Hemolysis

Definition ROLE OF COMPLEMENT
Complement is a group of serum proteins that interact with
The term immune hemolytic anemia describes a group of each other to bring about, among other events, complement-
disorders in which erythrocytes are destroyed prematurely dependent cell lysis. Complement can be activated by two dif-
by an immune-mediated process. Immune hemolysis results ferent routes: the classical pathway or the alternate (prop-
from antibodies, complement, or both attached to the red erdin) pathway. !
blood cell membrane, and is confirmed by a positive direct
antiglobulin test (DAT). Immune hemolytic anemia can CLASSICAL PATHWAY Activation of the classical pathway
occur as either an intravascular or an extravascular process. is initiated by immune complexes containing immunoglobu-
In intravascular hemolysis, red cells are destroyed within lin G (IgG1, IgG2, and IgG3) or IgM. The first complement
the vascular system. In extravascular hemolysis, they are component, Cl, consists of three subunits—Clgq, Clr, and
destroyed outside the vascular system in the mononuclear— Cls—as well as calcium (recognition unit). Clq initiates the
phagocyte system (primarily in the spleen and liver). complement cascade by interacting with the Fe portion of the

252
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 253

immunoglobulin (Fig. 13-1). Clq then causes the activation C5 convertase (C4b2a3b) cleaves C5 into the compo-
of Clr, which then activates Cls. (A bar across the top of a nents C5a, which is released into the plasma and acts as an
complement component denotes its active form as shown in anaphylatoxin and a chemotactic agent, and C5b, which binds
Figures 13-1 and 13-2.) C4 is the second complement protein C6 and C7 to the cell membrane. Membrane-bound C5b67
to be activated. This occurs when Cls cleaves C4 into its acti- causes binding of C8, resulting in immediate ion flux into the
vated components, C4a, which remains in the plasma, and cell and the beginning of cell lysis. The C5b678 complex can
C4b, of which a small number of molecules attach to the cell bind up to six C9 molecules, together forming the membrane
membrane, with the rest remaining in the plasma in the inac- attack unit, C5b6789, which causes cell lysis and accelerated
tive form. C2 attaches to C4b in the presence of magnesium movement of ions into the cell. With the binding of C9, the
and is then cleaved by Cls into a and b subunits. C4b2a com- rate of cell lysis is greatly accelerated (see Fig. 13-1).
bines with C4b and forms the enzyme C3 convertase (C4b2a), Complement activity is regulated by certain inhibitors
and C2b is released into the plasma. (Cls inhibitor, C3b inactivator, C4 inactivator) and by the
Amplification of complement activity now occurs with the instability of certain components (C4b2a, and C4b2a3b).'
action of C3 convertase on C3. C3 convertase (C4b2a) cleaves
C3 into its active components, C3a and C3b, and is able to cleave ALTERNATE (PROPERDIN) PATHWAY The alternate, or
hundreds of C3 molecules. C3a is released into the plasma and properdin, pathway of complement activation also results in cell
acts as an anaphylatoxin. C3b binds to the cell membrane and lysis, but by a different mechanism and group of proteins. The
combines with C4b2a to form another enzyme, C5 convertase alternate pathway bypasses the complement components Cl,
(C4b2a3b). Some of the C3b molecules attach to other sites on C2, and C4 and enters at C3. This pathway consists of a distinct
the cell, are inactivated (iC3b), or are cleaved by C3 inactivator group of proteins: complement component C3; factor B, which
to C3c, which 1s released into the plasma, and to C3d, an inactive is enzymatically cleaved into fragments Bb (biologically active)
subunit that remains attached to the cell. The components C4, and Ba; factor D, which cleaves factor B; properdin (P), a serum
C2, and C3 are referred to as the enzyme activation unit. protein that stabilizes the C3bBb complex; and factor H (C3b
inactivator accelerator), which aids in controlling activation of
Cl (Clq, Clr, Cls—recognition unit) the alternate pathway? (see Fig. 13-2).
Clq + IgG or IgM + Caté
mace

Clr = ‘ ‘ C8, factor B, factor D, Properdin(P)

C4a /C4 inactivator (factor |)
¢
7

Cle C4 Cap *--+C4e, C4d C83Bb (Priming C3 Convertase)

| Mgt

C2 C3b + factor B + Mgt?

factor D-——~>
C2a [ca
factor H
(Control proteins)
C4b2a (C3 Convertase) Bb factor |
Ba /
/

y
ace C3
C3 +C3bBb (C3 Convertase)
C3a ,C3 inactivator (factor |, factor H) C8b inactivator ae
YA
¢ Vi ee C3a
N

C3c, C3d «---C3b” C3c, C3d

Sees
<--- C3b + C&8bBb + Properdin

C4b2a3b (C5 Convertase, enzyme attack unit)

C4b2a3b (C5 Convertase)

.
|
C5 G5
ten
C5a
C5b + C6 + C7 C5b + C6 + C7

C5b67 + C8
<—
|<
ante+ C9 oTSi +~ @)
O a| QO foe)
o

=—— OO
<——
C5b6789 (membrane attack unit) oO a oy <4foe)©

|
Cell Lysis
<—o
Cell Lysis

Figure 13-1 @ Classical pathway of complement activation. Figure 15-2 @ Alternate pathway of complement activation.
254 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects

The alternate pathway may be triggered by certain micro-

organisms, polysaccharides, lipopolysaccharides, aggregates of
IgA, and cells or particles even in the absence of specific anti-
body. Present in the plasma are small amounts of a “priming”
Characteristic IgGl IgG2 IgG3
C3 convertase (C3bBb). The priming C3 convertase is produced
continuously, owing to spontaneous interaction of intact C3, % Total serum IgG 23-28 4-7 3a
factor B, factor D, and properdin, an event not requiring activat- Complement fixation Yes No
ing substances. This results in the formation of small amounts of (classic pathway)
C3b. C3b binds to the cell surface and, under appropriate condi- Binding to macrophage
Fe receptors
tions and in the presence of magnesium, causes the attachment
Placental transfer
of factor B. The bound factor B is cleaved by factor D, releas- Biologic half-life (days) 21
ing the Ba fragment and uncovering the C3 cleaving site on the
Bb fragment. The C3bBb complex can rapidly lose activity or
disassociate unless properdin is present. Properdin binds to the
C3b part of the complex and stabilizes it. The C3bBb fragment Antibody-Dependent Cellular Cytotoxicity
(C3 convertase) then cleaves more C3, resulting in C3a and C3b Another possible mechanism of direct (intravascular) lysis of
fragments. A complex of C3bBbP3b (C5 convertase) forms and immunoglobulin-coated red cells is antibody-dependent cellular
cleaves C5 into its fragments, C5a, and C5b. CSb, together with cytotoxicity (ADCC). Many white cell lines (macrophages/
C6, C7, C8, and C9, form the membrane attack unit, the same monocytes, neutrophils, and natural killer lymphocytes [NK
way as in the classical pathway, which results in cell lysis (see cells]) have receptors on their membranes that bind
Fig. 13-2). immunoglobulins and complement degradation products.° The
Control mechanisms also exist in the alternate pathway, cells with such receptors are collectively referred to as effector
just as they do in the classical pathway. Spontaneous dissolu- cells. The effector cell receptors specific for IgG1 or IgG3 are
tion of C3bBb may occur, or factor H with factor I protein called Fe receptors (FcR). They are called FcR because they
may compete with factor B, as may Bb for the C3b fragment, bind the Fe portion of these immunoglobulins (see Chap. 22 for
then blocking the formation of C3 convertase (C3bBb). Fac- a review of immunoglobulin structure). These effector cells also
tor H may increase the susceptibility of C3b to be destroyed have receptors for complement degradation products, C3b and
by C3b inactivator. iC3b. These complement receptors (CR) are called CRI and
CR3, respectively. ADCC results when the immunoprotein
(IgG3, IgG1, C3b, or iC3b) is bound to its respective FcR or CR
MECHANISMS OF IMMUNE HEMOLYSIS
of the effector cell. This interaction causes the release of lytic
INTRAVASCULAR HEMOLYSIS Intravascular hemolysis, as
enzymes.”* It should be noted that not all IgGl and IgG3
the name implies, occurs within the vascular system and
immunoglobulins are capable of mediating lysis.° It is very
results from activation of the classical complement pathway
likely that complement degradation products work synergisti-
via IgM or IgG antibodies (see Chap. 22 for an explanation of
cally with IgG3 or IgG1, or both, to enhance ADCC.”!©?
immunoglobulins). Intravascular hemolysis occurs when
antibodies that bind to antigenic determinants on red cells
Laboratory Findings
activate complement to completion (i.e., lysis). This occurs
Laboratory findings associated with all hemolytic anemia
only when the activation process is intense enough to over-
include the presence of anemia (commonly normocytic but
whelm the natural regulatory process that inactivates comple-
occasionally macrocytic); reticulocytosis (increased reticulocyte
ment. IgM is a very efficient activator of complement because
count) reflecting erythroid hyperplasia of the bone marrow; and
of its pentameric structure. A single molecule of IgM is capa-
accumulation of the products of red cell catabolism. Patients
ble of initiating complement activation by the classical path-
will show increased serum bilirubin (primarily indirect or
way.** The ability of IgG antibodies to activate complement
unconjugated, but also including direct or conjugated); and ele-
is dependent on several factors:
vated lactate dehydrogenase (LD), primarily isoenzyme LD-1.
1. The IgG subclass; IgG3 is the most efficient at activating In immune hemolytic anemia, the DAT will be positive. (See
complement, followed by IgGl then IgG2. IgG4 is under Extravascular Immune Hemolysis below for a discussion
not capable of complement activation.» The biologic of the DAT.) The laboratory findings in immune hemolysis are
properties of the IgG subclasses are summarized in summarized in Table 13-2. (For a review of hemoglobin catab-
Table 13-1. olism, see Chap. 3, Erythrocyte Senescence.)
2. The number and ___location of IgG molecules on the red cell Hemoglobin is released into the blood when red cells are
surface; at least two IgG molecules must be in close destroyed intravascularly. This condition of free hemoglobin
enough proximity to allow cross-linking of complement liberation in the blood is called hemoglobinemia. Free hemo-
receptors, which initiates complement activation.*» globin is filtered through the kidneys, resulting in hemoglo-
3. The physical ___location of the red cell antigens influences the binuria (free hemoglobin in the urine). Hemoglobinuria may
binding of IgG. be confused with hematuria (intact red cells in the urine),
4. The ability of the immunoglobulin to remain attached to especially when the urine is red. It is important to distinguish
the red cell surface (avidity) is important as well. between the two because hemoglobinuria is an indicator of
 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects _
i) Nn

| Table 13-2 Mechanisms of Immune Hemolysis

Intravascular Extravascular

Mechanisms IgM or IgG3, IgG1, IgG2 (two IgG IgM and/or IgG sensitization with/
molecules within close proximity of each without iC3b (inactivated complement)
other) activate complement to completion Cell-mediated phagocytosis
Antibody-dependent cellular
cytotoxicity (ADCC)
Organ Occurs within blood vessels Spleen: IgG alone or IgG +
iC3b-coated cells
Liver: iC3b alone or IgG +
iC3b-coated cells
Laboratory findings Hemoglobinemia Spherocytosis
Hemoglobinuria
Serum haptoglobin: marked decrease Serum haptoglobin: decreased
Indirect bilirubin: elevated Indirect bilirubin: elevated
Lactate dehydrogenase (LD): elevated Lactate dehydrogenase (LD): elevated
Positive direct antiglobulin test Positive direct antiglobulin test

hemolysis, whereas hematuria is not related to hemolysis. process does not always go to completion (C1 through C9). In
Microscopic examination of the urine may be helpful in iden- most cases, complement activation is stopped by an inhibitory
tifying intact red cells to distinguish hematuria from hemo- factor (control mechanism) at the C3b stage. C3b is cleaved to
globinuria. In chronic intravascular hemolysis, hemosiderin form iC3b. If activation is stopped, iC3b is further broken
may appear in the urine (hemosiderinuria). down into C3dg, which then remains attached to the red cell
In hemolysis, free hemoglobin in the blood is bound by membrane.”
a molecule called haptoglobin, which is consumed in the Red cells coated with IgG! or IgG3 are preferentially
process. Within hours of intravascular hemolysis, haptoglobin removed in the spleen® rather than the liver, because blood
is depleted. As little as 5 mL of lysed red cells can bind all of passing through the spleen becomes hemoconcentrated, alter-
the available haptoglobin. However, decreased haptoglobin is ing the ratio between free [gG in the plasma and cell-bound
not specific for intravascular hemolysis, since it is also seen IgG. Free IgG can bind to Fe receptors, blocking their ability
in extravascular hemolysis. In addition, haptoglobin is rapidly to bind the IgG that is attached to red cells. The condition of
synthesized in the liver, and can return to normal levels within hemoconcentration in the spleen shifts the ratio of free IgG to
24 hours. Because haptoglobin is an acute phase reactant pro- red cell—bound IgG in favor of the red cell—bound IgG. The
tein, concentrations may vary considerably, depending on activated form of complement (C3b) or its inactivated form
several factors, such as underlying disease processes. Conse- (iC3b) are not present in the free form in plasma; therefore,
quently, care must be taken when interpreting haptoglobin
levels as an indicator of hemolysis. Ji is advisable to have a
“baseline” haptoglobin level to compare to the haptoglobin INTRAVASCULAR HEMOLYTIC EVENT
level following suspected hemolysis. See Figure 13-3 for the
sequence of laboratory findings. Serum haptoglobin

Hemoglobinuria
EXTRAVASCULAR IMMUNE HEMOLYSIS Extravascular
hemolysis is the phagocytosis of red cells by fixed phagocytes
within the mononuclear phagocyte system (MPS), formerly
called the reticuloendothelial system. The two major organs of Level
——~> —
the MPS are the spleen and the liver. In the red pulp region of
Hemosiderinuria
the spleen, macrophages with Fc receptors (see ADCC, earlier)
line the splenic cords. Antibody-coated red cells interact with
the Fe receptors, resulting in complete or partial phagocytosis.
In the case of partial phagocytosis, part of the red cell membrane
is removed. If the red cell membrane is able to repair itself, the Time (days)
normal biconcave disk is converted to a sphere-shaped red cell
called a spherocyte. Spherocytes lack deformability and become Figure 13-3 @ Indicators of acute intravascular hemolysis. Within
a few hours of an acute hemolytic event, free hemoglobin is cleared
physically trapped in the spleen; those that do escape the spleen
from plasma and the serum haptoglobin falls to undetectable levels:
can be seen in the peripheral blood and their presence 1s indica- hemoglobinuria ceases soon after. If no further hemolysis occurs,
tive of immune-mediated hemolysis (Fig. 13-4). the serum haptoglobin level recovers, and methemalbumin disap-
As previously mentioned, both IgG and IgM antibodies pears within several days. The urinary hemosiderin can provide
are capable of activating complement. However, the activation more lasting evidence of the hemolytic event.
256 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

Table 13-3 Factors Influencing the

Presence and Extent Ofer
_ Immune Hemolysis |
Antibody
Immunoglobulin class and subclass
Concentration
Avidity
Thermal reactivity (determined by the nature of the
predominant noncovalent bonds formed at the time of the
antigen-antibody reaction)

Antigen
Number and density
Cellular distribution
Presence of soluble antigen
Figure 13-4 = Autoimmune hemolytic anemia (peripheral blood). Complement
Note (A) spherocytes and (B) polychromasia. Concentration of complement factors
Concentration and activity of regulating factors

the hemoconcentration of blood in the spleen does not con- Mononuclear Phagocytic System
Activity of phagocytic cells (influenced by underlying
tribute significantly to the destruction of complement-coated
disease processes, generation and activity of lymphokines
red cells.!° However, cells coated with both IgG and and interleukins, and any concurrent drug therapy)
C3b/iC3b are phagocytized more efficiently in the spleen than
if they are coated by IgG alone.'®
The liver has the largest concentration of macrophages
with receptors specific for immune complexes; thus, the liver 1. Alloimmune: The patient produces alloantibodies to for-
is the major site of removal for red cells coated with comple- eign red cell antigens introduced through transfusions,
ment or heavily coated with IgG.** There is very little pregnancy, or organ transplantation.
removal of red cells coated with small amounts of IgG in the 2. Autoimmune: The control mechanism preventing autoreac-
liver, because of the high concentration of free IgG located tive antibodies is lost and antibodies directed against the
there. Cells sensitized with both IgG and iC3b/C3d are patient’s own red cells develop.
removed in the liver and spleen. 3. Drug-induced: The patient produces antibodies directed at
a particular drug, its metabolites, or red cells coated with
Laboratory Findings the drug. These antibodies then destroy red cells.
Spherocytes are commonly seen on the peripheral blood smear
when extravascular immune hemolysis has occurred. Serum
ALLOIMMUNE HEMOLYTIC ANEMIA
bilirubin (primarily indirect) and LD are usually elevated, and
Alloimmunization is the process in which the immune system
urobilinogen may be increased in urine and stool specimens.
of an individual is stimulated by a foreign antigen and pro-
The DAT is usually positive. The DAT (also called the antihu-
duces the corresponding antibody. The antibody produced by
man globulin test, AHG, and previously called the direct
this immune response is termed an alloantibody. The anti-
Coombs’ test) detects antibody and/or complement coating
body coats the foreign red cells introduced into the circula-
patient red cells. In the DAT, patient red cells are washed to
tion, resulting in shortened red cell survival. Alloantibody
remove adherent proteins, and then reacted with reagent con-
production may result from:
taining high-titer, polyspecific antibodies against both IgG (all
subclasses) and complement, and examined for resulting red |. Transfusion of blood (antibodies are produced against for-
cell agglutination; this is called the polyspecific DAT. If this test eign donor red cell antigens)
is positive, testing is repeated with monospecific reagents that 2. Pregnancy (antibodies are produced against “foreign” anti-
recognize IgG and complement (C3d or C3b+C3d) separately, gens on fetal cells released into the maternal circulation)
to determine which is involved.'' The laboratory findings in 3. Organ transplantation (antibodies are produced against for-
immune hemolysis are summarized in Table 13-2. The factors eign antigens on the transplanted organ or “passenger
that influence immune hemolysis are listed in Table 13-3. cells” that may be released into the recipient).

Alloimmune hemolytic anemia is usually associated with

Classification of Immune Hemolytic blood transfusions. An antibody present in the recipient is
Anemia directed against a foreign red cell antigen located on
the transfused cells. This antibody destroys transfused
Numerous classifications of immune hemolytic anemias have red cells but not native red cells. Basically there are two
been proposed; however, three broad categories are usually types of transfusion reactions: acute hemolytic and delayed
used: hemolytic.
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects =257

ACUTE HEMOLYTIC TRANSFUSION REACTIONS Acute posttransfusion sample as early as 48 hours after the transfusion.
hemolytic transfusion reactions (acute HTRs) are character- This type of reaction is termed delayed, because it takes time for
ized by acute intravascular hemolysis and are associated with the patient to produce sufficient antibody to destroy the trans-
the ABO blood group antibodies. The immunoglobulins asso- fused cells. Characteristically the reaction may occur anywhere
ciated with the ABO blood group are IgM or both IgM and from 2 to 10 days after transfusion.'*
IgG. In individuals who have both types (IgM and IgG), the The symptoms of delayed HTRs are usually mild and
majority of antibody is IgM with a minor amount of IgG." nonspecific; therefore, delayed HTRs may not be recognized
Ordinarily, an individual possesses antibodies directed toward by clinicians.!° Symptoms include mild fever, mild jaundice,
the A and/or B antigens absent from his or her own red cells. and an unexpected fall in hemoglobin (or conversely, lack of
As previously stated, IgM antibodies are efficient activators expected rise in hemoglobin after transfusion). Laboratory
of complement, which results in immediate destruction of the findings are those associated with extravascular hemolysis, but
transfused cells. most cases are subclinical and discovered only serologically
Symptoms of acute HTRs are variable. Typical symp- (through direct antiglobulin test, antibody screening, and com-
toms are fever, shaking, chills, and pain or a burning sensation patibility tests).'°
along the infusion site. Other symptoms include nausea, vom- Treatment is rarely necessary, and investigation focuses
iting, lower back pain, hypotension, and chest pain. See Table on accurately identifying the antibody to ensure that blood for
13-4 for a list of clinical features. An incoherent, unconscious, future transfusions will be negative for the foreign antigen
or anesthetized patient cannot verbalize his or her symptoms that corresponds to the patient’s antibody. The antibodies
and is therefore at greater risk of receiving one or more units most commonly implicated in delayed HTRs are listed in
of incompatible blood undetected. Table 13-5, and the laboratory findings are summarized in
The laboratory findings in acute HTRs are those associ- Table 13-6.
ated with intravascular hemolysis (see Table 13-2). The treat-
ment of acute HTR focuses on prompt termination of the HEMOLYTIC DISEASE OF THE NEWBORN Hemolytic dis-
transfusion and treatment of any signs or symptoms of shock ease of the newborn (HDN) is an immune hemolytic disorder
with supportive measures. Stroma from hemolysed red cells in which fetal or newborn red cells are destroyed by maternal
can clog and damage renal glomeruli; thus, intravenous fluids alloantibodies. In HDN, maternal—fetal blood group incompat-
are administered to maintain renal function. ibility is always present. Maternal IgG antibodies directed
against “foreign” antigens on fetal red cells cross the placenta
DELAYED HEMOLYTIC TRANSFUSION REACTIONS and destroy the red cells in the fetal circulation. Only IgG
Delayed hemolytic transfusion reactions (delayed HTR) are antibodies can cross the placenta; IgM cannot, and does not
associated with antibodies to blood groups other than the ABO cause HDN.
blood group. Antibodies implicated in delayed HTR are usually Pregnant women can become immunized to “foreign”
IgG, which may activate complement, causing sensitization of fetal red cell antigens when fetal blood enters the maternal cir-
the red cells with C3 but seldom leading to intravascular hemol- culation during pregnancy or at delivery (fetal-maternal hemor-
ysis. Delayed HTRs are the most common type of transfusion rhage). The amount of whole blood exchanged is normally only
reactions. They are caused by an anamnestic or secondary | mL. There is some evidence that as little as 0.03 mL of red
immune response to the transfused red cells. This occurs in pre- cells can stimulate an immune response.'*'” Thus, it is possible
viously immunized patients whose alloantibody level (after the to become immunized from miscarriages and abortions. Blood
initial stimulation) has dropped to serologically undetectable transfusions can also stimulate an immune response, as men-
levels. As a result, the initial antibody screening and compatibil- tioned earlier in the discussion of alloimmune hemolytic
ity tests on the patient’s pretransfusion sample are negative.
When the patient is re-exposed to the foreign red cell antigen
(transfused), an anamnestic response is mounted by the recipi- ys Table 13-5 Antibodies Most
ent. The titer rises and the antibody may be detected in the . Commonly Implicated
in Delayed Hemolytic :
_ Transfusion Reactions Cae
Table134 Clinical Features of (DHTRs) :
: - Acute Hemolytic Antibody Blood Group System
-Transfusion Reactions.
Anti-Jk*
Fever Hemoglobinuria Anti-K
Chills Shock Anti-c
Chest pains Generalized bleeding Anti-E
Hypotension Oliguria Anti-Fy*
Nausea Anuria Anti-Jk°
Flushing Back pain Anti-C
Dyspnea Pain at infusion site Anti-e
258 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

table 13-6 Differential Diagnosis of 3 Table 13-7

_ Hemolytic Anemia — |
Parameter/Analyte Extravascular Intravascular
ABO HDN
Serial hemoglobin and Decreased Decreased Rh HDN
hematocrit Other
Reticulocyte count* Increased Increased
RBC morphology Spherocytes
Serum bilirubin (indirect) Increased Increased
Serum haptoglobin* Decreased* Decreased autoantibodies whose serologic reactivity is optimal at 4°C but
Serum LD (isoenzyme Increased Increased
that also react at temperatures between 25°C and 31°C. Two
ED 1):
Hemoglobinemia Absent Present types of cold AIHA have been described:
Hemoglobinuria Absent Present
1. Cold agglutinin syndrome (CAS)
*May not show increase immediately. 2. Paroxysmal cold hemoglobinuria (PCH).
‘Important to compare with a prehemolysis “baseline” value.
*More likely to be markedly decreased with intravascular hemolysis. Some drugs may induce the formation of autoantibodies that
LD = lactate dehydrogenase. may be difficult to distinguish from other cases of AIHA.
Drug-induced immune hemolytic anemia is the third type of
AIHA, representing approximately 12% of cases in various
anemia. If a pregnant woman has been previously immunized to studies.*! The frequency of the various types of AIHA is listed
a foreign red cell antigen and that antigen is present on the red in Table 13-9.
cells of the fetus, the exchange of blood that occurs early in the
pregnancy will be sufficient to stimulate an anamnestic response WARM AUTOIMMUNE HEMOLYTIC ANEMIA Warm
in the mother. The mother can produce increasing amounts of autoimmune hemolytic anemia (WAIHA) is one of the com-
antibody directed against the fetal red cells.'* The fetal red cells monest causes of hemolytic anemia in adults, with a slightly
will become coated with maternal antibodies and destroyed by higher frequency of disease in women than in men.**~ The
extravascular hemolysis. Responding to this increased red cell majority of individuals who develop WAIHA are older than
destruction, fetal hematopoietic tissue increases erythrocyte pro- AO years of age.?!
duction. The fetal bone marrow may not be able to keep up with WAIHA may be idiopathic, with no underlying disease
the increased need for red cells. Extramedullary hematopoiesis process (50% to 70% of cases), or it may be secondary to
is expanded, causing liver and splenic enlargement. The fetus another disease process (30% to 50% of cases),*° such as lym-
may not be able to compensate for the hemolysis, and an ane- phoid neoplasm, other neoplastic disorder, autoimmune or
mia characterized by increased numbers of nucleated RBCs chronic inflammatory disorder, and viral infection. The disor-
(erythroblasts) in peripheral blood may result. Hence, the term ders reported to be associated with WAIHA are listed in Table
erythroblastosis fetalis has been used to describe HDN. There 13-10. Development of WAIHA may precede the diagnosis of
are two major forms of HDN: that associated with ABO, and one of these disorders. In one series of patients with idiopathic
that associated with Rh(D) or other blood group antigens. Table WAIHA, 18% were diagnosed with a lymphoid neoplasm
13-7 lists the frequencies of the various types of HDN. Table during 2 years of follow-up.”
13-8 provides a comparison of ABO and Rh(D) HDN. (For Signs and symptoms usually do not appear until significant
further information on hemolytic disease of the newborn, see anemia has developed. Pallor, weakness, dizziness, dyspnea,
Harmening, DM: Modern Blood Banking and Transfusion Prac- jaundice, and unexplained fever are occasionally presenting
tices, ed 5. FA Davis Company, 2005). complaints. Hemolysis is usually extravascular and occurs pre-
dominantly in the spleen. The degree of anemia can be severe
AUTOIMMUNE HEMOLYTIC ANEMIA (hemoglobin less than 7.0 g/dL) or mild. The onset of WATHA
Autoimmune hemolytic anemia (AIHA) represents an abnor- is usually gradual and may be precipitated by a variety of fac-
mality within the immune system whereby the ability for self- tors, such as infection, especially viral infection,’ or after blood
recognition of an individual’s own red cell antigens is lost. transfusion or organ transplantation.**?? WAIHA may predis-
Recent experiments support that recognition of self occurs pose patients to thromboembolic disease. *°
during embryogenesis by inactivation of autoreactive B and T
lymphocytes. Under certain conditions (e.g., bacterial or viral Serologic Evaluation
infections), these autoreactive B and T lymphocytes escape the A positive DAT is present with a polyspecific antiglobulin
mechanism for tolerance of self.'? As a result, patients produce reagent. On further analysis with monospecific antiglobulin
antibodies that bind to their own red cells (autoantibody). AIHA reagents, the DAT result is frequently positive for both IgG
can be broadly divided into warm or cold types. The warm type and C3d (67% of cases). The remaining cases are positive for
(WAIHA) is the most common, accounting for approximately IgG (20%) or C3d (13%) alone.*
70% of all cases.*”?! This type involves autoantibodies whose The serum of a patient with WAIHA usually demon-
serologic reactivity is optimal at 37°C. Cold AIHA involves strates evidence of free autoantibody at low titer (e.g., weak
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 259

aie ABO Rh

Severity Mild Severe

Child affected First-born (40%-50% of cases) Usually second or subsequent
births (first-born: 5% of case)
Blood groups Mother: O Mother: Rh negative
Child: A or B Child: Rh positive
Anemia Uncommon, mild Severe
Stllbirths/hydrops fetalis Rare Frequent
Jaundice Mild Severe
Spherocytes on peripheral smear Usually present Absent
Direct Coombs’ test result Negative or weakly positive Positive
Maternal antibodies Inconsistent, inconclusive Always present
Antenatal diagnosis Unnecessary Necessary
Treatment (dependent on severity) Phototherapy Exchange transfusion (common
(common) newborn treatment)
Exchange transfusion (rare) Intrauterine transfusion (common
antenatal treatment)
Types of antibody ; IgG IgG
Prophylaxis None RhIG
Antenatal RhIG

reactivity). In 80% of the cases, the immunoglobulin is IgG is also lackingSo in Rh,,,, null cells.2? On rare occasions, other specifici-
alone or IgG together with IgA, IgM, and/or C3d.*! Comple- ties have also been reported.*'??!
ment proteins act synergistically with immunoglobulins to
cause red cell hemolysis. In fact, the severity of hemolysis is DAT-Negative AIHA
greater in the presence of complement in addition to IgG.*!? Occasionally (in 1% to 3% of patients) the DAT result is repeat-
Although not evaluated as a part of routine diagnostic testing, edly negative in a patient who has clear evidence of hemolysis
the presence of IgA, IgM, or both, may be found in addition with no other apparent cause.**** These patients represent a
to IgG if appropriate antisera are used.*! WAIHA can present small group that is referred to as having DAT-negative AITHA.*°
several difficult problems in serologic testing, which fall into More sensitive techniques for the detection of IgG or C3d, or
two categories: both, on red cells have shown that many of these patients have
increased levels of these immunoproteins on their cells. The
1. The patient’s red cells are strongly coated with autoanti-
routine direct antiglobulin test (DAT) can detect immunoglob-
body, which interferes with phenotyping.
ulin sensitization of as little as 200 molecules of IgG per red
2. Autoantibody present in the serum may mask an underly-
cell.*! More sensitive techniques are capable of detecting as
ing alloantibody.
few as 20 IgG molecules.*° When interpreting DAT results, it is
important to remember that a positive DAT alone is not indica-
Autoantibody Specificity
tive of immune hemolysis, but 1f hemolysis is present or sus-
The autoantibodies produced in WAIHA usually react with all
pected, it could be the result of immune mechanisms. The DAT
cells tested. Serologic studies have suggested that some autoanti-
result can be positive in up to 8% of hospitalized patients who
bodies are directed at Rh blood group antigens because of their
have no signs or symptoms of hemolysis.*°°! In most of these
lack of reactivity with Rh,,,, cells (cells which lack all Rh blood
group antigens).'? However, further analysis of these autoantibod-
ies with apparent Rh specificity has demonstrated that the reactiv-
ity is actually directed at another red cell membrane protein which

wie 15-9 TypesofAutoimmune —

Lymphoid neoplasms such as chronic lymphocytic leukemia,

(by -
Hemolytic Anemia
Hodgkin’s disease, non-Hodgkin’s lymphoma, multiple
myeloma, and Waldenstrom’s macroglobulinemia
Autoimmune disorders such as systemic lupus erythematosis,
rheumatoid arthritis, scleroderma, and pernicious anemia
Warm AIHA Other neoplastic disorders, including carcinomas of the ovary,
Cold agglutinin syndrome breast, lung, colon, pancreas, thymus, kidney, and uterus
Paroxysmal cold hemoglobinuria Viral infections, including hepatitis B and hepatitis A
Drug-induced Chronic inflammatory disorders including ulcerative colitis
260 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects

patients, the positive result reflects complement sensitization, of these alternative therapies is variable, and they are used
probably secondary to the disease process from which the only in selected cases.“ 25,50,51
patient is suffering.
IgA antibodies have also been reported to cause AIHA COLD AUTOAGGLUTININS
with characteristics of WAIHA or, less commonly, cold agglu- Normal Cold Autoagglutinins
tinin disease.*’*’ These would also result in a negative DAT Cold reacting autoantibodies (autoagglutinins) are present in
result since IgA does not fix complement in vivo.” all normal human sera.*?** The specificity of these cold
autoantibodies includes anti-I, anti-H, and anti-IH. Practically
Laboratory Diagnosis all adults have I and H antigens present on their red cells.
Typically, patients with WAIHA exhibit a moderate to severe Generally, most examples of anti-I, anti-H, and anti-IH have
normocytic anemia with increased reticulocyte count. The no clinical significance, and these autoantibodies are often too
blood smear can display classic signs of extravascular hemoly- weak to be detected via routine serologic testing, owing pri-
sis: polychromasia reflecting reticulocytosis (see Fig. 13-4) marily to their low concentration in the serum and their narrow
and spherocytosis. Occasionally, nucleated red blood cells may thermal range (4°C to 22°C). The characteristics of normal
be seen. On rare occasions, WAIHA is associated with reticulo- cold autoantibodies found in healthy adults with those of
cytopenia (decreased reticulocyte count). Reticulocytopenia pathologic cold autoantibodies are compared in Table 13-11.
associated with intense hemolysis indicates a lack of bone mar- The benign autoagglutinins differ in many ways from the
row response, and is associated with a high mortality rate. pathologic cold autoagglutinins that produce cold agglutinin
Patients also show accumulation of the products of red cell syndrome (or cold AIHA). The fundamental characteristic that
catabolism: hyperbilirubinemia (especially unconjugated), and differentiates benign autoagglutinins from pathologic autoag-
increased LD. glutinins is the thermal amplitude: pathologic cold autoagglu-
tinins may react at or above 30°C.»
Treatment
Therapy in WAIHA is aimed at treating the underlying disease
Pathologic Cold Autoantibodies
if one is present. Measures to support cardiovascular function
Pathologic cold autoantibodies can be divided into:
are important in patients who are severely anemic. Transfusion
is usually avoided if possible, as this may only accelerate the 1. Primary (idiopathic) cold agglutinin disease (primary CAD)
hemolysis instead of ameliorating the anemia. However, trans- 2. Cold agglutinin syndrome secondary to infection (sec-
fusion should be used in life-threatening situations. ondary CAD)
As all donor blood is invariably incompatible, it is general 3. Paroxysmal cold hemoglobinuria (PCH)
practice to use donor blood that is least reactive in the cross-
match and that is antigen negative for any clinically significant Cold Agglutinin Syndrome (CAS)
alloantibodies that may be present in the patient’s serum.*!** Primary CAD Primary cold agglutinin also called cold
Blood is transfused slowly, in small volumes (100 mL), and the hemagglutinin disease or idiopathic cold AIHA, represents
patient observed closely for any adverse reactions.**** Some approximately 16% of the cases of AIHA.*' Primary CAD is
hematologists advocate the use of phenotypically similar blood a chronic condition and occurs predominantly in older indi-
irrespective of its degree of incompatibility in the crossmatch. viduals, with a peak incidence after 50 years of age.*! It is
The rationale for this approach is that patients with autoim- found in all racial groups, affecting both men and women.
mune antibodies may be more likely to produce alloimmune Although the disease is often idiopathic, a careful evaluation
antibodies, which can be masked by the autoantibodies. How- of the patient may reveal the presence of a lymphoprolifera-
ever, one study indicates that the incidence of alloimmuniza- tive disorder,'’ other malignancy, or infection. Because of this
tion or adverse hemolytic transfusion reactions in patients with association, it is prudent to investigate patients for possible
WAIHA is no greater than the incidence found in other multi- malignancy when they present with a pathologic cold autoan-
transfused patient populations.*° tibody and no other obvious cause, such as infection. This is
Corticosteroids are usually the first line of treatment. illustrated by one series of 78 patients with persistent cold
Corticosteroids such as prednisone produce their effect by: agglutinin disease. On investigation, 28 of these patients were
found to have no underlying malignancy, 6 had chronic lym-
1. Reduction of antibody synthesis*’
2. Altered antibody avidity‘ phocytic leukemia, 31 had non-Hodgkin’s lymphoma and
13 had Waldenstrom’s macroglobulinemia.*©
3. Depression of macrophage activity,*® which reduces the
clearance of antibody-coated red cells”! CAD is a hemolytic anemia produced by an autoanti-
body that reacts optimally at 4°C, but has a wide thermal
Splenectomy is usually considered as the next step if corti- amplitude, reacting at temperatures greater than 30°C as
costeroid therapy is ineffective. Splenectomy decreases the well.*!»’°* The antibody is usually an IgM immunoglobulin,
production of antibody and removes the primary site of which quite efficiently activates complement.** Antibody
red cell destruction.*! Immunosuppressive drugs, intravenous specificity in this disorder is almost always anti-I,5*** less
immunoglobulin, antilymphocyte globulin, anti-CD20 commonly anti-i, and rarely anti-Pr.*+
(Rituximab)*” or plasma exchange may be used in patients CAD is rarely severe and is usually seasonal, as the cold
who do not respond to conventional therapy. The success rate winter months often precipitate the signs and symptoms of a
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 261

Characteristic Pathologic

Thermal amplitude Broad: up to 32°C

Spontaneous autoagglutination Significant degree that disperses on
warming to 37°C
Titer Zz xo ay ACE > 1:1000 at 4°C
Albumin enhancement None Reactivity enhanced
Clonality of antibody Polyclonal Idiopathic = monoclonal
Secondary to infection = polyclonal
Clinical significance None Causes cold AIHA
Usual antibody specificity Anti-! Anti-I
Direct antiglobulin test (DAT) Negative or weak positive 2 to 3+ with polyspecific antiglobulin
with polyspecific reagent
antiglobulin reagent

chronic hemolytic anemia. Acrocyanosis, also called mance of blood counts and preparation of blood smears may
Raynaud’s phenomenon’ (symptoms of cold intolerance, be difficult. The patient’s mean corpuscular volume (MCV)
such as pain and a bluish tinge in the fingertips and toes, from an automated cell counter will be erroneously high and
owing to vasospasm), is frequently the patient’s main com- the red blood cell count erroneously low due to clumping of
plaint, along with a sense of numbness in the extremities red blood cells. This causes unrealistic values in other calcu-
when exposed to the cold. These symptoms occur because the lated parameters, such as hematocrit, mean corpuscular
cold autoantibody agglutinates the individual’s red cells in the hemoglobin (MCH) and mean corpuscular hemoglobin con-
capillaries of the skin, causing local blood stasis.°? During centration (MCHC). The blood sample should be warmed to
cold weather, the temperature of an individual’s skin and 37°C for 30 to 60 minutes and reassayed on the automated cell
exposed extremities can fall to as low as 28°C, activating the counter to obtain accurate values. In severe cases, replace-
cold autoantibody. This activated cold antibody agglutinates ment of plasma in the prewarmed blood specimen with an
red cells and fixes complement as the erythrocytes flow equal volume of warm saline may be necessary to obtain
through the capillaries of the skin. When the erythrocytes accurate CBC results.
return to the body core (where the temperature is 37°C), the The tendency for spontaneous autoagglutination of red
cold agglutinin elutes off the red cells, leaving activated com- cells from these patients dictates that serum samples must be
plement behind. Hemolysis occurs from the completion of the maintained and separated at 37°C to obtain accurate results for
complement cascade or by removal of red cells sensitized the antibody titer and thermal amplitude studies.*? Similarly,
with C3b/iC3b by macrophages in the liver (see the earlier samples for the determination of DAT results must be collected
section on Intravascular Hemolysis for a review). If any red into ethylene diaminetetraacetic acid (EDTA) to inhibit any in
cells coated with C3b/iC3b escape destruction in the liver, vitro attachment of complement to the cells after collection.
their complement proteins are further degraded to C3d, for A simple serum screening procedure can be performed
which there are no receptors on macrophages.’ The patient’s by testing the ability of the patient’s serum to agglutinate nor-
DAT result will be positive with monospecific anti-C3d mal saline-suspended red cells at 20°C and 4°C. If this test
antiglobulin reagents. result is positive, further steps must be taken to determine the
This hemolytic episode is not associated with fever, titer and thermal amplitude of the cold autoantibody; if nega-
chills, or acute renal insufficiency, as would be characteristic tive, the diagnosis of CAD is unlikely.*”
of patients with paroxysmal cold hemoglobinuria (see later The peripheral blood smear in patients with CAD may
discussion). Hemoglobinemia and, less frequently, hemoglo- show agglutination (clumping of red cells) (Fig. 13-5). The
binuria may be detected after exposure to the cold. Patients clinical criteria for diagnosis of CAD are summarized in
also display weakness, pallor, and weight loss, which are Table 13-12.
characteristic symptoms of chronic anemia. CAS usually
remains quite stable, and when it does progress, it intensifies CAD Secondary to Infections Cold agglutinin syndrome can
gradually. Other clinical features of CAS may include jaun- also occur as a transient disorder that is secondary to infec-
dice and splenomegaly. tions. Episodes of cold autoimmune hemolytic anemia often
occur after upper respiratory infections. Approximately 50%
Laboratory Findings Most patients with CAD present with of patients suffering from pneumonia caused by Mycoplasma
reticulocytosis and a positive DAT result (polyspecific and pneumoniae have elevated titers (greater than 1:64) of cold
C3d). In some cases grossly visible agglutination of anticoag- autoagglutinins.*'°”°* Secondary CAS develops in the second
ulated whole blood samples occurs as the blood cools to room or third week of the patient’s illness, and a rapid onset of
temperature. As a result of this autoagglutination, perfor- hemolysis with symptoms of pallor and jaundice is usually
262 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects

infectious mononucleosis. The percentage of patients with

infectious mononucleosis who develop anti-i varies from 8% to
68%.°+ The antibody is usually a low-titer IgM cold agglu-
tinin with a narrow thermal range. A small number (1%) of
these patients who develop anti-i produce a high-titer, IgM cold
agglutinin with a wide thermal range,°’ which causes in vivo
hemolysis. Acute illness with sore throat and high fever, fol-
lowed by weakness, anemia, and jaundice, are characteristic
features of infectious mononucleosis. For a review of infectious
mononucleosis, see Chapter 15. The cold autoantibody speci-
ficity most commonly found in the various infections that cause
secondary CAD are listed in Table 13-13.

Treatment
Treatment of primary CAD, secondary CAD, and the anemia
Figure 15-5 ® Cold hemagglutinin disease (peripheral blood). associated with infectious mononucleosis is similar. In many
Note the autoagglutination of red cells. cases, the disease is self-limited and requires no treatment.
Patients with persistent disease may be instructed to avoid the
cold, keep warm, or move to a milder climate.® It is interesting
found. Resolution of the episode usually occurs in 2 to
3 weeks, as the hemolysis is self-limited.°* The offending cold to note that one ingenious physician contacted the National
Aeronautics and Space Administration (NASA) for an environ-
autoantibody is an IgM immunoglobulin with characteristic
anti-I specificity. Very high titers of the cold autoagglutinin mentally controlled spacesuit so his patient would not have to
are seen almost exclusively in patients with mycoplasma remain indoors during the acute phase of the disease.®* Corticos-
pneumonia. It has been reported that the cold agglutinin pro- teroids have been used, but have limited success.°! Plasma
duced in this infection is an immunologic response to the exchange has been used in acute cases to provide temporary
mycoplasma antigens, and these antibodies cross-react with removal of antibodies.*°°' Patients with severe disease unre-
the red cell I antigen.®*! sponsive to conventional therapy have been successfully treated
The antibodies produced in both primary CAD and CAD with anti-CD20 (Rituximab).” Splenectomy is ineffective
secondary to mycoplasma pneumonia have anti-I specificity. because extravascular hemolysis resulting from complement
They differ in that the autoantibody in primary CAS is invari- sensitization occurs predominantly in the liver.
ably monoclonal (IgM with kappa [k] light chains only), Transfusion is rarely required. If blood is needed, the
whereas the autoantibody produced secondary to infection is blood should be ABO/Rh compatible and lack any antigens
polyclonal (IgM with both k and lambda [A] light chain for which the patient has an alloantibody. Blood should be
types).° The monoclonality of the autoantibody in primary warmed using a blood warmer and transfused slowly, with
CAS suggests a possible underlying lymphoproliferative constant monitoring of the patient for adverse reactions.”
disorder.
Paroxysmal Cold Hemoglobinuria
Infectious Mononucleosis Paroxysmal cold hemoglobinuria (PCH) is the least common
Infectious mononucleosis may also be associated with a type of AIHA, representing only 1% to 7% of patients.?!
hemolytic anemia resulting from a cold autoagglutinin. Many It occurs most commonly in children, associated with viral
studies have reported an association of anti-i production in disorders such as measles, mumps, chickenpox, infectious
mononucleosis, and the poorly defined ‘flu syndrome.””°
Although PCH is transient and self-limited, severe hemolysis
may occur.

* Clinical signs of an acquired hemolytic anemia, sometimes

with a history of acrocyanosis and hemoglobinuria upon
exposure to cold
* A positive DAT result using polyspecific antisera Type of Infection Cold Autoantibody Specificity
* A positive DAT result using monospecific C3 antisera
* A negative DAT result using monospecific IgG antisera Mycoplasma Anti-I
* The presence of reactivity in the patient’s serum owing to a pneumonia
cold autoantibody Infectious Anti-i
* A cold agglutinin titer of 1:1000 or greater in saline at 4°C mononucleosis
with visible autoagglutination of anticoagulated blood at Lymphoproliferative Anti-I, i, or Pr
room temperature disorder
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 263

of Paroxysmal Cold Hemoglobinuria and Cold Agglutinin

PCH Cold Agglutinin Syndrome

Patient population Children or young adults Elderly or middle-aged

Pathogenesis Following viral infection Idiopathic, lymphoproliferative
disorder or following
Mycoplasma pneumoniae infection
Clinical features Hemoglobinuria: acute attacks Acrocyanosis, autoagglutination
upon exposure to cold (symptoms of blood at room temperature
resolve in hours or days)
Severity of hemolysis Acute and rapid Chronic and rarely severe
Hemolysis Intravascular Extravascular or intravascular
Autoantibody IgG (anti-P specificity, IgM (anti-i/I, monophasic)
biphasic hemolysin)
DAT 3+ (polyspecific)/neg 3+ (polyspecific)/neg
IgG/3-4+ IgG/3-4+
C3 monospecific C3 monospecific
Thermal range Moderate (< 20°C) High (up to 30-31°C)
Titer (4°C) Moderate (< 1:64) High (> 1:1000)
Donath—Landsteiner test Positive Negative
Treatment Supportive (disorder terminates Avoid the cold
when underlying illness resolves)

Originally, PCH was described in association with The other aliquot is cooled at 4°C for 30 minutes and then
syphilis, in which an autoantibody was formed in response to incubated at 37°C for another 30 minutes. Both samples are
Treponema pallidum organism, the causative agent of the dis- then centrifuged and observed for hemolysis. A positive test
ease.’' However, with the discovery and use of antibiotics, result is present when hemolysis is seen in the sample placed
PCH is no longer commonly associated with syphilis. at 4°C and then at 37°C, and no hemolysis in the control
Red cell destruction in PCH is the result of a cold-reacting sample. The Donath—Landsteiner test is summarized in
IgG autoantibody (always polyclonal) termed an autohe- Table 13-15.
molysin. This autohemolysin binds to the P antigen on the
patient’s red cells at lower temperatures. Hemolysis occurs Treatment Protection from cold exposure is the only useful
when the red cells are rewarmed on return to the normal body therapy for PCH. For acute postinfection forms of PCH, the
temperature. The autohemolysin fixes complement, and the hemolysis usually terminates spontaneously after resolution of
sensitized cells undergo complement-mediated intravascular the infectious process.” If anemia is severe, transfusions may
hemolysis.” The PCH autoagglutinin attaches only to red cells be required. The same transfusion protocol as in CAD applies.
at cooler temperatures and then activates complement in Characteristics of warm and cold autoimmune hemolytic ane-
warmer temperatures. Thus, the antibody is called a biphasic mias are reviewed and compared in Table 13-16.
hemolysin. It is also called the Donath—Landsieiner antibody,
and its specificity is anti-P.*’ MIXED AUTOIMMUNE HEMOLYTIC ANEMIA In the past
As the name of PCH implies, paroxysmal or intermittent two decades, a number of reports have drawn attention to
episodes of hemoglobinuria occur on exposure to the cold. the occurrence of mixed AIHA, in which patients exhibit
These acute attacks may be characterized by a sudden onset
of fever, shaking chills, malaise, abdominal cramps, and back
pains.”? All the signs of intravascular hemolysis are evident,
including hemoglobinemia, hemoglobinuria, and hyperbiliru-
binemia (see Fig. 13-3). This results in a severe and rapidly Whole Blood Whole Blood
Control Test
progressive anemia. Polychromasia, nucleated red blood
cells, and poikilocytosis are demonstrated in the peripheral Procedure
blood smear. The symptoms and signs may resolve in a few 1. 30 min He
hours or persist for days. Splenomegaly and renal insuffi- 2. 30 min BRE
ciency may also develop. PCH and cold agglutinin syndrome 3. Centrifuge
are compared and contrasted in Table 13-14. and observe
The Donath—Landsteiner test is the classic diagnostic Results
test for PCH, developed by the two physicians for whom it is Positive No hemolysis Hemolysis
named. A blood sample drawn from the patient is split into Negative No hemolysis No hemolysis
two aliquots maintained at different temperatures. One Inconclusive Hemolysis Hemolysis
aliquot, used as the control, is kept at 37°C for 60 minutes.
264 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

unknown.* A positive DAT develops in approximately 12% to

Table 13-16 Comparison of Warm 15% of the patients receiving a-methyldopa, and 1% to 3% of
and Cold Autoimmune these patients go on to develop AIHA. The antibodies produced
Hemolytic Anemias by patients suffering from this disorder react weakly with all
cells tested or demonstrate specificities similar to those found
WAIHA Cold AIHA in WAIHA. Hemolysis is extravascular and the DAT result is
strongly positive with anti-[gG and negative with anti-C3.
Optimal == 32,€ <3, ()2@
reactivity Patients may continue to have a positive DAT result for up to
Immunoglobulin IgG IgM (exception: 2 years after discontinuation of the drug.
class PCH-IgG)
Complement May bind Binds complement DRUG ADSORPTION (HAPTEN) MECHANISM This is the
activation complement
second most common mechanism of drug-induced hemolytic
Hemolysis Usually Usually intravascular
extravascular anemia. The drugs implicated in this response include the
Frequency 70%-15% 16% of cases penicillins and the cephalosporins.’* This mechanism requires
of cases (PCH: 1%-2%) two components (Fig. 13-6). First, the drug is nonspecifically
Specificity Frequently Rh li system adsorbed to the patient’s red cells and remains firmly
(PCH: anti-P)
attached. Second, once adsorbed, the drug must be able to
elicit an antibody response. The drug antibody is usually IgG
and reacts only with drug-treated red cells. Large doses of
autoantibodies having the characteristics of both warm and intravenous penicillin (10 million units daily) are needed to
cold autoantibodies.’”*’> Less than 10% of cases of AIHA are produce an immune response.*' Approximately 3% of patients
considered mixed.°° Patients with mixed-type AIHA usually
present with a severe, acute condition.’° They may exhibit signs
of extravascular hemolysis from IgG antibodies and intravascu-
lar hemolysis from IgM or complement activation. This situa-
tion of both warm and cold autoantibodies may be expected
when one realizes that a number of the lymphoproliferative and
collagen diseases may be associated with either form of autoan-
tibody.*’° Approximately half of the cases of mixed AIHA are
SR RE Ta(Autoimmune) Mechanism
idiopathic and the remainder are associated with autoimmune Methyldopa
diseases such as systemic lupus erythematosis.°°°! Ceftriaxone
Chlorpromazine
DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA Ibuprofen
The administration of drugs may lead to the development of a Levodopa
Mefenamic acid
wide variety of hematologic abnormalities, including immune Nomifensine
hemolytic anemia. Drug-induced immune hemolytic anemia Procainamide
represents approximately 12% of cases of immune hemolytic Thioridazine
anemia in various studies.*'”’ Historically, three mechanisms
Drug Absorption Mechanism
have been described that lead to the development of drug-
Cephalosporins
induced immune hemolytic anemia, and a fourth mechanism Diclofenac
that leads to the development of a positive DAT but is not Penicillins
associated with hemolysis. Sufficient new data have emerged
Immune Complex Mechanism
to perhaps reclassify these mechanisms. Nevertheless, it is
Acetaminophen
instructive to review the traditional mechanisms. Antihistamines
Cephalosporins
AUTOIMMUNE MECHANISM This represents the most com- Chlorpromazine
mon drug-induced immune hemolytic anemia, accounting for Diclofenac
Isoniazid
approximately 70% of all cases.’ The antibodies produced Quinidine
by this mechanism are considered “true autoantibodies,” be- Quinine
cause they react against intrinsic red blood cell antigens, not the Rifampin
drug or the drug—erythrocyte complex. The drugs implicated Streptomycin
in this response include the antihypertensive drug a-methyldopa Sulfonamides
Stibophen
(Aldomet) and related drugs (L-dopa, procainamide)”
Tetracycline
(Table 13-17). Drug-induced AIHA by this mechanism is diffi-
cult to diagnose because it mimics WAIHA. It has been sug- Membrane Modification Mechanism (Protein Adsorption)
gested that the autoantibodies produced in response to these Cephalosporins
drugs are the result of altered red cell antigens that are not rec- Note: Some drugs may acteemore detone mechanism.
ognized as self; however, the exact mechanism is. still
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 6
ie) Nn

Laboratory findings include evidence of intravascular

RBC Antibody hemolysis with hemoglobinemia and hemoglobinuria. The

aT DAT result is positive with complement components only,

because the immune complex has disassociated. In vitro
agglutination reactions are generally observed during sero-
logic testing only when the drug is added to the patient’s
ont
serum and test red cell mixture.
The primary treatment is to stop the drug if the patient is
anemic because of active hemolysis. Corticosteroids may also
be given. An interesting note is that some of the drugs produc-
ing hemolysis by this mechanism are also associated with the
development of drug-induced immune thrombocytopenia
(ITP). The antidrug immune complex also adsorbs onto the
Figure 15-6 m Drug adsorption mechanism. (From Petz, LD, and
platelets.*° However, it is rare to find a patient with simulta-
Garratty, G [Eds]: Acquired Immune Hemolytic Anemias. Churchill neous hemolysis and thrombocytopenia caused by antibodies
Livingstone, New York, 1980, with permission.) to a single drug.

MEMBRANE MODIFICATION MECHANISM (PROTEIN

on high-dose intravenous penicillin develop drug antibodies ADSORPTION) As the name implies, the drug modifies the
causing a positive DAT, but only 5% of these patients have red cell membrane so that normal plasma proteins are non-
actual clinical hemolysis.7! specifically adsorbed onto the patient’s red cells (Fig. 13-8).
Laboratory findings include signs of extravascular hemol- Cephalosporins are also the drugs most commonly implicated
ysis. The disorder develops over a period of 7 to 10 days. The in this response.*' The red cells become coated with numer-
DAT results are strongly positive with anti-lgG and negative ous plasma proteins such as albumin, fibrinogen, and globu-
with anti-C3. lins. Approximately 3% of patients receiving the drug
develop a positive DAT result owing to the nonspecific
IMMUNE COMPLEX*MECHANISM The immune complex immunoglobulin adsorption by the red cells. Hemolytic ane-
mechanism is the least common drug mechanism in drug- mia has not been reported in association with this mechanism
induced hemolytic anemia. The most common drug involved in of drug-induced positive DAT.
this response is quinidine.” Other drugs associated with the The preceding classification used for the drug-induced
immune complex mechanism are listed in Table 13-17. The immune hemolytic anemias provides a convenient mechanistic
patient responds to these drugs by producing an antibody (IgG or approach to how drugs may be implicated in immune hemoly-
IgM, or both) against the drug that binds to the drug, forming an sis. Recent reports have demonstrated immune hemolytic ane-
antibody-drug immune complex (Fig. 13-7). The antibody—drug mia from certain drugs with more than one mechanism. More
complex then adsorbs onto the patient’s red cells, and comple- recently it has been suggested that only a single mechanism
ment is activated. The antibody—drug immune complex is merely may be responsible for all drug-related immune hemolysis
adsorbed onto the red cell membrane (not bound to it) and easily (unifying theory).*** The four mechanisms of drug-related
disassociates, leaving activated complement behind. Only a anemia are compared in Table 13-18. The antibody character-
small amount of the drug is necessary to produce this response. istics of the various types of autoimmune hemolytic anemias
are contrasted in Table 13-19.

Drug +

X fir Complex

a
Complement
RBC
Membrane
Modification

yt .
Non-specific

Drug r% K F protein
adsorption
Antibody \. tyx =*
ys
Proteins oF. \d
@<@

Figure 13-7 ™ Immune complex mechanism. (From Petz, LD, and Figure 13-8 ® Membrane modification mechanism. (From Petz,
Garratty, G [Eds]: Acquired Immune Hemolytic Anemias. Churchill LD, and Garratty, G [Eds]: Acquired Immune Hemolytic Anemias.
Livingstone, New York, 1980, with permission.) Churchill Livingstone, New York, 1980, with permission.)
266 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects

Prototype Immunoglobulin Biologic Frequency of

DAT Results Hemolysis
Mechanism Drugs Class

Positive (often to Eluate often Small doses of

Immune complex Quinidine IgM or IgG
formation complement negative drug may
(innocent fragments only; cause acute
however, IgG intravascular
bystander)
may be present) hemolysis with
hemoglobinemia
and hemoglobinuria;
renal failure is
common
Drug adsorption Penicillins IgG Positive (strongly) Eluate often 3% 4% of patients
due to IgG negative on large doses
sensitization (10 million units)
daily of penicillin,
which is one of
the most common
causes of
drug-induced
immune hemolysis,
usually
extravascular
in nature
Membrane Cephalosporins | Numerous Positive due to Eluate negative No hemolysis;
modification plasma proteins a variety of however, 3% of
(nonimmuno- (nonimmuno- serum proteins patients receiving
logic protein logic the drug develop a
adsorption) sensitization) positive DAT
Autoimmunity Methyldopa IgG Strongly positive Eluate positive 0.8% develop a
(Aldomet) (due to IgG (warm hemolytic anemia
sensitization) autoantibody that mimics a
identical to WAIHA (depends
antibody on the dose
found in of the drug); 15%
WAIHA) of patients receiving
Aldomet develop a
positive DAT

Nonimmune Hemolytic Anemia survive a childhood infection invariably suffer from an

ongoing debilitating disease. Over the past 20 years, control
Acquired nonimmune hemolytic anemias represent a diverse measures have significantly reduced the incidence of the
group of conditions that lead to the shortened survival of red disease, but recently, the incidence of the disease has again
cells by various mechanisms. Often a number of mechanisms risen to epidemic proportions. The increased incidence of
are operative at the same time; for example, malaria leads to infections has resulted from (1) the organism becoming
mechanical destruction of red cells and, in addition, immuno- resistant to many antimalarial drugs (e.g., chloroquine) and
logic factors play a role in shortened red cell survival. Classi- (2) the mosquito vector having become resistant to insecti-
fications may be made along either causative or mechanistic cides.** There are four species of malaria that can infect
lines. A classification incorporating both approaches is pro- humans: Plasmodium vivax, P. falciparum, P. ovale, and
vided in Table 13-20. P. malariae. Plasmodium vivax and P. falciparum are
responsible for most infections producing disease in
Intracellular Infections humans. In the case of P. falciparum, the disease can have
a rapid and often fatal course. Plasmodium malariae and
MALARIA P. ovale infections are uncommon. Plasmodium ovale
Malaria is the most common protozoal infection in humans. infections are confined to certain areas of Africa.
It has a high incidence in the tropical and subtropical The number of patients presenting with malaria is also in-
regions of the world, accounting for a fair percentage of the creasing in countries such as the United States, Europe, and
anemia in those regions. It has been estimated that more Australia because of increased travel to endemic tropical and
than 400 million people suffer from the disease worldwide, subtropical regions. It is estimated that from 10,000 to 30,000
resulting in the deaths of more than 1.5 million annually.** travelers from industrialized countries contract malaria each
Most fatalities occur in nonimmune children; those who yeane
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 267

teristics inAutoimmune Hemolytic =

Warm Reactive Cold Reactive Drug-Related
Autoantibody Autoantibody PCH Autoantibody

Immunoglobulin Polyclonal IgG; Polyclonal IgM in infection Polyclonal IgG Polyclonal IgG
characteristics IgM, and IgA may Monoclonal k chain
also be present; rarely IgM in cold agglutinin
IgA alone disease
Complement Variable Always Always Depends on
activation mechanism of
drug, antibody,
and RBC
interaction
Thermal 20°C-37°C; 4°C—32°C optimum 4°C; 4°C-20°C; 20°C-37°C;
reactivity optimum 37°C occasionally to 37°C biphasic optimum 37°C
hemolysin
Titer of free Low (< 1:32) High (> 1:1000 at 4°C) Moderate to Depends on
antibody May only be low (< 1:64) mechanism of
detectable using drug, antibody,
enzyme treated cells and RBC
interaction
Reactivity of eluate Usually panreactive Nonreactive Nonreactive Panreactive with
with antibody Aldomet-type
screening cells antibody.
Nonreactive in
all other
circumstances
Most common Anti-Rh precursor =| Anti-P Anti-e-like;
specificity -common Rh -j Aldomet, antidrug
-LW -Pr
-En*/Wr?
-U
Site of RBC Extravascular: Extravascular: predominantly Intravascular Intravascular and
destruction predominantly liver, rarely intravascular spleen
spleen with some
liver involvement

LIFE CYCLE The malarial parasite has a complex life cycle. activation with increased monocyte/macrophage activity,
The insect vector is the Anopheles mosquito, of which numer- which promotes extravascular hemolysis of both infected and
ous species can transmit the parasite. Figure 13-9 illustrates noninfected red cells in the spleen.*’**
the malarial life cycle. Anemia associated with malaria is normocytic nor-
mochromic. Leukopenia is present in many cases, as is
CLINICAL PRESENTATION The most commonly reported thrombocytopenia (particularly in individuals with P. falci-
symptoms are fever, malaise, headache, chills, and sweats. parum infections). Diagnosis of malaria is made by exami-
Some patients show classic periodic episodes of fever nation of a peripheral blood smear. Blood should be taken
and chills, correlating with rupture of infected erythrocytes. just prior to the onset of fever because the parasitemia is
Nausea, vomiting, and diarrhea may be present. Quite often greatest at this time. However, this is possible only if classic
classic periodic episodes of fever and chills are absent, and periodic episodes of fever and chills are present. It is best
these symptoms are mistakenly attributed to a viral infection, to make blood smears from a fingerstick. If anticoagulated
allowing the malaria to go untreated. It is always advisable to blood must be used, smears should be made as soon as
inquire if the patient has been overseas and which countries possible to prevent changes in erythrocyte and parasite
have been visited. morphology. Examination of an unfixed Giemsa- or
Splenomegaly is present in 40% to 50% of patients with Wright-stained blood smear (thick preparation) is per-
acute malaria. It is present in virtually all patients with formed to ascertain the presence of malarial parasites.
chronic malaria, accounting for the high incidence of Staining is usually performed at a pH of 7.2 to enhance the
splenomegaly in the tropics.*® blue staining of the parasites’ cytoplasm. Determination of
the species of parasite may be made on this smear if sch-
LABORATORY DIAGNOSIS Hemolysis in malaria occurs izonts are present (by the number of merozoites within the
intravascularly as a result of direct red cell destruction by schizont). If only trophozoites are present, the examination
the parasite. In addition, malarial infection causes immune of a thin blood smear is necessary. Often more than one
268 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

Cause Examples Mechanisms

Infections
Intracellular Malaria Physical disruption and immune
Babesiosis Physical disruption
Extracellular Bartonella Direct action on RBC membrane
and MPS sequestration
Clostridium Enzymatic action on RBC membrane
Bacterial sepsis: meningococcal, Physical disruption secondary to DIC
pneumococcal
Viral Unknown
Mechanical
Macroangiopathic Cardiac prosthesis Physical disruption because of shear stress
March hemoglobinuria Physical disruption
Microangiopathic Hemolytic uremic Physical disruption
syndrome (HUS)
Thrombotic thrombocytopenic Physical disruption
purpura (TTP)
Chemical and physical agents
Oxidative agent Dapsone at high dosage Direct oxidation of RBC membrane components
Nonoxidative agents Lead Alteration of RBC membrane components
Venoms Possible direct effect on RBC membrane by enzymes
Osmotic effect Water (drowning or water irrigation Osmotic lysis
during surgery)
Burns Localized dehydration
Acquired membrane disorders Vitamin E deficiency; abetalipo- RBC membrane oxidation; lack of membrane
proteinemia deformability
Liver disease Lipid abnormalities of RBC membrane lead to
decrease in deformability
Renal disease Retained metabolic products cause membrane
changes, leading to a decrease in deformability
Hypersplenism Sequestration of normal cells

DIC = disseminated intravascular coagulation; MPS = mononuclear phagocyte system.

parasite is present in a single cell (Fig. 13-10). Plasmodium tickborne in humans, but has also been transmitted by blood
falciparum gametocytes have a characteristic banana or transfusion.?! In the United States, cases are most common in
crescent shape, assisting in the identification. Occasionally New England and the upper Midwest, owing to the presence of
it is possible to see irregular purple inclusions called infected ticks and their hosts in those areas.”* Infection tends to
Maurer’s dots in the red cell cytoplasm, which are probably be self-limited, although in elderly, immune-compromised, or
breakdown products of hemoglobin. Plasmodium vivax asplenic patients it can follow an acute and fatal course.??
gametocytes are round, ameboid forms that expand and Patients usually present with a history of malaise, headache, and
distort the red cell. Bluish purple inclusions called fever, sometimes associated with vomiting and diarrhea. In
Schiiffner’s dots are often seen in red cells infected with P. splenectomized patients this condition can progress to rigors,
vivax and P. ovale (Fig. 13-11). The features of the differ- acute intravascular hemolysis with associated hemoglobinemia,
ent malarial parasites infecting humans are summarized in hemoglobinuria, jaundice, and renal failure.
Table 13-21.
Immunoassays help screen large numbers of patients for LABORATORY DIAGNOSIS Diagnosis of the disease is made
the presence of infection. Flow cytometry has also been used via examination of the peripheral blood, where parasites very
to show the presence of malarial parasites in red cells. similar to P. falciparum are seen in the red cells (Fig. 13-12).
Features that distinguish babesiosis from malaria are the forma-
BABESIOSIS tion of tetrads of merozoites (Maltese cross), absence of pig-
Infection by the organism Babesia represents a zoonotic ment granules in infected erythrocytes, and the presence of
infection, as humans are not natural hosts for the parasite. The extracellular merozoites.”” A history of possible exposure to
disease is carried by ticks (Lxodes scapularis) and normally ticks and a lack of recent travel to areas where malaria is
infects cattle, deer, and rodents.” The disease is usually endemic help in making the correct diagnosis. Serologic tests
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 269

Rupture of schizont
to release merozoites
into bloodstream
Sporozoites platon GS — =
asexual mutipcaton, Ss —> E=t—
-@-
forming schizonts
a Merozoites enter red blood
Exoerythrocytic cell, forming ring forms
cycle called trophozoites

Sporozoites enter Erythrocytic @ | Veaie oie

liver cells cycle Ke Rupture of schizonts
releasing merozoites (b)
*Infectious Merozoites enter more red
mosquito bites a cells infecting them (c)
human, injecting (a,b, &c) stages occur
sporozoites into ae cists

ra Siam

HUMAN
MOSQUITO Some merozoites
form 9 & O' gametocytes
NG Mature is after several erythrocytic
gametocytes cycles

~——
Sporozoites stored Gametocytes
in salivary glands ingested by
‘ awaiting new Gametocyte union Anopheles
human host forms ookinetes mosquito
which produce (sexual cycle)
thousands of
sporozoites

Figure 15-9 @ Malarial life cycle in humans and mosquitoes. Beginning of cycle is indicated by an asterisk.

Figure 1S: 10 m Ringed forms of Plasmodium falciparum in red Figure 15-11 @ Late stages of Plasmodium vivax malaria.
blood cells (RBCs). Note that the same RBCs may be infected with Schuffner’s dots. Note and contrast the platelet on the RBC (center)
more than one ring. with the ring form of malaria toward the periphery (arrow).
270 Chapter 13. Hemolytic Anemias: Extracorpuscular Defects

Table 13-21 Characteristics of Malarial Parasites Infecting Humans —

P. falciparum P. vivax P. malariae P. ovale

Incubation period (days) 6-10 10-12 * 13-16 10-12

Asexual life cycle (h) 48 48 TD 48
RBCs infected All Reticulocytes Senescent Reticulocytes
Secondary exoerythrocytic No Yes Yes Yes
development
Duration of relapses in Not applicable 3-5 yr Up to 40 yr 3-5 yr
untreated patients
Level of parasitemia 50%-60% 2%-S% 2%-3% 2%-3%
Ring form Small, delicate may Large irregular, poor Large thick, promi- Large irregular, poor
have two chromatin outline nent chromatin dot outline
dots, often on edge One chromatin dot
of RBC
Maurer’s dots Schiiffner’s dots Schiiffner’s dots
Schizonts Rarely seen in Large, about same Small “daisy-head” Irregular arrangement
peripheral blood size as RBC 6—16 merozoites 4-16 merozoites
8-32 merozoites 12-25 merozoites
Gametocytes Crescent or Round and Round, same
sausage shape expanded RBC size as
RBC

for antibodies to Babesia by immunofluorescent assay or testing does not appear to be any intermediate host. The organisms
by polymerase chain reaction (PCR) have been described.”** adhere to the red cell surface and appear as gram-negative rods
in the acute phase of the disease. In the recovery phase, they
assume a coccoid appearance.
Extracellular Infections The disease has two clinical phases. The first is the
BARTONELLOSIS (OROYA FEVER)
hemolytic phase (Oroya fever), which may not occur in all
This disease is restricted to northern areas of South America, patients. When it does occur, there is a rapid onset with marked
including Peru, Ecuador, and Columbia. The name Oroya fever intravascular hemolysis. Red cells are also sequestered in the
derives from the city of Oroya in the Peruvian Andes, where spleen and liver.*° The anemia can be quite severe, and blood
many railroad construction workers were affected by the disease smears show many nucleated red cells and a reticulocytosis.”°
in the late 1800s. It is also referred to as Carrion’s disease,” Antibiotic therapy, including penicillin, streptomycin, and tetra-
named after the medical student who died as a result of a self- cyclines, is effective in treating patients in this stage of the
experiment designed to determine the nature of the infection. infection.*° The second stage of the disease (verruca peruviana)
Bartonellosis has a high fatality rate in nonimmune is nonhematologic and involves the development of verrucous
patients and is caused by the organism Bartonella bacilliformis. nodes (warty tumors) over the patient’s face and extremities.
Infection is transmitted by the sand fly (Phlebotomus), and there
CLOSTRIDIUM PERFRINGENS (WELCHID
This organism is a gram-positive, spore-forming bacillus that
is responsible for the development of gas gangrene. Infections
Ss with this organism are generally located in deep tissues where
anaerobic conditions required for the organism’s survival
exist. The organism is normally present in the environment
and may infect tissues exposed by trauma and surgical proce-
dures. There is a high incidence of the infection in septic
abortions.”’ The organism is responsible for extensive tissue
damage resulting from the release of enzymes and toxins.
Septicemia caused by C. perfringens may produce an acute
intravascular hemolytic process resulting from the release of
an alpha (a) toxin or lecithinase. This process, combined with
phospholipases and possibly proteinases also produced by the
organism, acts on the red cell membrane to cause its destruc-
tion and subsequent lysis of the cell.°* Hemolysis is often
severe, with marked hemoglobinemia and hemoglobinuria.
Figure
Acute renal failure may develop quite rapidly, and the prog-
13-12 ® Comparison of babesiosis (left) and malaria (right).
nosis is generally poor.””'®? Microspherocytes, hemolysed
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 27]

“ghost cells” and left shift in neutrophils with toxic changes

are common findings in the peripheral blood smear. Thrombo-
cytopenia is present in most cases.
Improvements in the maintenance of aseptic conditions
during and following surgery and the decrease in criminal
abortions have caused this form of hemolytic anemia to
become quite uncommon.
Other organisms that have been associated with
hemolytic anemia are listed in Table 13-22.

Mechanical Etiologies
The passage of red cells through the vascular system subjects
the cell to a wide range of environmental conditions. As red
cells travel around the body, shear forces are highly variable
Figure 13-15 @ Peripheral blood showing red cell fragmentation
and are influenced by:
with thrombocytopenia. (A) Polychromasia and (B) nucleated RBCs
1. The surface conditions of the blood vessel from a patient with thrombotic thrombocytopenia purpura (TTP).
2. The size of the vessel lumen
3. The rate at which the cell is moving (Fig. 13-14). The reticulocyte count and serum LD level are
4. The number of other cells present at the same time. Other usually elevated.** Leukocytes are usually normal, and platelets
environmental conditions the cel! is exposed to include are often reduced because of their interaction with the abnor-
changes in pH, electrolytes, and protein concentration. mal surface.'”* Decreased haptoglobin, mild hemoglobinemia,
mild hemosiderinuria,'" and occasionally hemoglobinuria are
When these factors cause mechanical rupture of the cell mem-
present (depending on the amount of red cell destruction).
brane, intravascular hemolysis results, accompanied by the
Treatment of cardiac hemolysis can range from supportive
presence of red cell fragments or schistocytes on peripheral
therapy, which may include iron supplementation and transfu-
blood smear (Fig. [os
sion, to surgically correcting the faulty valve or vessel.!°
CARDIAC PROSTHESIS
MARCH HEMOGLOBINURIA
Historically, hemolytic anemia associated with prosthetic
This form of hemolytic anemia was first described in the late
heart valves was a frequent complication ofcardiac corrective
1800s in a young German soldier who demonstrated frank
surgery. Innovative changes in design and composition of
hemoglobinuria following a field marching exercise.'!°° The
valves has reduced mechanical hemolysis to a rare and minor
anemia has been described in individuals involved in strenu-
complication.'°!
ous and sustained physical activity.'"’ Similar traumatic red
The primary cause of hemolysis is mechanical trauma to
cell destruction has been reported in a practitioner of karate!
red blood cells, resulting from turbulence of flow through the
and a conga drum player.'°”’ The cause of the anemia is com-
prosthesis.'°* The severity of the anemia is highly variable in
plex, involving:
patients with heart valve prostheses. Mild, compensated
hemolysis is common; overt anemia is unusual, and rarely is |. Direct physical disruption of red cells as they flow through
the anemia severe enough to require transfusion.'°! the capillaries of the feet or hands
The peripheral blood smear shows many fragmented 2. [ron loss in sweat
cells (schistocytes), helmet cells, and occasional spherocytes 3. Adaptation to a right-shifted oxygen dissociation curve.!!°

Viruses Protozoa

Bartonella bacilliformis Coxsackie Babesia microti

Clostridium perfringens Cytomegalovirus B. divergens
Escherichia coli Epstein-Barr Plasmodium falciparum
Haemophilus influenzae Herpes simplex P. malariae
Mycobacteria tuberculosis Influenza A P. ovale
Mycoplasma pneumoniae Rubeola P. vivax
Neisseria meningitidis Varicella Toxoplasma
Salmonella sp.
Shigella sp.
Streptococcus sp.
Vibrio cholera
Yersinia enterocolitica
 Chemical and Physical Agents
OXIDATIVE HEMOLYSIS
Oxidative stress on the red cell resulting from either drugs or
chemicals may affect either the globin chains or the heme group
of the hemoglobin molecule. Most oxidizing agents affect the
hemoglobin molecule by denaturing the globin chains, produc-
ing Heinz bodies, or by oxidizing the heme group, producing
methemoglobinemia (see Chap. 10). Both of these processes
will eventually lead to membrane damage and decreased red cell
deformability, with eventual extravascular hemolysis in the
spleen.''* Glucose-6-phosphate dehydrogenase (G6PD) defi-
ciency increases the susceptibility of red cells to oxidant stress
(see Chap. 10). However, strong oxidants can induce hemolysis
even in normal individuals. Examples include naphthalene (moth-
Figure 15-14 ™ RBC fragmentation in microangiopathic hemoly- balls), phenol, cresol (Lysol or penetrating oil), and aniline."°
sis from a patient with a prosthetic cardiac valve (mechanical
hemolysis); note the presence of schistocytes (arrows).
NONOXIDATIVE HEMOLYSIS
ARSENIC Industrial processes involving the action of acids
and metals may give rise to the production of arsenic gas.
Patients with march hemoglobinuria usually demonstrate a Continued exposure to the gas gives rise to intravascular
normal hemoglobin, although there may be an increase in the hemolysis with anemia and hemoglobinuria.''’ Marked
reticulocyte count. Hemoglobinemia and hemoglobinuria are methemalbumin formation causes the serum of affected
episodic and present only after exercise. This obvious associ- patients to turn a characteristic brown and often masks the
ation with exercise is helpful in distinguishing the hemoglo- presence of any hemoglobinemia. Current Occupational
binuria from other causes such as paroxysmal nocturnal Safety and Health Administration (OSHA) requirements have
hemoglobinuria (see Chap. 8). Fragmented red cells are not a minimized this hazard in the workplace.
feature of this condition.
Treatment involves wearing cushion-soled shoes or run- LEAD Anemia is associated with either acute or chronic lead
ning on softer surfaces. poisoning which includes some component of hemolysis.''*!'°
The red cells of patients exposed to lead have a shortened sur-
MICROANGIOPATHIC HEMOLYTIC ANEMIA vival.'*? Lead poisoning is usually a problem of young chil-
Microangiopathic hemolytic anemia (MAHA) refers to a dren living in deteriorated housing where they eat chips of
group of clinical disorders that are characterized by fragmen- lead-based paint. Children affected by lead poisoning may
tation of the red cells as they pass through abnormal arteri- show a normocytic to microcytic, hypochromic blood picture,
oles, resulting in intravascular hemolysis.''' Most often the with classic punctate basophilic stippling (Fig. 13-15).
abnormalities in the microcirculation are caused by the depo- Adults are more likely to acquire lead poisoning in an
sition of fibrin strands resulting from intravascular activation occupational setting. These patients commonly present with
of the coagulation system (see the discussion of disseminated neurologic or renal disease with variable anemia.
intravascular coagulation [DIC] in Chap. 27).
In MAHA, the mechanical process leading to fragmenta-
tion of the red cells occurs as the blood flow forces the cells
to negotiate a blood vessel whose lumen is restricted by
microthrombi.''* The red cells are physically torn as they are
forced along the narrow confines of the blood vessel. Schisto-
cytes and other poikilocytes are seen on the peripheral blood
smear, as well as decreased platelets in some cases. The
degree of hemolysis correlates with the amount of thrombosis
present.'* In addition to the intravascular destruction of the
red cells, the fragments produced lack deformability leading
to an increase in extravascular hemolysis.
In addition to DIC, MAHA may be associated with inva-
sive carcinoma, malignant hypertension, cavernous heman-
giomas, complications of pregnancy, and kidney or liver
transplantation.''* Manifestations of MAHA are also promi-
nent in two related clinical entities: hemolytic uremic syn-
Figure 15-15 m Peripheral blood from a patient with lead poison-
drome (HUS) and thrombotic thrombocytopenic purpura ing. Note the normocytic, hypochromic red cells, with the classic
(TTP) (see Chap. 25). punctate basophilic stippling.
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 273

COPPER Very high levels of copper ions have been associ-

ated with intravascular hemolysis. These levels may occur as
a result of suicide attempts in which copper sulfate solution is
ingested'*! or in Wilson’s disease.'?? The cause of hemolysis
is unknown, although it has been shown that high levels of
copper ions can affect a number of intracellular enzymes
(e.g., pyruvate kinase and hexokinase).!”? The anemia may be
associated with the presence of spherocytes.

VENOMS Hemolysis may follow spider bites, bee and wasp

stings, and some venomous snake bites. In the United States,
bites of the brown recluse spider are known to cause DIC with
hemolysis after 24 to 48 hours.'** The venomous bites of pit
vipers (rattlesnakes, cottonmouths, water moccasins, and cop- he 2 “Se

perheads) also cause DIC with hemolysis.'?> Cobra bites

cause hemolysis directly through the action of phospholipases Figure 13-17 @ Spur-cell anemia (acanthocytosis) associated with
on red cell membranes.'”° severe liver disease.

OSMOTIC EFFECTS accidentally used in hemodialysis or as an intravenous fluid.

BURNS Patients who have suffered severe burns to more Similarly, distilled water used as an irrigant during urologic
than 15% of their body may show evidence of intravascular procedures can enter the circulation through the raw tissue
hemolysis. The hemolytic process is thought to result from bed in amounts sufficient to cause hemolysis.
the direct effect of the heat on the red cells in the affected
area. Red cells that are heated to temperatures in excess of Acquired Membrane Disorders
47°C undergo changes, including fragmentation, budding,
and microspherocyte formation; blood collected within A number of mechanisms can be implicated in producing
24 hours of such heating shows evidence of these changes changes to red cell membranes, which can result in the short-
(Fig. 13-16). Because the cells are osmotically and mechani- ened life span of the cell. Any change that compromises the
cally fragile, they are rapidly removed from the circulation, and red cell’s deformability or its resistance to oxidative stress can
blood collected after that time is often normal in appearance. potentially contribute to a hemolytic process. For example,
spur-cell anemia, observed primarily in patients with alco-
DROWNING AND OTHER WATER-RELATED OSMOTIC holic cirrhosis, is a condition in which the red cells assume a
INJURY Water that is solute free or hypotonic can cause characteristic spherical shape with a number of fine, finger-
osmotic hemolysis. This occurs most commonly in cases of like spike projections (acanthocytes) (Fig. 13-17). Lipid
near-drowning in fresh water, when sufficient water is inhaled disorders can also result in a loss of red cell deformability. In
and absorbed through the lungs to cause a decrease in plasma abetalipoproteinemia, the cells also assume the classic shape
osmolality with some degree of hemolysis.'*? This can of acanthocytes (Fig. 13-18). In end-stage renal disease,
also occur when hypotonic solution or distilled water is many of the cells take on the appearance of burr cells or

Hiei
ie
ie

Figure 13-16 m Peripheral blood from a patient with extensive Figure 15-18 @ Acanthocytosis from a patient with abetalipro-
burns. Note the typical (A) microspherocytes and (8) membranous teinemia. (From Hyun, BH, et al: Practical Hematology. A Laboratory
fragments. (From Bell, A: Hematology. In Listen, Look and Learn. Guide with Accompanying Filmstrip. WB Saunders, Philadelphia,
.) 1975, with permission.)
Health and Education Resources, Inc., Bethesda, MD, with permission
274 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

Case Study 2 |
A 38-year-old man was diagnosed with pneumonia 3 weeks
previously. He returned to the hospital because he was expe-
riencing severe weakness and shortness of breath on slight
exertion. The patient was pale and jaundiced. Laboratory
findings revealed severe normocytic anemia. The peripheral
blood smear demonstrated marked agglutination of erythro-
cytes. The crossmatch and antibody screen tests were both
strongly positive at the room temperature phase (20°C).
On further testing, the patient’s DAT was also strongly pos-
itive, with complement proteins. The Donath—Landsteiner
test was negative. Thermal amplitude studies revealed
immunoglobulin reactivity up to 34°C.
Figure 13-19 @ Renal disease (peripheral blood). Note the presence
of (A) burr cells, (B) thorn cell, (©) blister cell, and (D) schistocyte. QUESTIONS
(From Bell, A: Hematology. In: Listen, Look and Learn. Health and
Education Resources, Inc., Bethesda, MD, with permission.) 1. What is the most likely diagnosis?
2. What other laboratory test values may be affected by the
agglutination of RBCs? What is the remedy?
echinocytes (Fig. 13-19), which have numerous small spines 3. What is the treatment for this patient?
over their entire surface. Other conditions in which
echinocytes are seen include pyruvate kinase deficiency and ANSWERS
bleeding associated with peptic ulcer disease.
1. Cold agglutinin syndrome, secondary to pneumonia
(probably Mycoplasma).
2. Certain complete blood count (CBC) values from an
automated instrument will be erroneous because of the
Case Study 1 agglutination of RBCs at room temperature. The blood
should be warmed to 37°C and rerun through the
A 26-year-old white woman diagnosed with rheumatoid instrument.
arthritis came to the emergency department with complaints 3. This cold agglutinin syndrome would be expected to be
of weakness and shortness of breath. Laboratory findings self limited; meanwhile the patient should avoid the cold.
revealed a severe normocytic anemia. The patient had
received many red cell transfusions over the past 8 years,
but had not received any transfusions in over a year. The
peripheral blood smear demonstrated mild anisocytosis,
spherocytosis, and moderate polychromasia. The cross-
match and antibody screen tests were weakly positive at the
Case Study 3
antiglobulin phase. On further testing, the patient’s direct
A 50-year-old African American man had recently returned
antiglobulin test (DAT) was also weakly positive, in poly-
from a trip to Africa to visit relatives. He complained of
specific and IgG phases. The effort to obtain compatible
headaches, fatigue, and general malaise over the past 2 weeks.
blood for transfusion was complicated because the patient’s
The patient then developed a high fever followed by severe
serum reacted weakly with all cells tested.
chills, which persisted for approximately a day. The fever and
chills subsided, and the patient thought he was getting better
QUESTIONS until last night, when he experienced another bout of fever and
1. What is the most likely diagnosis? chills. Laboratory findings revealed the patient was anemic and
2. Is this hemolysis likely to be intravascular or extravas- slightly leukopenic. The laboratory noted that odd, ring-like
cular? Why? inclusions were seen in approximately 10% of his red cells,
3. What other laboratory tests may be requested to aid in along with rare blue, crescent-shaped forms that appeared to be
the diagnosis and what are the expected results? extracellular. On further testing the DAT was negative. The
crossmatch and antibody screening tests were also negative.
ANSWERS
QUESTIONS
1. Warm autoimmune hemolytic anemia, secondary to
rheumatoid arthritis. 1. What is the most likely diagnosis?
2. Extravascular hemolysis. Immune hemolysis with IgG, 2. What other laboratory tests should be requested to aid in
complement negative, occurs in the spleen. the diagnosis?
3. Indirect bilirubin (increased), LDH (increased), reticulo- 3. What is the vector for this disease? What are the reasons
cyte count (increased). for a recent increased incidence of this disease?
continued
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 275

ANSWERS
1. Malaria, most likely Plasmodium falciparum.
2. Thick smear preparation, stained with Giemsa or
Wright’s stain; it is best to use fingerstick blood rather
than anticoagulated blood.
3. The vector is the Anopheles mosquito. The incidence of
malaria is increasing because the parasite has developed
resistance to antimalarial drugs, and the mosquito vector
has developed resistance to some insecticides.

Quess ons
1What are the mechanisms of immune hemolysis? sg Ss Which is not a characteristic of warm autoimmune
a. IgG or IgM antibodies that activate the classical com- hemolytic anemia? ;
plement pathway a. Variable anemia
b. Antibody-dependent cellular cytotoxicity (ADCC) b. Reticulocytosis and spherocytosis
mediated by NK cells, monocytes/macrophages, and c. Positive result for Donath—Landsteiner test
granulocytes d. DAT result usually positive for both IgG and C3d
c. Complete or partial phagocytosis of antibody-coated . What are features of cold agglutinin syndrome?
erythrocytes a. Usually an IgM antibody
d. All of the above b. Reticulocytosis and positive DAT
to . Which would best distinguish hemolytic anemia caused c. Tendency for spontaneous autoagglutination of RBC
by immune mechanisms from other hemolytic anemias? samples
a. Presence of spherocytes on peripheral blood film d. All of the above
b. Increased reticulocyte count . What is the principle of the Donath—Landsteiner test?
c. Enlarged spleen a. Antibody binds red cells at 37°C and causes lysis
d. Positive DAT at 4°C.
. Which is true concerning autoimmune hemolytic anemia? b. Antibody binds red cells at 4°C and causes lysis
a. Majority of cases are of the “cold” type at 37°C.
b. Seen in transfusion reactions c. Antibody binds red cells at 4°C or 37°C and causes
c. Is demonstrated in hemolytic disease of the newborn immediate lysis.
d. Antibodies are produced against one’s own erythro- d. Antibody binds red cells at 4°C or 37°C but causes
cyte antigens lysis only at 4°C.
What is the process in which the immune system pro- . What are causes for nonimmune hemolytic anemia?
duces antibodies to foreign red cell antigens introduced a. Infections
into their circulation through transfusion, pregnancy, or b. Mechanical, chemical, and physical agents
organ transplantation? c. Acquired membrane disorders
a. Alloimmune hemolytic anemia d. All of the above
b. Autoimmune hemolytic anemia 10. Which of the following organisms is (are) associated
c. Drug-induced immune hemolytic anemia with hemolytic anemia?
d. None of the above a. Mycoplasma pneumoniae
. What causes hemolytic disease of the newborn (HDN)? b. Clostridium perfringens
a. Maternal IgG antibodies, formed as a result of a previ- c. Babesia microti
ous blood exposure or pregnancy, cross the placenta d. All of the above
and attach to fetal cells. . Which of the following, as measured via an automated
b. Fetal IgG antibodies cross the placenta and attach to hematology instrument, would most likely be affected by
maternal red cells. a cold agglutinin?
c. Maternal IgM antibodies, formed as a result of a pre- a. Hemoglobin
vious blood exposure or pregnancy, cross the placenta b. Hematocrit
and attach to fetal cells. c. Platelet count
d. Fetal IgM antibodies attach to fetal red cells and cross d. Leukocyte count
the placenta to enter the mother’s circulation.
276 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects

12. Which of the following drugs causes a hemolytic anemia c. Aldomet

resulting from production of “true autoantibodies” rather d. Quinidine
than antibodies to the drug or to the drug-erythrocyte
complex? See answers at the back of this book.
a. Penicillin
b. Cephalosporin

group; the reaction may occur from 2 to 10 days after

transfusion and is generally the result of an anamnestic
response to transfused red cells.
= Complement is a group of serum proteins that interact
with each other to bring about complement-dependent mw Hemolytic disease of the newborn (HDN) is an immune
cell-mediated lysis. hemolytic disorder caused by maternal—fetal blood group
incompatibility; maternal IgG antibodies with ABO, Rh,
@ Intravascular hemolysis occurs when antibodies bind to
or other blood group specificity cross the placenta and
antigenic determinants on red cells and activate the clas-
destroy antigen-containing fetal red cells.
sical complement pathway.
mw Warm autoimmune hemolytic anemia (WAIHA) accounts
mwAntibody-dependent cellular cytotoxicity (ADCC) is a
for 70% of autoimmune hemolytic anemias, and the DAT
form of direct (intravascular) lysis of immunoglobulin-
is positive for both IgG and C3d in 67% of cases, in 20%
coated cells; effector cells contain receptors for IgG1 and
with IgG alone, and in 13% with C3d alone.
IgG3, and complement proteins C3b and iC3b, which
facilitate cell lysis. m The specificity of normal cold autoantibodies includes
anti-I, anti-H, and anti-IH, which react at temperatures
# Hemoglobinemia, hemoglobinuria, and decreased haptoglo-
from 4°C to 22°C.
bin levels are common findings in intravascular hemolysis.
m Cold agglutinin syndrome (CAS) represents 16% of
w Extravascular hemolysis is the phagocytosis of red cells
autoimmune hemolytic anemias; it occurs at a wide ther-
by fixed phagocytes within the mononuclear phagocyte
mal range (4°C to more than 30°C); antibody specificity is
system (MPS); the two major organs of the MPS are the
an IgM toward anti-I, anti-I, and anti-Pr.
spleen and liver.
m Secondary CAS, caused by infections to Mycoplasma
= Common laboratory findings in extravascular hemolysis
pneumoniae and infectious mononucleosis, has antibody
may include the presence of spherocytes, increased serum
specificity toward anti-I and anti-I, respectively.
indirect bilirubin, and urine urobilinogen.
m The four species of malaria that can infect humans
min alloimmune hemolytic anemia, patients produce
through the mosquito vector are Plasmodium falciparum,
alloantibodies to foreign red cell antigens introduced
P. vivax, P. ovale, and P. malariae; P. falciparum causes
through transfusions, pregnancy, or organ transplantation.
severe disease with an often fatal course.
min autoimmune hemolytic anemia (AIHA), patients
= Bartonellosis is caused by the organism Bartonella bacil-
develop antibodies to their own red cell antigens.
liformis and is transmitted by the sand fly; the disease is
m In drug-induced hemolytic anemia, patients produce antibod- characterized by a hemolytic phase and a tumor phase.
ies directed at a particular drug, its metabolites, or red cells
= Microangiopathic hemolytic anemia refers to a group of
coated with the drug; the four major drug-induced mecha-
disorders that are characterized by fragmentation of the
nisms include immune complex, drug adsorption, membrane
red cells as they pass through abnormal arterioles, result-
modification, and methyldopa-induced mechanism.
ing in intravascular hemolysis.
m Acute hemolytic transfusion reactions are characterized
A variety of chemical and environmental agents can cause
by acute intravascular hemolysis and associated with the
hemolysis of red cells. These include oxidating agents,
ABO blood group antibodies.
arsenic, lead, copper, and insect or snake venoms.
mwAcute hemolytic transfusion reactions are characterized
m Spur-cell anemia is a condition in which the red cells
by acute intravascular hemolysis and associated with the
assume a characteristic shape, with a number of fine, fin-
ABO blood group antibodies.
gerlike spike projections resembling acanthocytes; it is
# A delayed hemolytic transfusion reaction is characterized frequently caused by alcoholic cirrhosis.
by exposure to red cell antigens other than the ABO blood
 Chapter 13 Hemolytic Anemias: Extracorpuscular Defects 209

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 Chapter

Hypoproliferative
Anemia
Anemia Associated with Systemic
Diseases
Carmen J. Julius, MD
Carl R. Schaub, MD

Introduction OBJECTIVES
Anemia of Chronic Disease At the end of this chapter, the learner should be able to:
The Inflammatory Response
and Body Defense l. Describe anemia of chronic disease.
Mechanisms Zz. List the three major types of diseases that cause anemia of chronic disease.
Etiology and
Pathophysiology 3 . Identify laboratory findings characteristic of anemia of chronic disease.
Characteristics 4 . Define the pathophysiology of anemia of chronic disease.
Treatment
>: Describe the treatment of anemia of chronic disease.
Anemia Associated
with Renal Disease 6 . Describe the pathophysiology of anemia associated with renal insufficiency.
and Renal Failure
in. Describe the treatment of anemia of renal insufficiency
Etiology and Pathophysiology
Characteristics 8 . Describe the characteristics of the red blood cells in anemia associated with liver
Treatment disease.

Anemia Associated with . Describe the etiology of anemia associated with alcoholism.
Liver Disease
Etiology and . List the three categories of causes of the anemia associated with malignancy and
some of the mechanisms under each category.
Pathophysiology
Characteristics . Describe the pathophysiology and characteristics of anemia of human immunodefi-
Treatment ciency virus infection and the acquired immunodeficiency syndrome.
Anemia Associated with . List the mechanisms of anemia of infancy and anemia associated with prematurity.
Alcoholism/Alcohol Abuse
Etiology and . List the causes of “anemia” of improperly drawn laboratory samples.
Pathophysiology . Describe the way(s) in which “anemia” of improperly drawn laboratory samples may
Characteristics be detected.
Treatment
Anemia Associated with
Endocrine
Disease/Disorders
Adrenal Insufficiency
Thyroid Disease
Hyperparathyroidism
Hypogonadism
Pituitary Dysfunction

280
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 281

Anemia Associated with

Malignancy
Etiology and Pathophysiology
Characteristics
Treatment
Anemia Associated with
Human Immunodeficiency
Virus Infection and the
Acquired
Immunodeficiency
Syndrome
Pathophysiology and
Characteristics
Treatment
Anemia of Infancy
Etiology and Pathophysioiogy
Characteristics
Treatment
Anemia Associated with
Prematurity
Etiology and Pathophysiology
Characteristics
Treatment
“Anemia” Associated with
improperly Collected
Laboratory Samples:
Preanalytical Anemia
Etiology and Pathogenesis
Characteristics
Prevention
Summary
Case Study 1
Case Study 2
Case Study 3

Introduction should be part of the initial laboratory evaluation of most

patients.
Anemias associated with inflammation or with chronic and sys- Many disease entities are outlined in the following
temic diseases are frequently referred to as hypoproliferative pages. All have as their common theme the role of systemic
anemias because they are not accompanied by an appropriate disorders (1.e., nonhematologic disorders) in producing ane-
proliferative response by the bone marrow. A hypoproliferative mia by suppressing red blood cell production by the bone
type anemia is usually normocytic, and is characterized by a marrow.
decreased or (inappropriately) normal reticulocyte count. It is
important for the physician and the medical technologist to rec- Anemia of Chronic Disease
ognize the characteristics of these anemias in patients with
systemic diseases and to understand the laboratory studies Anemia of chronic disease (ACD) is the second most common
required to diagnose them. Anemia in a patient without other cause of anemia in humans, after iron deficiency anemia, and is
clinical symptoms at presentation may be the first indication of the commonest cause of anemia in hospitalized patients.'* It
a systemic disease (e.g., an occult malignancy). Likewise, a occurs in patients with chronic infections, chronic inflamma-
sudden change in the complete blood count (CBC) of a patient tory diseases or malignant tumors present for more than | to
who has been diagnosed with an inflammatory or systemic dis- 2 months; more recently it has also been described in other
ease may indicate a new complication. For this reason, a CBC acute diseases. Its occurrence in patients with chronic or acute
282 Chapter 14. Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

immune activation has suggested a variety of other, perhaps modern era, when many infections can be rapidly and effec-
more descriptive names ranging from “anemia of inflamma- tively treated with antibiotics, it is the chronic, the unsus-
tion” to “cytokine-mediated anemia,” but ACD seems to have pected, or the antibiotic-resistant infection that will more
persisted as the accepted terminology, and will be used here. commonly cause ACD. All of the chronic inflammatory disor-
ACD is defined by a modest normocytic or sometimes ders (also called autoimmune, collagen vascular, connective
microcytic anemia that presents with a decreased serum iron tissue or rheumatologic diseases) have the ability to produce
concentration despite ample reticuloendothelial iron stores ACD because they are, by nature, inflammatory diseases.
(measured by serum ferritin or bone marrow iron stains). Anemia is the most common hematologic abnormality seen in
Thus, the diagnosis of ACD should be made only when these patients with rheumatoid arthritis, systemic lupus erythemato-
findings are present, and the use of ACD as a wastebasket sus, mixed connective tissue disease, scleroderma, dermato-
term should be resisted. Anemias associated with other some- myositis, and Sjégren’s syndrome.® Chronic rejection after
times chronic diseases such as renal failure, liver disease, or solid organ transplantation is also commonly accompanied by
endocrine diseases are not included in ACD, and are dis- ACD.>° Recently, an anemia in acutely ill patients in the inten-
cussed separately below. sive care setting has been described which otherwise has all
the characteristics of ACD®; surprisingly, not all patients with
ACD need have a truly chronic disease (Table 14-4).
The Inflammatory Response and Body In some patients the diagnosis of ACD may provide an
Defense Mechanisms initial clue leading to the detection of a previously undiscov-
ered infection, chronic inflammatory disease, or occult malig-
THE GENERAL INFLAMMATORY RESPONSE (FIRST
nancy. This justifies the use of the CBC in health screening
LINE OF DEFENSE)
and in long-term serial evaluation of patients.
Inflammation is one of the body’s responses to tissue injury
ACD is due primarily to a decrease in red blood cell
from physical agents, foreign organisms, and immune reactions
(RBC) production by the bone marrow, with secondary con-
in the host. Inflammatory and hemostatic responses occur
tribution by decreased RBC survival. The pathophysiology of
simultaneously to control any damage at the injured area (see
ACD is multifactorial and complex, but is ultimately driven
Chaps. 26 and 27). The coagulation cascade and the comple-
by the immune response. Cytokines produced by inflamma-
ment, fibrinolytic, and kinin systems interact to modulate
tory cells produce:
inflammation (see Chaps. 24, 26, and 27).
During the inflammatory process, complement can be 1. Dysregulation of iron homeostasis
activated by the antibody-induced classical pathway, or 2. Impaired proliferation of erythroid precursors
directly by microorganisms via the alternative pathway. 3. Decreased erythropoietin response to anemia
The presence of C3a, C5a, and other chemotaxins attracts 4. Shortened RBC lifespan’
phagocytes to the site of injury, where they recognize and
Each of these mechanisms contributes to ACD (Table 14-5).
phagocytose foreign substances or organisms. Neutrophils,
monocytes, and macrophages possess receptors for comple-
DYSREGULATION OF IRON HOMEOSTASIS
ment that can induce both exocytosis of granules (containing
Dysregulation of iron homeostasis, also called reticuloen-
proteolytic enzymes, free ion radicals, and other inflamma-
dothelial iron block, refers to the inability to use iron properly.
tory metabolites) and endocytosis of complement-coated
It is primary in the pathogenesis of ACD.*° Pro-inflammatory
foreign substances.
cytokines such as interleukin-1 (IL-1) and tissue necrosis
Inflammation will continue as long as injury and damage
factor-alpha (TNF-a) divert iron from the circulation into stor-
continue. When the source of inflammation is persistent and
age sites in the reticuloendothelial system.'° This effect of
the condition is chronic, mediators from the humoral and cell-
reducing the availability of iron for hematopoiesis, despite
mediated immune responses contribute to the onset of anemia
otherwise ample iron stores in reticuloendothelial storage
(Fig. 14-1). The functions of the various types of T-cell lym-
sites, produces a functional iron-deficiency state. A decrease in
phocytes in cell-mediated immunity are listed in Table 14-1.
intestinal iron absorption and impaired iron reutilization by the
The cytokines that contribute to inflammation are listed in
hepatocytes’! also occurs in ACD patients.
Table 14—2 and the functions of macrophages are summarized
The dysregulation of iron homeostasis in ACD is prob-
in Table 14-3.
ably mediated by hepcidin, a recently described iron regulat-
ing protein and acute phase reactant.'? It acts to decrease iron
Etiology and Pathophysiology absorption from the small intestine and block iron release
from macrophages.'*'* In animal models, inflammation,
Anemia of chronic disease occurs in patients with chronic (or probably acting through production of interleukin-6 (IL-6),
occasionally acute) immune activation. This includes chronic increases the release of hepcidin, which causes decreased
infections (bacterial, fungal, parasitic, and mycobacterial) serum iron.'* Because iron is important for the growth of
such as chronic osteomyelitis or tuberculosis; chronic inflam- many bacteria, this probably acts as a defense against infec-
matory diseases such as rheumatoid arthritis, systemic lupus tion. Hepcidin plays a central role in the development of
erythematosus, vasculitis, sarcoidosis, or inflammatory bowel ACD by decreasing the availability of iron for hemoglobin
disease; and malignant tumors, particularly carcinomas. In the synthesis.
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases WN 83

Ag presentation
to T cell :

MHC

IL-2 ~~
yas,
S

! |
B Humoral Immunity | C Cellular Immunity |
|
| |
| Lymphokines T-suppressor/cytotoxic |
| (CD8) |
| |
| CSF T cell. >|
| { MAF == oy |
| ieogtc De) | AgClass| Altered |
| 3 | BFU-E Nite
AD
self-cell ||
| | |Suppression
| | and Md ; |
, GM-CFU : :
production ee
| increase |
| Activated macrophage |
| | y * phagocytosis |
| | Leukocyte ¢ monokine |
| | production production |

| y |
|
| | IL-1 |

! |
Lysozyme and |
| lactoferrin |
| | release |
ais oe ae Le AL ene ER Ea, a a
Figure 14-1 @ Mechanism of humoral and cellular immunity. A. The antigen phagocytized by the APC is digested, and small antigenic frag-
ments or epitopes are associated with the class Il MHC and presented to aT cell with a receptor specific for the antigen. Formation of the
antigen-receptor complex between the two cells and IL-1 secreted by the APC provide the signals for the T cell to be activated and secrete
IL-2 for its autostimulation and proliferation to effector T-helper cells. 8B. Humoral immunity. The T-helper effector cell (CD4) and some of its
lymphokines provide the necessary signals for the B cell with the same antigen specificity to be activated and proliferate to B memory and
antibody-producing plasma cells. C. Cellular immunity. Some of the lymphokines from the activated CD4 celis and the complex formed
between antigens associated with class | MHC on altered self-cell and T-cytotoxic cell (CD8) receptors cause the activation of the CD8 cells,
which mediate the cytotoxic killing of the altered self-cells. Some of the other lymphokines produced also play significant roles in
hematopoiesis and activation of phagocytic cells. (Ag = antigen; APC = antigen-presenting cell; MHC = major histocompatibility complex;
IL-1 = interleukin-1; IL-2 = interleukin-2; CSF = colony-stimulating factor; BFU-E = burst-forming unit-erythroid; GM-CFU = granulocyte/
macrophage-colony-forming units; PMN = polymorphonuclear neutrophils; MAF = macrophage-activating factor.)

Macrophages in the bone marrow demonstrate SUPPRESSION OF ERYTHROPOIESIS AND THE ROLE
increased iron stores despite the low serum iron. The iron is OF CYTOKINES
trapped within the reticuloendothelial system and is unable The bone marrow is morphologically normal in ACD (except
to be fully utilized in erythropoiesis. Serum ferritin is for increased iron stores), but is unable to increase erythro-
increased reflecting the increased reticuloendothe lial iron poiesis in response to the anemia.*”'’'* Increased cytokines
stores (see Chap. 6). This results in the paradoxical ferroki- including IL-1, TNF-a and interferon gamma (IFN-y) act to
netics typical of ACD: ample iron stores with a functionally suppress erythropoiesis by the bone marrow by increasing
iron-deficient or sideropenic state. This also increases the apoptosis (cell death) in erythroid precursors.’ Sera from
amount of free erythrocyte protoporphyrin (FEP), which is patients with chronic inflammatory disorders (e.g., rheuma-
a porphyrin ring without iron,'® reflecting decreased heme toid arthritis, systemic lupus erythematosus, and juvenile
production. rheumatoid arthritis) inhibit erythropoiesis in vitro.'°?° The
284 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

inhibition of early erythrocytic progenitors, burst-forming

Table 14-1 Thymus-Derived unit-erythroid (BFU-E) and colony-forming unit-erythroid
Lymphocytes (CFU-E), is proportional to the severity of the anemia and to
indicators of inflammation such as the presence of acute
Helper (Inducer) T Cells phase reactants.
Aid B-cell maturation in bone marrow
IFN-y is the most potent inhibitor of erythropoiesis; levels
Supply activating (permissive) signals to B cells
Activate the generation of different antibody classes of IFN-y are inversely related to both hemoglobin level and
Activate effector T cells reticulocyte count.”!”? Evidence also suggests that TNF-a may
be involved in the pathogenesis of ACD by suppression of ery-
Effector T cells thropoiesis.> Even very low concentrations of TNF-a inhibit
Two types, producing delayed hypersensitivity and cytotoxic
effects. Responsible for:
erythroid progenitor growth in normal bone marrow cultures.
1. Immunologic surveillance for malignant cells This was demonstrated at concentrations similar to those found
2. Eradication of established viral and fungal infection in affected patients.’ Other cytokines may exert a direct toxic
3. Eradication of intracellular bacterial infection effect on erythroid precursors by inducing production of
4. Destruction of parasites
free radicals such as nitric oxide or superoxide anion by
Suppressor (Immunoregulatory) T Cells macrophages in the bone marrow. *
Control inflammation produced by T cells
Control antibody production DECREASED ERYTHROPOIETIN RESPONSE
Balance ratio of immunoglobulin classes
Erythropoietin response is decreased in ACD in two ways.
Block aactivation of osand B- cell Clones reactive
1 cs ‘self”
First, the increase in production of erythropoietin by the kid-
Source: From ae JM: The Cell-Mediated IImmune Renn neys is inadequate for the degree of anemia present.’ Patients
Cutter Biologicals, Emeryville, CA, 1982, p 3 with permission.
with ACD have been found to have lower levels of erythro-
poietin than patients with a similar degree of anemia due to
iron deficiency.*>’ This decreased erythropoietin expression
is probably mediated by IL-1 and TNF-a.* Second, even
when increased erythropoietin is present, the marrow is

Table 14-2 Cytokines That Contribute to Inflammation

Cytokine + target Cell Activity

IL-1 ecelisas coei na bacos eee Lymphocyte activation, macrophage

and tissue cells activation, acute-phase reaction
IL-2 T lymphocytes Stimulates proliferation of activated T cells
IL-3 (multilineage Stem cells Stimulates differentiation of bone marrow
colony-stimulating factor) stem cells
IL-4 (B-cell growth factor) B lymphocytes B-cell proliferation
IL-5 B lymphocytes, eosinophils, B-cell growth and differentiation,
precursor cells eosinophil differentiation
IL-6 B lymphocytes, hepatocytes Stimulates antibody production
and acute-phase reactants
MIF Macrophages Inhibits migration
MAF Macrophages Activates macrophages and enhances
their functions
ECE Phagocytes Promotes chemotaxis to site of injury
LIF Phagocytes Inhibits migration
GM-CSF Stem cells Stimulates differentiation of
granulocyte-monocyte precursor cells
M-CSF Stem cells Stimulates differentiation of monocytes
G-CSF Stem cells Stimulates differentiation of granulocytes
Interferon gamma (INF-y) Macrophages Activates macrophages for cytotoxic
functions; Induces MHC II molecules
on APCs
TNF-a Macrophages, granulocytes Activates macrophages, granulocytes,
and cytotoxic cells
IL= inter]atc MIF ==penton migrationFee iiitery factor; MAF = ee activation factor;
MHC= major histocompatibility complex; LCF = leukocyte chemotaxis factor; LIF = leukocyte inhibition factor;
GM-CSF= granulocyte/macrophage-colony-stimulating factor; M-CSF = monocyte- colony-stimulating factor; G-CSF=
granulocyte-colony-stimulating factor; APC = antigen-processing cell; TNF-c = tumor necrosis factor-alpha.
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 285

‘ ‘Table 14-5 Mechanisms Involved in

; _ Anemia of Chronic
Disease
Production of IL-1
Fever Dae of Iron Homeostasis
Neutrophil activation Inflammatory cytokines (IL-1 and TNF-a) divert iron into
Release of acute-phase reactants storage sites
Lymphocyte activation and proliferation Hepcidin decreases iron absorption and blocks iron release
Lymphokine production
Antibody production Suppression of Erythropoiesis by Cytokines
Cytokines (IL-1, TNF-a and IFN-y) increase apoptosis (cell
Antigen Processing and Presentation death) in erythroid precursors
Tumor Destruction
Production of TNF-a Blunted Erythropoietin Response
Cytotoxic action Cytokines (IL-1 and TNF-a) decrease erythropoietin release
Toxic factors by kidneys

Phagocytic Activities Decreased RBC Survival

Fe and C3 receptors Extracorpuscular defect
Microcidal and bacteriostatic activities
Protection from parasites
Removal of particulate substances and damaged cells
Lymphokine receptors (MAF, MIF)
unable to respond adequately with increased erythropoiesis
Inflammation and Tissue Alterations for the reasons described in the preceding text.**
Fibroblast activation
Collagenase synthesis
Synthesis and secretion of factors DECREASED RED BLOOD CELL SURVIVAL
Lysozyme, plasminogen activators, elastase, complement
AND LIFE SPAN
_ components —
eee a ee Finally, RBC survival is modestly decreased in ACD through
TNF-a= tumor necrosis factor-PaiphaenMAF = ere activating an extracorpuscular mechanism that is probably cytokine
factor; MIF = macrophage migration inhibitory factor.
related, but this seems to make only a minor contribution to
the anemia present.
It is becoming increasingly evident that ACD, regarded
by early investigators as a “simple anemia,” is far from sim-
ple (Fig. 14-2). The effect of cytokines on granulocytes,
macrophages, and erythroid progenitor cells appears to be
responsible for ACD. Therefore, mechanisms responsible for
host protection against infection and tissue injury become

Table ive Diseases That Cause ans,

detrimental to the host indirectly. Although the exact mecha-
nism is still being researched, cytokines including IL-1, TNF-a
Anemia of Chronic and IFN-y, along with the iron-regulating protein hepcidin,
‘Disease - have emerged as key pathogenic factors.” Synergy among
many cytokines has also been postulated.''”? Any disease that
Chronic Infections involves tissue injury and inflammation can, if present for a
Bacterial (pneumonia, bacterial endocarditis, osteomyelitis)
period of at least | to 2 months, result in ACD.
Fungal
Mycobacterial (tuberculosis)
Viral
Parasitic Characteristics
Chronic Inflammatory Diseases In ACD, the anemia is usually mild in degree (hemoglobin-
Rheumatoid arthritis HGB 9.5 to 11 g/dL); however, in about 20% of patients, the
Systemic lupus erythematosus
anemia will be more severe (HGB <8 g/dL). In about two-
Vasculitis
Sarcoidosis thirds of patients, the mean corpuscular volume (MCV) will be
Inflammatory bowel disease normal; in one-third of patients, the anemia will be modestly
microcytic (MCV 72 to 79 femtoliters [fL]). Because this is a
Cancer hypoproliferative anemia, the reticulocyte count is (inappropri-
Carcinoma
Lymphoma, leukemia, myeloma ately) normal to decreased.
Iron studies show a decreased serum iron as well as low
Chronic Rejection After Solid Organ Transplantation normal to decreased transferrin (and total iron binding capac-
ity [TIBC]). Transferrin saturation is usually normal, but may
“Acute” Anemia of Chronic Disease®
be decreased in 20% of patients.’
286 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

: oo
jHl y.
ae Ge oe

Lipopolysaccharide
emergence of malignant Q
cells, or autoimmune %
dysregulation

|
Immune effector
Hepcidin

mechanisms ©
Stomach

Duodenum

Transferrin-
bound iron

Decreased Fe?+
Increased absorption
ferritin o Transferrin

O32
receptor

Sincreased eS
) © Fe2+02.0
@ ©

/ @
Degradation and Macrophage
phagocytosis of Divalent metal
senescent ef transporter 1

& 6
erythrocytes @ ©0

~ Ferroportin 1

&
S) pk kee

oD :
Decreased
Fe?+

© Inhibit
erythrop

Hone Bone Erythroid

marrow __ Progenitor cell

Key

Activates
SSS Secretes/produces
Stimulates
Inhibits
Increases uptake
Decreases

Figure 14-2 @ Pathophysiological mechanisms underlying anemia of chronic disease. In A, the invasion of microorganisms, the emergence of
malignant cells or autoimmune dysregulation leads to activation of T cells (CD3+) and monocytes. These cells induce immune effector mecha-
nisms, thereby producing cytokines such as interferon-y (from T cells) and tumor necrosis factor-a (TNF-«), interleukin-1, interleukin-6 and
interleukin-10 (from monocytes or macrophages). In B, interleukin-6 and lipopolysaccharide stimulate the hepatic expression of the acute-phase
protein hepcidin, which inhibits duodenal absorption of iron. In C, interferon-y, lipopolysaccharide, or both increase the expression of divalent
metal transporter 1 on macrophages and stimulate the uptake of ferrous iron (Fe?*). The anti-inflammatory cytokine interleukin-10 upregulates
transferrin receptor expression and increases transferrin-receptor-mediated uptake of transferrin-bound iron into monocytes. In addition, activated
macrophages phagocytose and degrade senescent erythrocytes for the recycling of iron, a process that is further induced by TNF-a through
damaging of erythrocyte membranes and stimulation of phagocytosis. Interferon-y and lipopolysaccharide down-regulate the expression of the
macrophage iron transporter ferroportin 1, thus inhibiting iron export from macrophages, a process that is also affected by hepcidin. At the same
time, TNF-a, interleukin-1, interleukin-6 and interleukin-10 induce ferritin expression and stimulate the storage and retention of iron within
macrophages. In summary, these mechanisms lead to a decreased iron concentration in the circulation and thus to a limited availability of iron for
erythroid cells. In D, TNF-a and interferon-y inhibit the production of erythropoietin in the kidney. In £&, TNF-a, interferon-y and interleukin-1
directly inhibit the differentiation and proliferation of erythroid progenitor cells. In addition, the limited availability of iron and the decreased bio-
logic activity of erythropoietin lead to inhibition of erythropoiesis and the development of anemia. Plus signs represent stimulation and minus signs
inhibition. (From Weiss G, and Goodnough LT: Anemia of chronic disease. N Engl } Med 352:1013, 2005, with permission. Copyright © 2005
Massachusetts Medical Society. All rights reserved.)
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 287

The serum ferritin concentration is usually increased, of choice is correction of the underlying infection, inflamma-
reflecting the block in iron utilization with accumulation of tory disorder, or malignancy. If this is unsuccessful, or if
storage iron. Because serum ferritin is also an acute-phase a more rapid correction of hemoglobin levels is required,
reactant, it may be a poor indicator of iron stores in chronic recombinant human erythropoietin (rHuEpo) therapy or red
inflammatory diseases. Other acute phase reactants will also cell transfusion may also be considered.
be elevated in these patients, including C-reactive protein Use of rHuEpo is sometimes effective in ACD and may
and fibrinogen. Nonspecific measures of inflammation decrease the need for transfusion. rHuEpo seems to partially
including increased “sed rate” (erythrocyte sedimentation counteract the antiproliferative effects of cytokines.*?*°
rate)*®*' and lymphopenia (decrease in the absolute lympho- Patients with decreased serum erythropoietin levels are most
cyte count below 1500 cells/uL) are also commonly seen. likely to benefit from rHuEpo administration.***
Serum erythropoietin level is generally normal in ACD, but Despite the low serum iron levels, there is not a true iron
if decreased, may predict a better response to treatment with deficiency in ACD. Instead, there is a lack of iron availability.
recombinant human erythropoietin (rHuEpo).*?** Treatment of this anemia with iron in the absence of a true
In ACD patients with more severe anemia and microcy- superimposed iron deficiency is ineffective, and in some cases
tosis, making the distinction from iron-deficiency anemia may can actually be harmful. An excess of iron could increase the
become important. This can usually be accomplished by mea- virulence of an infecting organism and exacerbate an under-
suring transferrin or TIBC (low normal to decreased in ACD, lying infection.
increased in iron deficiency) and serum ferritin (increased in Transfusion offers another treatment possibility in
ACD, decreased in iron deficiency) (Table 14-6). Some patients with more severe anemia and no sign of remission in
authors have proposed using soluble transferrin receptor to their disease,'' but only after a proper evaluation of body iron
make this distinction (increased in iron deficiency),*** but stores, possibly including a bone marrow evaluation, has
one study found soluble transferrin receptor no more useful taken place.
than TIBC in distinguishing ACD from iron deficiency.*° Free
erythrocyte protoporphyrin (FEP) is elevated because limited
iron is available to the red cell precursors for incorporation Anemia Associated with Renal Disease
into the porphyrin ring. Thus the RBC cannot complete pro- and Renal Failure
duction of the heme moiety for incorporation into the hemo-
Renai disease is associated with a wide variety of hematologic
globin molecule.
abnormalities. These include anemia, abnormal platelet func-
Difficult cases may require bone marrow examination. In
tion, abnormal white blood cell function,’ and coagulopathy.
ACD, iron stain on the bone marrow aspirate will show normal
The latter usually results from the direct effect of uremia on
to increased iron in marrow macrophages but decreased iron in
platelet and coagulation factor function. Anemia almost invari-
erythroid precursors (sideroblasts).*”** This again reflects the
ably accompanies significant chronic renal insufficiency or renal
reticuloendothelial iron block with decreased availability of
failure.
iron for erythropoiesis.

Etiology and Pathophysiology

Treatment
The anemia of renal insufficiency is caused by:
The degree of anemia seen in patients with ACD correlates
with the severity of the inflammation present, but is generally 1. Inadequate erythropoietin production by damaged kidneys
mild and frequently does not cause symptoms. The treatment 2. Suppression of erythropoiesis by the uremic toxins that
accumulate in renal failure
3. A slightly to moderately reduced RBC lifespan.***?

Other contributing factors may include hyperparathyroidism,

blood loss (in dialysis tubing or from gastrointestinal hemor-
rhage), iron or folate deficiency, or ACD, but these should be
considered complications of anemia of renal insufficiency
Normal y IDA
rather than fundamental causes.** The mechanisms involved
Serum iron (ug/dL) 65-170 in anemia of renal insufficiency are outlined in Table 14-7.
TIBC (ug/dL) 250-450 The primary cause of anemia of renal insufficiency is
Serum ferritin (ug/dL) 12-300 decreased production of erythropoietin by failing kidneys.
Transferrin saturation (%) 20-50 Measured erythropoietin levels may be decreased, normal, or
FEP (ug/dL of RBCs) 16-65
increased, but even “increased” levels are low relative to what
RE marrow iron deposits 2-3+
Sideroblasts (%) 40-60 would otherwise be expected in a patient with a similar
Reticulocytes (%) 0.5—2.0 degree of anemia. Thus the erythropoietin production by the
diseased kidneys is insufficient for the severity of the anemia
ACD = anemia of chronic disease; IDA = iron-deficiency anemia;
. present, and administration of rHuEpo becomes a primary
FEP = free erythrocyte protoporphyrin; RE = reticulo-endothelial
treatment for anemia of renal insufficiency.
288 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

Table 14-7 Mechanisms Involved in

Anemia of Renal
Insufficiency
Inadequate Erythropoietin Produced by Damaged
Kidneys
Erythropoietin is inadequate for the degree of anemia
Decrease in erythroid precursors in the bone marrow

Suppression of erythropoiesis by uremic toxins

Dialyzable toxins such as polyamines

Reduced RBC Lifespan

Extracorpuscular defect related to uremic toxins

Other Complicating Causes of Anemia

Hyperparathyroidism Figure 14-3 ™ Peripheral blood from a patient with renal disease.
Blood loss (related to dialysis or GI hemorrhage) Note the burr cells (arrows). (From Bell, A: Hematology. In Listen,
Iron deficiency Look and Learn. Health and Education Resources, Inc., Bethesda, MD.
Folate deficiency With permission.)
Anemia of chronic disease
Dilutional anemia related to fluid retention

of renal insufficiency, with the exception that anemia tends to

be less severe in patients with polycystic kidney disease.°>°°
The anemia of renal insufficiency may be severe depend-
There is also evidence that substances present in uremic ing on the severity and duration of the renal failure, and is
plasma suppress erythropoiesis both in vivo and in vitro.*? It usually normocytic with a decreased or (inappropriately) nor-
has been observed that patients treated with peritoneal dialy- mal reticulocyte count (i.e., this is a hypoproliferative ane-
sis show improvement in anemia, even though the dialysis mia). On peripheral blood films erythrocytes are normocytic
would not be expected to improve erythropoietin production and normochromic; burr cells, sometimes seen in uremia,
by the kidneys.** This suggests that there is a dialyzable fac- may be present (Fig. 14—3).
tor that suppresses erythropoiesis in these patients, indepen-
dent of the relative erythropoietin deficiency. In addition,
plasma from renal failure patients has been shown to suppress Treatment
erythropoiesis by bone marrow cells in culture.*°*’ The exact
identity of the offending substance(s) remains unclear, but Recombinant human erythropoietin (rHuEpo) has been very
candidates include polyamines such as spermine, spermidine, successful in treating the anemia of renal insufficiency, such
putrescine, and cadaverine, as well as parathyroid hormone that many patients treated with rHuEpo are able to become
and inflammatory cytokines.** transfusion independent.*'*’ Anemia and its accompanying
Finally, some renal failure patients show a mild to mod- mortality and cardiovascular morbidity are reduced with
erate shortening of RBC survival that probably contributes to rHuEpo therapy.**°° Five to ten percent of patients show resis-
the anemia of renal insufficiency. The mechanism is unclear, tance to treatment with rHuEpo, the most common cause of
but seems to be extracorpuscular. In transfusion studies, red which is a superimposed iron deficiency.” This is treated with
cells drawn from renal failure patients and administered to iron supplementation.
normal subjects show normal survival, while red cells drawn rHuEpo has been used in both dialysis and predialysis
from normal subjects show shortened survival in renal failure patients with renal disease with good efficacy.°'©
patients.**°° Uremic toxins are again suspected as the
hemolytic factors.
Anemia Associated with Liver Disease
A wide array of hematologic disorders is encountered in patients
Characteristics
with liver disease. The morphology and degree of anemia differ
Anemia develops in virtually all patients with chronic renal somewhat depending on the cause and duration of the liver dis-
insufficiency, and is common when the creatinine clearance ease and on the presence or absence of other associated effects
falls below 20 to 30 mL/min.*! However, recent epidemio- (Table 14-8). Most cases of anemia associated with liver disease
logic studies have found that significant anemia is common in are Seen in patients with chronic liver disease.
patients with more modest degrees of renal insufficiency.>?*?
The severity of anemia correlates with the patients’ serum
Etiology and Pathophysiology
creatinine, creatinine clearance, and glomerular filtration
rate.>°* Many kidney diseases cause renal failure, but the In chronic liver disease, the anemia may be severe but overall
severity of the anemia is not related to the underlying cause does not correlate with the degree of hepatocellular failure or
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 289

‘Table 14-9» RBC Morphology iin

Pere Liver Disease
Direct Effects Morphology Pathophysiology
Toxic effects of ethanol
Vacuolization of marrow hematopoietic precursor cells Target cells T Membrane lipids
Decreased marrow cellularity Spur cells T Membrane lipids
Macronormoblastic changes unassociated with Macrocytosis | Folate, 7membrane
folate deficiency or vitamin B,, deficiency lipids
Acute and chronic blood loss Reticulocytosis Anemia
Gastrointestinal bleeding (alcoholism) Macronormoblasts: a Membrane lipids —
Liver disease—associated coagulopathic states
Viral suppression of erythropoiesis *Bone marrow eeentobincts

Indirect Effects
Dilutional anemia
Hypersplenism, erythrocyte sequestration Patients with chronic liver disease or liver failure and
Hemolytic anemia
Spur-cell anemia (acanthocytosis)
end-stage cirrhosis have decreased capacity for the production
RE macrophage activity of coagulation factors by the liver, especially the vitamin
Malnutrition K-dependent factors (see Chaps. 24 and 26). This may be
Protein, folate, iron, and vitamin deficiencies a cause of chronic blood loss or severe bleeding associated
Anemia of chronic disease with hemostatic challenges (such as surgery). Microcytic,
hypochromic anemia may develop as a result of acute or, more
often, occult chronic blood loss similar to the mechanisms
severity of liver disease. Target cells and acanthocytes are seen of iron-deficiency anemia as outlined elsewhere (see Chap. 6).
on the peripheral blood smear in cases of chronic liver disease Patients with chronic liver disease may also have hyper-
and are a consequence of the altered lipid production seen with splenism, a condition found in 15% to 20% of patients with
liver dysfunction (Fig. 14-4 and Chap. 3). Macrocytosis with cirrhosis and advanced liver disease.°’ This condition is a
the formation of target cells is seen on the peripheral blood cause of platelet sequestration leading ultimately to thrombo-
smear in cases of chronic liver disease with a level of obstruc- cytopenia and associated chronic bleeding tendencies,
tive jaundice or hepatocellular cholestasis affecting lipid especially gastrointestinal and mucosal surface bleeding.
metabolism. Target cells are due to increased membrane lipid Hypersplenism can also lead to decreased red cell survival as
(cholesterol and phospholipids) content while acanthocytes— RBCs are trapped in this major reticuloendothelial system
usually seen in cases of severe, chronic, liver disesase®™— organ.
are due to excess membrane cholesterol (Table 14-9).
Hemolytic anemia may occur because of the acanthocytes
Characteristics
(“spur cells”) due to the RBC membrane’s rigidity or lack of
permeability®° decreasing the deformability of the cell as it Overall, anemia associated with chronic liver disease alone
passes through the spleen. (without complications) is mild to moderate and normo-
cytic, normochromic. It is hypoproliferative, as demon-
strated by absent reticulocytosis, and may show variable
RBC morphology including moderate numbers of round
macrocytes. Increasing macrocytes may even cause the
MCV to be modestly elevated.
The anemia may be complicated by hemolysis or
decreased red blood cell survival due to destruction of acan-
thocytes,® in which case reticulocytosis may be present, or by
the simultaneous effects of iron deficiency (from blood loss)
or folate deficiency (predominantly from poor dietary habits).
Serum ferritin is increased in chronic liver disease reflecting
a chronic inflammatory response. Decreased plasma iron may
be due to the same mechanism seen in ACD or may be due to
decreased transferrin synthesis related to liver cell synthetic

x dysfunction.
Macrocytosis can be seen in patients with cirrhosis,
obstructive jaundice, and other types of liver disease, often
without accompanying anemia.® These macrocytes are
Figure 14-4 m Peripheral blood smear from an individual with
round, slightly flattened RBCs with a slight increase in size
severe liver disease. Target cells (arrows) are caused by altered lipid
metabolism with subsequent effects on red blood cell membrane. and volume. The MCV in liver disease is usually between
290 Chapter 14. Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

100 and 110 fL. These macrocytes are due to abnormal mem- precursors in the bone marrow is observed in alcoholic
brane cholesterol and phospholipid content. They are not, patients.°7' >> This toxic change is often associated with
however, the macro-ovalocytes seen in megaloblastic anemia abnormal hemoglobinization of the red blood cell cytoplasm.
(see Fig. 7-4).°° Bone marrow examination shows macronor- The mitochondria in red blood cell precursors—altered by
moblastic RBC precursors but no megaloblastic changes in alcohol’s effects—develop abnormal heme synthesis with sec-
early red blood cell precursors or the myeloid series cell ondary accumulation of iron. Surrounding the nucleus, these
changes such as giant “band” forms, or hypersegmented neu- abnormal iron-encrusted mitochondria form ringed sideroblasts
trophils that are typical of a true megaloblastic anemia.*®* (demonstrated upon iron staining of bone marrow aspirates or
The survival of these macrocytic red cells is not decreased. biopsies) in as many as one-fourth of hospitalized alcoholic
This type of macrocytosis does not respond to vitamin B,, or patients.” This phenomenon, also seen in chloramphenicol
folate therapy but usually resolves only after improvement, if (drug) toxicity, results in red blood cell membrane damage.”
any, in liver disease and function. Chloramphenicol (an older, discontinued antibiotic), which has
Oval megaloblastic macrocytes caused by folate defi- severe bone marrow effects associated with RBC precursor
ciency are another cause of macrocytosis, may also be seen in maturation arrest,’””* shares similar features with heavy and
some patients with liver disease, and are caused by poor nutri- prolonged alcohol abuse which too may result in overall,
tion, usually as a result of chronic alcohol intake.°* Alcoholism decreased bone marrow cellularity.’*’? Alcohol and its metabo-
is acommon cause of end-stage liver disease (see below). lites then are suppressants of hematopoietic-progenitor cell
Reticulocytes are also a cause of macrocytes in liver dis- production”’’”*”**° but fall short of causing a complete matura-
ease. These macrocytic cells are younger red blood cells that tion arrest or bone marrow aplasia. Thus, alcohol use leads to a
demonstrate polychromasia as seen on the peripheral blood hypoproliferative anemia.
smear. They are associated with blood loss or complications Macrocytosis is very common in chronic alcoholics and
seen in liver disease (see above). Therefore, macrocytes in may not show megaloblastic features.°**°*' Even in alcoholics
liver disease have different morphologic features depending who are well nourished or who have only mild liver disease,
on their cause. macrocytosis may persist unaccompanied by any megaloblastic
The anemia of chronic liver failure is often complicated changes.**® The mechanism of macrocytosis due to alcoholism
by portal hypertension, hypersplenism, and increased plasma alone is unknown.
volume as is seen with splenomegaly. The RBC mass is Megaloblastic anemia, when seen in alcoholics, may be
“pooled” in the venous system, sequestered in the spleen, or associated with direct effects of alcohol on folate metabolism
diluted in an increased plasma volume. These factors can (e.g., antifolate effect of ethanol, ethanol-induced increased
even yield a falsely decreased hematocrit, which may cause urinary folate excretion, ethanol-induced interruption of nor-
difficulty in evaluating the degree of anemia. mal enterohepatic circulation—usually seen in cirrhosis, or
ethanol-induced intestinal folate malabsorption).°+”? How-
ever, it is usually related to dietary folate deficiency due to
Treatment
malnutrition in these patients.**Anemia of chronic disease
Correction of any dietary deficiencies (lack of iron or folate) is also common in alcoholics as in other patients with chronic
can help the anemia of chronic liver disease. However, reso- liver disease. The element of malnutrition may give rise to
lution of the liver disease itself corrects the macrocytosis, decreased hemoglobin production or synthetic capacity by the
acanthocytosis, and anemia. In most instances, advanced liver liver affected by alcohol use/abuse.
disease and hypersplenism, when present, are irreversible. In With increasing chronic liver damage and disease, the
these cases, dietary supplementation (iron, vitamin B,,, and alcoholic patient is prone to hypersplenism and all of the
folate) can be efficacious in preventing any hematologic same effects of cirrhosis and end-stage liver disease outlined
sequelae; however, bizarre red cell morphology and low- in the preceding text.
grade anemia may persist due to the liver disease alone.
Characteristics
Anemia Associated with Alcoholism/ The anemia associated with alcoholism is usually macrocytic
Alcohol Abuse and hypoproliferative with similar morphologic changes as
described above with other liver disease. The change in cell
One of the most common causes of liver disease is excessive size persists until 1 to 4 months after the patient stops con-
alcohol ingestion (see Table 14-8). A wide array of hemato- suming ethanol.*
logic abnormalities occur as complications of acute and Iron deficiency due to chronic blood loss (e.g., bleeding
chronic alcoholism.°*° from esophageal varices formed as a consequence of cirrho-
sis) and folate deficiency related to poor nutrition may occur
Etiology and Pathophysiology together in severe, chronic liver disease due to alcohol use. In
this case, the MCV may even be normal, but it will be as a
Ethanol has direct toxic effects on red blood cell precursors, result of the average of two disparate populations of RBCs.
bone marrow cellularity, and peripheral red cell morphol- The red blood cell distribution width (RDW) will be greatly
ogy. Vacuolization of erythroblasts and red blood cell increased.
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 291

Treatment gastritis leads to decreased or absent production of intrinsic

factor—the binding factor released by stomach-lining cells
Treatment of the underlying liver disease by abstinence from which binds vitamin B,, from the diet and permits its absorp-
alcohol is essential to the reversal of the anemia. This will tion from the gastrointestinal tract at the level of the ileum
reverse the direct toxic effects on the bone marrow, and in a (small intestine). Without intrinsic factor, even an otherwise
few months the MCV and RBC morphology should return to well-nourished Addison patient may become vitamin B,,
normal if no irreversible liver disease has occurred. deficient, thus ultimately demonstrating a macrocytic/
megaloblastic anemia. The etiology of the anemia in this
instance is vitamin B,, deficiency similar to nutrient defi-
Anemia Associated with Endocrine ciency megaloblastic anemia (see Chap. 7). Treatment with
Disease/Disorders intramuscular injections of vitamin B,, may be helpful in this
case as a means of bypassing the diseased/altered gastroin-
The spectrum of endocrine diseases and their effects on metab-
testinal tract, thereby guaranteeing entry of vitamin B,, into
olism is wide. Patients with endocrine disease can have distur-
the body by the intramuscular as opposed to the oral route.
bances in protein synthesis, energy metabolism, growth, and
mineral metabolism. Many of the same hormones are involved
in the regulation of hematopoiesis. Therefore, the resulting Thyroid Disease
anemia is not due to the mechanisms involved in the anemia of
chronic disease. Diseases of the adrenal, thyroid gland, Thyroid hormones affect the basal metabolic rate—the rate of
parathyroid glands, gonads, and pituitary gland are most often oxygen and energy consumption—of the human body. Erythro-
involved in the development of anemia. poietin production by the kidney depends on tissue oxygenation,
which is influenced by the metabolic rate and thus by thyroid
hormones (e.g., tri-iodothyronine [T;] and tetra-iodothyronine
Adrenal Insufficiency [T,]). Thyroid dysfunction will either increase or decrease ery-
thropoiesis by increasing (as hyperthyroidism) or decreasing (as
Primary chronic adrenocortical insufficiency is sometimes also
hypothyroidism) the demand for oxygen.
called Addison disease. Patients with adrenocortical insuffi-
Hyperthyroidism is seldom associated with anemia, and
ciency may develop’ weakness and fatigability during the
may sometimes even be accompanied by a mild erythrocytosis.
course of everyday events for many other reasons besides
On the other hand, anemia is much more commonly seen
anemia—most often because of corticosteroid or steroid hor-
with hypothyroidism. A hypoproliferative anemia characterized
mone insufficiencies. While some of these cases are still caused
by the absence of a reticulocyte response, it is generally mild and
by disseminated tuberculosis and even systemic amyloidosis,
normocytic or modestly macrocytic.** HGB levels may be as low
most are idiopathic (i.e., of unknown cause)—the majority are
as 9.0 g/dl. Hypovolemia may accompany hypothyroidism. This
probably caused by autoimmune adrenalitis, a chronic, destruc-
may artifactually increase the HGB level by hemoconcentration,
tive inflammatory disorder of the adrenal glands themselves.*°
thus decreasing the apparent severity of the anemia.*’ The ane-
Idiopathic adrenalitis may cause isolated adrenocortical
mia can be corrected with the treatment of the hypothyroid state
insufficiency (steroid deficiency) resulting in a mild, normo-
by the administration of exogenous thyroid hormone.
cytic, normochromic anemia with absent reticulocytosis—a
In some hypothyroid patients, iron deficiency anemia
hypoproliferative anemia. The anemia may not be evident
may develop as a result of menorrhagia—an effect of hypothy-
because of the accompanying decrease in plasma volume,
roidism, achlorhydria (usually a gastrointestinal effect seen
which manifests concurrently with the adrenal insufficiency.
with generalized autoimmune disease associated with and
Thus, the decreased RBC mass is masked by the presence of
causing hypothyroidism), or a concurrent nutritional defi-
a decreased plasma volume.
ciency.*’** These share characteristics of nutrient deficiency
The etiology of the anemia of adrenal insufficiency is
anemias described elsewhere in the text (see Chaps. 6 and 7).
unclear but indicates some contribution to normal erythropoi-
Even more rare, acquired hemolytic anemia due to hypothy-
etic activity by glucocorticoids.*’ Thus, anemia accompanies
roidism, a derangement of lipid metabolism, and a subsequent
the decrease in glucocorticoids seen in adrenocortical insuffi-
alteration of RBCs has also been described.*
ciency. Treatment with an exogenous source of corticosteroids
The anemia associated with hypothyroidism should
will usually correct this mild anemia.
therefore be considered in the differential diagnosis of macro-
Autoimmune Addison disease (a subset of the idiopathic cytosis or mild, macrocytic anemia and could even, with a
cases) may also be associated with other autoimmune more severe anemia, mimic a vitamin B,, or folate deficiency.
endocrine diseases including diabetes mellitus, hypoparathy- However, the absence of megaloblastic changes in the periph-
roidism, hypogonadism, autoimmune thyroid disorders, and eral blood favors hypothyroidism over nutrient deficiency.
pernicious anemia. These polyglandular (multiple organ) syn-
dromes occur because of destructive autoantibodies directed
against parenchymal cells of the above-mentioned glands or Hyperparathyroidism
organs. The autoantibodies cause destruction of the gland or
organ resulting in subsequent hypofunction.*° For instance, Hypoproliferative anemia occurs in primary hyperparathy-
destruction of specialized gastric-lining cells in atrophic roidism in between 5 and 20 percent of patients.”’°? The
292 Chapter
p 14 Hypoproliferative
ypop Anemia: Anemia Associated with Systemic Diseases

anemia is mild to moderate in degree, normocytic, nor- It also secretes gonadotropins that control the production of
mochromic, and is associated with a decreased or (inappropri- androgens and estrogens as well as adrenocorticotrophins that
ately) normal reticulocyte count; it is not associated with control the production of adrenal cortical hormones. Thus,
neutropenia or thrombocytopenia. Anemic patients tend to pituitary function has far-reaching consequences. To correct
have more severe hyperparathyroidism with higher levels of this anemia, patients usually require the exogenous replace-
parathyroid hormone, serum calcium, and alkaline phos- ment of thyroid hormone, androgens, and/or estrogens, as
phatase. They are also more likely to show bone cysts, subpe- well as the administration of corticosteroids. All glands
riosteal bone resorption, and marrow fibrosis.” affected by the pituitary dysfunction are bypassed by this
The pathogenesis of anemia of hyperparathyroidism is exogenous hormone replacement necessitated because the
unclear. Parathyroid hormone may decrease proliferation of “master gland” is not functional.
erythroid precursors in the bone marrow.°? Bone marrow A summary of endocrine disorders including the
examination in these patients also tends to show more exten- type/morphology of the anemia, the pathogenesis, and the
sive marrow fibrosis in anemic patients, but myelophthisic treatment is outlined in Table 14-10.
changes in the peripheral blood are not seen. ”'
The treatment of this anemia involves the treatment of
the underlying hyperparathyroidism. Patients undergoing Anemia Associated with Malignancy
parathyroidectomy generally show improvement of their ane-
Anemia is very common in patients with both hematologic and
mia, usually to normal.”
nonhematologic malignancies (Fig. 14-5). The type of abnor-
Patients with renal failure frequently develop secondary
mality depends on a multitude of factors, including the type of
hyperparathyroidism. In these patients, it is difficult to sort
cancer, the site or sites in the body involved by the malignancy,
out the contribution of the anemia of hyperparathyroidism
the patient’s treatment with chemo- or radiation therapy, and the
from that of anemia of renal insufficiency (see preceding
extent of involvement of the hematopoietic tissues (i.e., bone
text). However, it has been noted that effective medical treat-
marrow) by the primary malignancy (Table 14-11). Anemia
ment of these patients’ hyperparathyroidism sometimes
may be the presenting symptom of an otherwise occult (uniden-
improves anemia and reduces the requirement for rHuEpo.”*
tified) malignancy. Anemia may be caused by decreased RBC
production, increased red blood cell destruction, or the toxic
Hypogonadism effects of the treatment of the malignancy. The focus in this
chapter and section is on decreased RBC production due to
Androgens are other hormones that have important roles as direct effects of malignancy on the hematopoietic bone marrow.
stimulants of erythroid activity, both physiologically and ther-
apeutically. For instance, the higher normal range for HGB
concentrations in men as compared to women reflects the Etiology and Pathophysiology
effect of androgens. Healthy men have HGB levels up to
DIRECT EFFECTS
1.0 g/dL higher than those of healthy women.
When the bone marrow is infiltrated by a malignant tumor
Androgens seem to promote erythropoiesis in at least
(also called myelophthisis), it can lead to anemia (i.e.,
two ways: (1) They increase the production of erythropoietin
myelophthisic anemia). Leukoerythroblastosis, which refers
by the kidney and (2) they stimulate the marrow in conjunc-
to the presence of both immature white blood cells (WBCs)
tion with erythropoietin. They are occasionally used pharma-
and immature—including nucleated—RBCs in the peripheral
cologically to treat refractory anemias. An example of this is
blood, is frequently seen on the peripheral blood smear along
Oxymetholone.
with teardrop RBCs (Fig. 14-6). The term can refer to bone
Men with hypogonadism as well as boys and elderly men
marrow infiltration by malignant cells or to marrow replace-
have hemoglobin levels similar to those seen in adult females.
ment by other processes (e.g., fibrosis, military tuberculosis,
In these instances, serum androgen (i.e., testosterone) levels are
osteopetrosis, etc.). Although leukoerythroblastosis can also
decreased while follicle-stimulating hormone and luteinizing
be seen in reactive and congenital conditions, it is the result
hormone levels (i.e., pituitary hormones) are increased in an
of myelophthisic processes in approximately two-thirds of
attempt by the pituitary to stimulate the nonfunctioning gonads.
cases.” Indeed, most individuals would reserve the term
In a mature man with gonadal hypofunction, the decreased
leukoerythroblastosis for the myelophthisic variant of this
androgen levels result in a mild normocytic, normochromic ane-
disorder only. In most cases of malignancy involving the bone
mia accompanied by a reticulocytopenia that may be reversed
marrow, some degree of reactive, space-occupying fibrosis
with the administration of exogenous androgens.
accompanies bone marrow infiltration by malignant cells.
Leukoerythroblastosis with “teardrop” RBCs is often a
Pituitary Dysfunction clue to marrow infiltration by the tumor with associated bone
marrow fibrosis (Fig. 14~7) as opposed to a reactive process
A mild, normocytic, normochromic anemia is seen in patients that usually demonstrates leukoerythroblastosis and normal
with hypopituitarism. Reticulocytosis is absent in this RBC morphology (Fig. 14-8). Marrow fibrosis for any
chronic, hypoproliferative anemia. The pituitary gland is reason, including desmoplastic (i.e., characterized by or caus-
responsible for the secretion of thyroid-stimulating hormone. ing the growth of fibrous tissue) reaction associated with
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 293

Endocrine Disorder/Disease Type of Anemia Pathogenesis (Cause of) Treatment

Hypopituitarism N/N Loss of trophic function Exogenous replacement

of pituitary hormones of thyroid hormone
Androgens and/or
estrogens
Administration of
corticosteroids
Adrenal insufficiency Idiopathic (unknown cause) Exogenous source of
In Addison disease - related corticosteroids
to destructive autoantibodies
directed against parenchymal
cells of the organ (autoimmune)
Hypothyroidism Mild macrocytic Hypothyroidism Exogenous thyroid
hormone
Hyperparathyroidism N/N Increased parathyroid hormone Parathyroidectomy or
medical treatment
Hypogonadism N/N Defective secretion of/from Androgens
«
the gonads

N/N = normocytic/normochromic anemia.

metastatic tumor to the bone marrow, can lead to “‘teardrop”- Blood loss can be another direct effect of malignancies'©°
shaped RBCs and leukoerythroblastosis. Marrow invasion as can chronic or acute disseminated intravascular coagula-
can occur with solid tumors, particularly metastatic small cell tion (DIC) (see Chap. 27). The latter is the result of activation
carcinoma of the lung; breast carcinoma; prostate cancer; gas- of the coagulation system by metastatic carcinoma with high
trointestinal tumors; and, in some instances, lymphoma.’*”” cell “turnover” or death and significant release of tissue
The anemia is due to a “crowding out” of normal bone mar-
row elements by the malignancy. Essentially, the bone mar-
row space loses both its hematopoietic cellularity and its
proper microenvironment, resulting in a hypoproliferative
anemia. Extensive marrow invasion/involvement can lead to
pancytopenia.
Direct Effects
Replacement of marrow by malignant cells
Primary hematologic malignancy
Ineffective erythroid production
Qualitative reduction in erythropoiesis
Metastatic marrow infiltration
Quantitative reduction in erythropoiesis
Replacement of marrow by fibrosis
Acute and chronic blood loss

Indirect Effects
Anemia of malignant disease/anemia of chronic disease
Anemia of associated organ failure (e.g., renal, hepatic)
Malnutrition and vitamin deficiency
Microangiopathic hemolytic anemia
Immune hemolytic anemia

Treatment-Associated Anemia
Immediate
Chemotherapy
Radiation therapy
Late
Secondary myelodysplasia or leukemia
Idiopathic
Depleted marrow reserve
Figure 14-5 m Peripheral blood from a patient with disseminated
Microangiopathic hemolytic anemia
carcinoma. Note presence of (A) schistocytes and (B) helmet cells.
and (for example postmitomycin chemotherapy)
(From Bell, A: Hematology, In: Listen, Look, and Learn, Health
n.)
Education Resources, Inc., Bethesda, MD, with permissio
294 Chapter 14. Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

SO

“Sar
,

Figure 14-6 m Leukoerythroblastosis, a peripheral blood picture

that often accompanies marrow infiltration by tumors (myeloph-
thisic anemia). Note the presence of immature (A) red and Figure 14-8 ™ Peripheral blood smear from a neonate with leuko-
(B) white cells. erythroblastosis caused by severe blood loss associated with birth
(a reactive condition). (A) Left-shifted granulocyte precursors and
(B) nucleated red blood cell precursors are seen. Although some
(QO “burr” cells are seen, no teardrop-shaped red blood cells are
factor. DIC may be associated with microangiopathic
identified.
hemolytic anemia (see Chap. 13 and Table 14-12).

INDIRECT EFFECTS
Malignant tumors elicit a chronic inflammatory response that Radiation therapy is also toxic to bone marrow stem
commonly causes anemia of chronic disease. cells when bone marrow is included within the field of irradi-
ation. Although radiation therapy may be focused directly on
TREATMENT/THERAPY-ASSOCIATED EFFECTS a metastatic lesion within bone, hematopoietic bone marrow
Transient pancytopenia is expected in any patient undergoing may be secondarily involved during radiation therapy for
aggressive treatment for hematologic or nonhematologic lesions within other organs.
malignancies. Most combination chemotherapy protocols use The sequence of recovery in bone marrow is quite vari-
one or more of the alkylating agents, which are directly toxic able. Both erythroid and granulocytic regeneration usually
to rapidly dividing cells, including both rapidly proliferating precede megakaryocyte regeneration'®'; however, the half-life
tumor cells and normally dividing hematopoietic cells. When of RBCs is considerably longer than the half-lives of the other
given frequently and in intervals, alkylating agents cause a two cell lines. Therefore, RBC recovery in the peripheral
dramatic decrease in the peripheral blood cell counts. blood often lags behind white blood cell and platelet recovery.
The anemia in this instance is hypoproliferative due to the
immediate toxic damage to hematopoietic precursors by
chemotherapeutic reagents or radiation therapy.

Morphology Pathophysiology

Leukoerythroblastosis Myelophthisis, marrow

fibrosis
Teardrop RBC Marrow fibrosis
Schistocytes* Microangiopathic hemolytic
anemia
Helmet cells* Microangiopathic hemolytic
anemia
Burr cells* Microangiopathic hemolytic
anemia
Hypochromia Chronic blood loss, J Fe
Figure 14-7 @ Peripheral blood smear from an individual with
myelophthisic anemia. Note left-shifted granulocyte precursors (solid *Disseminated carcinomas; may be associated with disseminated
arrow). Teardrop-shaped red blood cells (open arrows) are indicative of intravascular coagulation.
marrow fibrosis in this type of leukoerythroblastic reaction. Nucleated Fe = iron.
red blood cell precursors were seen in other fields
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 295

Characteristics Pathophysiology and Characteristics

The anemia of malignancy/myelophthisis is usually classi- Direct infection of bone marrow stem cells was originally pos-
fied as normocytic, normochromic, and hypoproliferative. tulated as the cause of anemia in HIV infection,''’ yet subse-
As stated earlier, many other processes can occur to change quent studies have shown that hematopoietic stem cells are
the severity or morphology of the anemia of malignancy. not a reservoir for HIV!!! and are even resistant to HIV
However, the most important change seen in anemia of infection.''* A direct effect may be seen in some precursors,
malignancy is leukoerythroblastosis with “teardrop”-shaped specifically monocytes'!> and megakaryocytes!'®''’; however,
RBCs, indicating bone marrow replacement and/or bone erythroid and granulocytic cells are not persistently or consis-
marrow fibrosis and a poor prognosis because of metastatic tently HIV infected when emerging from the bone marrow.''°
involvement of hematopoietic bone marrow by malignant The hematologic picture seen in HIV-positive patients is a
tumor. Any polychromasia seen in this type of anemia is due normocytic, normochromic, hypoproliferative anemia with a
to “crowding out” of normal red blood cell precursors from decreased reticulocyte count.'“ Early studies in HIV-infected
the bone marrow by tumor and not to the premature release patients revealed bone marrow specimens with dyserythro-
or increased production of precursors from bone marrow, as poiesis and megaloblastic maturation.'™ Vitamin B,, malabsorp-
would be seen in anemia with reticulocytosis. As such, the tion has also been seen in HIV-infected individuals.!'*"°
level of reticulocytosis is often far below that expected for Because of the already documented effects of IL-1 and
the severity of anemia and so fits with the hypoproliferative TNF-a on erythropoiesis, current theory suggests that the
profile of this anemia. inflammatory process is predominantly responsible for the
anemia associated with AIDS. The normocytic, nor-
mochromic anemia is thus similar to ACD but can be slightly
Treatment more severe in degree. AIDS patients may demonstrate a
In severe cases of bone marrow replacement, transfusions are reticuloendothelial system iron blockade and iron studies sim-
needed to correct the patient’s anemia. Chemotherapy or radi- ilar to ACD.'” For the most part, the anemia of HIV infection
ation therapy protocols are often used to reduce the burden of and AIDS is a subset of ACD.
malignancy involving the bone marrow while at the same
time worsening anemia in the short term. Eradication of Treatment
malignancy from the bone marrow offers the only chance for
reconstitution of the marrow by hematopoietic precursors and Treatment in HIV infection includes antibiotics (prophylactic
for endogenous correction of the anemia. or with infections), supportive care, and antiretroviral ther-
apy. Azidothymidine (AZT) therapy was shown to exacerbate
the anemia of HIV infection'*'; however, AZT was shown to
Anemia Associated with Human inhibit HIV replication and has helped to prolong life in many
Immunodeficiency Virus Infection AIDS patients. Anemia and granulocytopenia are the major
and the Acquired Immunodeficiency adverse effects associated with AZT.'” Patients with less
advanced infection at the initiation of AZT therapy are less
Syndrome likely to develop severe anemia and gianulocytopenia.
Anemia may also be associated with human immunodefi- AZT therapy can cause a macrocytosis that can change
ciency virus (HIV) infection and the acquired immunodefi- the features of the anemia of AIDS.'** It does this because it
ciency syndrome (AIDS). HIV infection is a devastating is a deoxyribonucleotide synthesis inhibitor acting at a simi-
condition caused by direct destruction of CD4 T lympho- lar point in the deoxyribonucleic acid (DNA) synthetic path-
cytes (helper-inducer cells) by HIV. By reducing the CD4 way as would a vitamin B,, or folate deficiency. It has also
T-cell count, HIV renders the host immunocompromised and been associated with a worsening of the reticuloendothelial
susceptible to many opportunistic pathogens. Most HIV- system iron blockade seen in HIV infection and AIDS.'°° Just
infected patients develop one or more cytopenias during the as in any ACD, when the hematologic picture changes, one
course of the disease.!°*'” should suspect complicating factors. In AZT treatment, this
Anemia develops early and becomes more severe as the means the emergence of macrocytosis in an otherwise normo-
disease progresses. Approximately 85% of patients with cytic, normochromic ACD.'** This may also occur with
AIDS are anemic.!°3 Anemia has been noted in preliminary stavudine-based regimens although the macrocytosis is not as
findings of HIV-infected individuals even before secondary marked.'**
infections ensue or medications are started.'°° Infections with When AZT therapy is stopped, increases in hematocrit
Mycobacterium avium, M. intracellulare, herpes simplex and reticulocyte count are observed. To treat the severe ane-
virus, Pneumocystis carinii, and cytomegalovirus contribute mia seen with retroviral therapy, AZT therapy often must be
to the state of chronic infection and chronic inflamma- discontinued or the drug dosage reduced. The FDA-approved
tion.!°""° The anemia associated with HIV infection can be the use of recombinant erythropoietin to treat severe anemia
worsened by concurrent conditions, such as one or more in patients with AIDS.'>'*° Recombinant erythropoietin
opportunistic infections, as HIV infection persists and (rHuEpo) may reduce or eliminate the need for red cell trans-
progresses. fusions in AIDS patients receiving AZT therapy.
296 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

The introduction of highly active antiretroviral therapy same time, a gradual switch to renal production takes place.'**
(HAART) has greatly reduced the anemia of HIV infec- This switch is not complete until about | to 2 months after
tion.'?7° Potential mechanisms include a reduction in oppor- birth.'**
tunistic infections, a lessening of the effects of ACD, and an Therefore, the more responsive kidney cannot and does
overall improvement in nutritional status related to a lessen- not play a part in responding to anemia for some time after
ing of the overall HIV infection.'*? While some have found birth. A contributing factor is the increased rate of clearance
that HAART lessened the effects of AZT on the anemia of of erythropoietin from neonatal plasma as well as_ its
HIV,!* others hold that AZT may still exacerbate the anemia increased volume of distribution in most infants on birth and
of HIV despite its use in HAART.'*° Persistent anemia and early life!*?'*!4? in addition to rapid neonatal growth in the
transfusion dependence despite HAART is now considered a first few weeks of life requiring a concomitant expansion of
poor prognostic indicator.'°°'?! RBC mass'** in the face of a shorter lifespan of the neonatal
Therefore, in HIV infection, decreased production of RBC as compared to the adult RBC.'** This leads to a relative
RBCs can be attributed to several possible mechanisms— decrease in erythropoietin and a poor response of erythropoi-
including therapeutic interventions. These are outlined in etin to anemia. Varying levels of anemia are due to the many
Table 14-13. physiologic factors that are present during the first | to
2 months of life (Table 14-14).
The anemia is usually well tolerated in term infants.
Anemia of Infancy Indeed, it is commonly referred to as the “physiologic” ane-
mia of infancy because it occurs in the absence of any recog-
Etiology and Pathophysiology
nized nutritional deficiency and is characteristic of even the
Infants are born with HGB values of 17.0 g/dL or higher. Dur- healthiest of infants.'°*'*!
ing the first month of life, all infants experience a decrease in
their circulating RBC mass. The concentration of HGB
declines rapidly from this highest (birth) level to the lowest Characteristics
value of any time during the first few years of life.'°* The The decrease in RBC mass leads to a mild, normocytic, nor-
nadir of the HGB level approaches 9.0 g/dL within the first 10 mochromic anemia with reticulocytopenia which corrects
to 12 weeks of life.'°? specifically as the production of erythropoietin increases.
This decline in red blood cell mass is due to many factors. Other cell lines such as platelets and WBCs are not affected.
The lack of permeability of the placenta to maternal system
erythropoietin is one contributing cause,'**'°° yet the most
prominent is the predominance of erythropoietin production Treatment
by the neonatal liver.'*’'*? The neonatal liver is less sensitive
to anemia and tissue hypoxia than is the kidney.'*”'*° At term Vitamin and hematinic supplementation do not alter the
birth, 70% to 75% of the erythropoietin produced in response decrease in bone marrow erythropoiesis.'*! If anemia causes
to anemia continues to be produced by the liver.'** At the symptoms (e.g., clinical evidence of hypoxia), packed RBC
units are the product of choice for transfusion. However, in the
absence of symptoms, no treatment is required for this physio-
logic anemia of infancy as it usually corrects on its own.

Suppression of erythropoiesis caused by cytokines IL-1

and TNF-a
Phagocytosis of erythroblastic cells by bone marrow
histiocytes
Antibodies to HIV, which circulate in bone marrow and Rapid growth
destroy red cell precursors Erythropoietin production by the neonatal liver (liver
Production of complement protein, C3, and IgG, which form produces 75% of erythropoietin at term)
immune complexes on red cell membranes, causing Increased rate of clearance of erythropoietin from neonatal
hemolysis plasma
Infections of red cell precursors by parvovirus B19 and Increased plasma volume/increased volume of distribution of
Mycobacterium, halting the maturation process in the erythropoietin
bone marrow Short half life of neonatal red blood cells
Inhibitory serum protein, which results in a decrease in Slow transition (1-2 months) to erythropoietin production by
BFU-E not present in normal individuals kidneys
Defective erythropoietin response to anemia Nutritional deficiencies (usually appearing 2 or more months
Treatment with AZT, which inhibits DNA synthesis of blood after birth in the absence of supplementation)
cells and results in a macrocytic anemia; may also be seen Chronic blood loss or “Lab draw” anemia (usually seen in
with Stavudine (see Glossary) hospitalized premature neonates)
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 297

Anemia Associated with Prematurity Vitamin and hematinic supplementation is not as impor-
tant during the first 1 to 2 months of life as it is after this time.
Etiology and Pathophysiology Therefore, if a premature neonate is likely to be hospitalized
The anemia associated with prematurity is a subset of the for a long period of time, it is advisable to supplement his diet
anemia of infancy. Premature infants experience the same with iron and certain vitamins, including vitamin E.'°*'*’ This
alterations in the production, plasma distribution, and clear- can prevent an anemia of prematurity from becoming worse
ance of erythropoietin as do term neonates. While some or from persisting as a nutrient deficiency anemia (e.g., iron
have related the premature infant’s blunted erythropoietin deficiency or vitamin B,,/folate deficiency).
response to the erythrocyte’s amount of fetal HGB and the rHuEpo has emerged as a possible treatment for the ane-
Shape of the infant’s oxygen—hemoglobin dissociation mia of prematurity.'**'°°>-!>4 Progenitor cells committed to
curve,'** the decreased response of erythropoietin to oxy- erythroid differentiation are present in infants with anemia of
prematurity, and these maintain normal responsiveness to ery-
gen tension is still the key factor to this anemia.!*+!45
In fact, preterm infants demonstrate earlier and more severe
thropoietin.'° Although some early evidence suggested that
the use of rHuEpo led to a reduction in the requirement for
declines in HGB values as compared to full-term
blood transfusion in the premature infant,'°*'°°'°’ much of
infants.'*°'*” As such, HGB levels may approach nadirs
the current evidence fails to support this finding in the anemia
of 8.0 g/dL in >1.0 kg and 7.0 g/dL in preterm infants
of prematurity.'*?!°*'°° The exact subgroup of premature
weighing less than 1.0 kg.!*”
neonates that is best helped by this treatment is still subject to
Although adequate oxygenation may be maintained in
investigation.'**'*’ Reviews show that rHuEpo can stimulate
symptom-free infants with anemia of prematurity through a
erythropoiesis.'°'°’ The failure to mount an appropriate,
host of adaptive physiologic mechanisms,'** an added factor
endogenous erythropoietin response may explain the HGB
(or complicating factor) in this clinical scenario is blood
levels seen in premature infants in the months following
loss.'** Many premature infants experience respiratory dis-
birth.'* Iron supplementation with rHuEpo use,'*”!°* as is
ease or problems necessitating ventilatory assistance and long
employed in the treatment of the anemia of renal disease/
hospital admissions while others require a long hospital stay
renal failure, is indicated here, as these neonates too may
because of the need for nutrient supplementation by the use of
become iron-deficient after stimulation with pharmacologic
special feeding tubes.or intravenous catheters. Whatever the
doses of erythropoietin.'*?'®'7° Since the spectrum of use of
reason, the more ill the infant, the longer the hospital-based
*Hupo is not clear and has not succeeded in the elimination
care and the more frequent the blood sampling from the pre-
or marked reduction of RBC transfusions in premature
mature infant. Therefore, in addition to all of the factors out-
neonates, the focus has shifted to making transfusion of these
lined for the anemia of infancy, frequent blood drawing for
patients as safe as possible by a host of mechanisms. '4?!7))!7?
laboratory specimens is added as a contributing factor to the
anemia of prematurity (see Table 14-14). It stresses the oth-
erwise physiologic anemia present in these infants and makes
the anemia of prematurity much more severe than the benign “Anemia” Associated with Improperly
“physiologic” event that is the anemia of infancy'*? because Collected Laboratory Samples:
the small or critically ill, premature infant requires special, Preanalytical Anemia
and sometimes intensive, care.
Etiology and Pathogenesis
Characteristics A final “anemia” deserves mention. That is, the anemia associ-
ated with improperly collected laboratory samples. It is not an
The anemia is normocytic and normochromic with reticulocy- anemia at all, but rather a specimen adequacy problem. It is a
topenia. The severity of anemia varies with the degree of laboratory problem—a preanalytical problem but has the poten-
blood loss from laboratory sampling in concert with the level tial to become a true clinical patient management problem.
of contribution of the other causes of anemia in the neonatal Most laboratory specimens are drawn from the venous
patient (see Table 14-14). Other blood constituents such as system of a patient. They are usually drawn from direct
platelets and WBCs are not greatly affected. venipuncture. Certain patients (e.g., hematology/oncology
patients or intensive care unit patients) have central venous
catheters or peripheral intravenous (IV) lines in place because
Treatment
of the nature of their primary disease or their hospital care.
If signs of hypoxemia are present, RBC transfusion is indi- Due to many circumstances, it is often easier to collect a spec-
cated.'*3 Indeed, replacement of blood drawn for laboratory imen from the indwelling catheters in these patients than to
studies is the predominant indication for RBC transfusions access a new vein by venipuncture.
given to neonates.'*° These are small volume RBC transfu- Care must be taken to avoid a dilutional effect on the
sions'33"'5! because the key is the maximum amount of RBCs specimen due to catheter “flush” fluid or concurrent IV fluid
for the minimum transfused volume necessary. RBC transfu- therapy. Blood removed directly from a catheter is not true,
sions prevent any untoward effects of “high-output” conges- whole blood but a mixture of catheter solution (usually an
tive heart failure as a result of the infant’s anemia.'** anticoagulant) and the patient’s whole blood. This “anemia”
298 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

is a dilutional one. In this case, whole blood entering the

catheter, on its way to the specimen collection vial, is diluted
by the solution within the catheter itself. The same can also
Case Study 1 |

happen when procuring a sample near, above, or through a

A 22-year-old woman was admitted from the emergency
peripheral IV line site.
department with fever, dysuria, and lower back pain. Imme-
diate laboratory results revealed the following:

Characteristics CBC
Urinalysis
In these cases, a normocytic, normochromic “anemia” will be Urine reagent strip: WBC: 11.8 X 10°/L
seen when the laboratory specimen is analyzed. Usually, other 4+ urine protein RBC: 29 < 1077/5
peripheral blood cells (platelets and WBCs) will also be 1+ hemoglobin Heb: 8.3 g/dL
decreased because the entire blood sample has been diluted. A Urine microscopic: Het: 25%
comparison with previous complete blood count (CBC) values a Ae:| ae oe
(the laboratory delta check) is most helpful here, as a sharp or Moderate leukocytes MCHC: 30%
significant change in the patient’s hemoglobin or hematocrit is Casts: few hyaline RDW: 14.7%
noted most of the time. This reflects the comparison of a previ- and few granular Platelets 315 x
ous, properly drawn sample—or one drawn from the patient : 10°/L :
prior to the placement of an IV line or a central venous Blood Chemistry eae es
ee BUN 113 mg/dL 1+ poikilocytosis
catheter—to one that is improperly drawn and therefore diluted. Cre ee mg/dL. i+ oeois Re

Prevention Blood cultures were drawn and eventually produced only a

few colonies of coagulase-negative Staphylococcus (probably
To avoid this problem, one should withdraw 5 to 10 mL of blood skin contaminants). Urine cultures grew >100,000 colony-
from a central venous catheter before collecting a hematology forming units per milliliter of E. coli. Antibiotic treatment was
specimen. The 5 to 10 mL includes the diluting fluid and reduces begun. Blood was still present in the patient’s urine after
the chance for error due to specimen dilution. 2 days, and bleeding was noted from intravenous infusion
When accessing a peripheral IV line for a hematology sites. Coagulation tests were ordered before the patient was to
specimen, one should disconnect the line for 3 min and dis- have a kidney biopsy performed. These showed:
card at least 5 mL of blood before collecting any specimen. Platelet count: 296 * 10°/L
Finally, when performing a venipuncture on a patient with a PT: 11.7 seconds (normal range: 10.0-12.9 seconds)
peripheral IV line in place, one should perform the procedure
APTT: 32 seconds (normal range: 23-35 seconds)
on the opposing arm, and, if this is not possible, the venipunc-
Bleeding time: > 20 minutes
ture should be performed downstream (i.e., peripheral) to the
IV line or site so as not to draw a dilute sample. (PT is prothrombin time; APTT is activated partial throm-
The failure of a delta check in the absence of patient symp- boplastin time)
toms and in the presence of concurrent and proportional This patient was suffering from renal failure and a urinary
decrease in white blood cell and platelet counts identifies this tract infection (UTI). The admitting CBC demonstrated a
laboratory specimen “anemia” for what it is—a preanalytical moderate anemia with some burr cells typical of renal dis-
variation. This allows for its correction by collection of another, ease. The anemia could be the product of several mecha-
PROPER and representative sample from the patient before any nisms. It was most likely a result of the anemia of chronic
clinical evaluation or treatment for anemia is instituted. disease related to her UTI coupled with anemia of renal
insufficiency. This may have been complicated by blood
loss due to abnormal platelet function related to her renal
Summary failure (see Chaps. 24 and 25).

This chapter discusses the characteristic features of the anemias QUESTIONS

associated with many systemic, nonhematologic disorders.
Most of these disorders are manifested by a hypoproliferative, 1. Classify the anemia with regard to RBC indices.
normocytic, normochromic anemia. Anemia may be the first 2. List the laboratory parameters in this case that coincide
with anemia of renal insufficiency.
sign of a previously undetected systemic disease, and changing
3. How would you prove that anemia of chronic disease is
RBC parameters may indicate new, complicating
; factors in an
pian present?
otherwise stable anemia associated with a systemic disease. 4. Why would chronic iron loss be a concern in this
These are anemias that the health-care worker should be patient?
familiar with owing to their frequency in all patient groups
seeking medical care. Indeed, this group of anemias will be
the most frequently encountered by the allied medical health
professional in his or her daily activities and practice.
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 299

The patient was transported to the recovery room where,

Case Study 2 over 2 hours, her blood pressure was noted to be unstable.
She was also noted to have a distended, tender abdomen.
A 42-year-old woman presented to her physician with pain Laboratory tests revealed:
in both of her wrists and in the proximal joints of both of her
hands. She had also recently begun experiencing right knee WBC: 10.0 X 10°/L
pain. Her laboratory results revealed the following: Heb: 12.4 g/dL
Het: 38%
WBC: 10.0 X 10°/L
Hgb: 8.5 g/dL Platelets: 150 < 10°/L

Het: 25% She was given 3 to 4 L of 0.9% saline, which stabilized her
blood pressure enough to allow her to be transferred back to
MCV: 90 fL
surgery. Laboratory tests then revealed:
RDW: 12.5% (normal 11%-14%)
WiBEz6 OP a6 7/15
Erythrocyte sedimentation rate (ESR): 100 mm/h (normal
0—10 mm/h) Hgb: 7.5 g/dL
Rheumatoid factor: Positive Het: 20%
Platelets: 100 < 10°/L
QUESTIONS The evening shift medical technologist noted a significant
1. What is the patient’s diagnosis? change in the patient’s hemoglobin level over the course of
2. What could cause the increase in ESR? 1 to 2 hours in the recovery room by the use of a delta check
3. Why was the patient anemic? (1.e., a significant change in laboratory values). The technol-
ogist called the recovery room to report the now lower value
and to ask about proper specimen collection. The call was
transferred to the operating room, where the patient was
now undergoing an emergency laparotomy. The technolo-
gist reported the value to the physician (surgeon), who
Case Study 3 explained that the patient’s abdomen was now full of blood
(approximately 2 L) because of a damaged artery that was
A 29-year-old woman with cholecystitis (inflammation of not “tied off’ during surgery. The surgeon had just sutured
the gallbladder) was taken to elective surgery, where she the artery and was closing the abdominal wound after evac-
underwent a cholecystectomy. Her preoperative laboratory uating and suctioning the blood from the cavity.
values revealed:

WBC: 10.0 X 10°/L QUESTIONS

Hgb: 12.5 g/dL 1. Why was the patient’s hemoglobin level normal when
Het: 38% she had a distended abdomen full of blood?
2. What caused the hemoglobin value to decrease?
Platelets: 150 < 10°/L
3. What other tests would be abnormal in this patient?
continued

Os estioms
1. Which of the following lists of laboratory findings would c Suppression of erythropoiesis by cytokines
be most characteristic of the anemia of chronic disease? d. All of the above
a. Normocytic, hypochromic anemia; high serum iron . What are the main factors responsible for reducing ery-
level: increased TIBC; decreased ferritin levels. thropoiesis in anemia of chronic disease?
b. Normocytic, normochromic anemia; low serum iron a. Cytokines from macrophages and lymphocytes
level: decreased TIBC; increased ferritin levels. b. Antibodies from B lymphocytes
c. Microcytic, hypochromic anemia; normal serum iron c. Erythropoietin from the kidney
level: decreased TIBC; normal ferritin levels. d. Polyamines associated with renal failure
d. Macrocytic, normochromic anemia; low serum iron
4. What is the treatment for anemia of chronic disease?
level; decreased TIBC; decreased ferritin levels.
a. Blood transfusion
i) Which of the following are causes for the anemia of b. Iron therapy
chronic disease? c. Treatment of the underlying inflammatory process
a. Shortened RBC lifespan d. Human recombinant IL-1
b. Impaired iron metabolism
300 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases

_ Which diseases do not cause anemia of chronic

‘nN c. Target cells, macrocytes, and acanthocytes
disease? d. Marked anisocytosis and poikilocytosis, and dysfunc-
a. Malignancy and neoplastic processes tional leukocytes
b. Connective tissue disorders 10. Leukoerythroblastosis with teardrop-shaped red blood
c. Bacterial and fungal infections cells is indicative of:
d. Endocrine, kidney, and liver disease a. Bone marrow fibrosis
. What is the primary cause of anemia associated with b. Bone marrow stress
renal disorders and renal failure? c. Abnormal lipid metabolism
a. Blood loss from dialysis d. Renal disease
b. Decreased erythropoietin production 11. The anemia of infancy is:
c. Hemolytic processes a. Physiologic
d. Vitamin B,, and folate deficiencies b. Well tolerated
. What is a typical appearance of anemia associated with c. Due to a switching from liver production of erythro-
liver disease? poietin to that of renal production
a. Spherocytic red blood cell morphology d. All of the above
b. Hypochromic, microcytic red blood cell morphology 12. The anemia of prematurity is:
c. Macrocytic, normoblastic red blood cell morphology . A subset of the anemia of infancy
d. Macrocytic, megaloblastic red blood cell morphology of . Often complicated by blood loss/frequent blood drawing
. Macrocytosis in liver disease is caused by all of the . Well tolerated
following except: @) a and b
(av

a. Abnormal lipid metabolism oO . all of the above

b. Difect effects of alcohol ibe), Delta check of hemoglobin values for a patient helps with:
QO . Iron deficiency
a. anemia of infancy
d. Vitamin B,, and folate deficiency b. anemia of endocrine disorders
. What are the typical hematologic findings associated c. anemia due to improperly collected laboratory samples
with anemia from endocrine dysfunction? d. anemia due to liver disease/alcoholism
a. Mild normocytic, normochromic anemia
b. Anemia, abnormal platelets, and coagulopathy See answers at the back of this book.

(3) blunted erythropoietin response to anemia, and

~’} SUMMARY CHA (4) decreased red blood cell (RBC) life span.
w Hepcidin, an iron regulatory protein and acute phase reac-
mw Anemias produced by inflammation and systemic diseases
tant, acts to mediate the dysregulation of iron metabolism
are perhaps the most common hematologic abnormalities
in ACD by decreasing iron absorption from the small
encountered in the clinical laboratory; these are generally
intestine and blocking iron release from the RE system;
hypoproliferative anemias characterized by decreased or
hepcidin is increased in response to release of the
normal reticulocyte counts.
cytokine interleukin-6 (IL-6).
mw The four major systems that serve to mediate the inflam-
m Cytokines that have been reported to inhibit erythro-
matory response include the coagulation, complement,
poiesis in ACD include interleukin-1 (IL-1), tumor necro-
fibrinolytic, and kinin systems.
sis factor-alpha (TNF-a), and interferon gamma (INF-y).
m Anemia of chronic disease (ACD) occurs in patients with
w ACD is characterized by a normocytic anemia with a normal
chronic (or occasionally acute) immune activation.
or low mean corpuscular volume (MCV), low serum iron,
# ACD most commonly occurs in patients with (1) chronic decreased total iron-binding capacity (TIBC), and normal to
infections such as osteomyelitis or tuberculosis, (2) chronic increased serum ferritin level; acute phase reactants and the
inflammatory diseases such as rheumatoid arthritis or sys- erythrocyte sedimentation rate (ESR) are usually increased.
temic lupus erythematosus, or (3) malignant tumors such as
m Renal disease may be associated with anemia, abnormal
carcinomas.
platelet and white blood cell function, and coagulopathy.
@ Possible etiologies for the ACD include (1) dysregulation
@ Chronic renal failure is almost invariably associated with
of iron homeostasis (decreased serum iron with ample
reticuloendothelial iron stores), (2) suppression of
significant anemia. This is especially true when the crea-
tinine clearance falls below 20 to 30 mL/min, but anemia
erythropoiesis by cytokines from inflammatory cells,
is also common with more modest renal insufficiency.
continued
 Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases 30]

@ The major causes for the anemia of renal insufficiency are dysfunction, adrenal insufficiency, Addison disease, thyroid
(1) decreased production of erythropoietin by damaged disease, hyperparathyroidism, and hypogonadism.
kidneys, (2) suppression of erythropoiesis by the uremic
m Anemia seen in malignancies is dependent on the type of
toxins that accumulate in renal failure, and (3) slightly to
cancer, the site of infiltration, the patient’s therapy, and
moderately decreased RBC lifespan. Other causes of ane-
the extent of bone marrow involvement.
mia may complicate the anemia of renal insufficiency.
# Myelophthisis refers to infiltration of the bone marrow by
m The anemia of renal insufficiency is hypoproliferative
a malignant tumor; the most characteristic peripheral
(normal to decreased reticulocyte count); it is normocytic
blood morphology associated with myelophthisis is
and normochromic, with the presence of burr cells.
leukoerythroblastosis (the presence of immature myeloid
m Chronic liver disease may show RBC morphology that precursors and nucleated RBCs) with teardrop-shaped red
includes macrocytosis, target cells, acanthocytes, and cells.
thrombocytopenia if hypersplenism is present; a micro-
m= Chemotherapy agents result in a transient pancytopenia
cytic, hypochromic anemia is evident in the presence of
resulting from alkylating agents that are directly toxic to
chronic blood loss with iron deficiency.
rapidly dividing cells.
m The bone marrow in chronic liver disease shows nor- m Anemia associated with HIV infection is characterized as
moblastic or macronormoblastic maturation in RBC a normocytic, normochromic, hypoproliferative anemia
precursors. RBCs have decreased survival because of with a decreased reticulocyte count and dysplastic bone
increased membrane lipids and lack of deformability marrow features; 85% of patients with acquired immun-
(e.g., acanthocytes or “spur cell” anemia).
odeficiency syndrome (AIDS) are anemic.
mwAnemia caused by alcoholism is the result of ethanol m@Anemia of infancy is physiologic and due to the switch
toxicity to precursor cells in the bone marrow. The bone from liver to renal erythropoietin production.
marrow shows macrocytosis, vacuolization of erythrob-
lasts, and abnormal hemoglobinization of erythroid series m Anemia associated with prematurity is a subset of the ane-
precursors. The peripheral blood shows macrocytosis and mia of infancy and even though influenced by erythropoi-
may show spur cells. etin production shift from liver to kidney, is also affected
by blood loss anemia due to or associated with the inten-
w A dimorphic blood picture may be seen in anemia of alco- sive care rendered to many of these premature infants.
holism because of iron deficiency caused by chronic
blood loss; the MCV is normal and the RDW increased.
mw Anemia of improperly collected laboratory samples is
often first suspected by a failed “delta” check on a given
m Anemia associated with endocrine disease is usually nor- patient/patient sample. It may be related to sample dilu-
mocytic and normochromic and may be caused by pituitary tion with intravenous fluids.

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vak, JL (Ed): Hematol Oncol Clin maturity: Factors governing the erythro- 160. Meyer, MP, et al: Recombinant erythro-
North Am 8:945, 1994. poietin response. N Engl J Med poietin and blood transfusion in
126. Phair, JP, et al: Recombinant human 296:647, 1977. selected preterm infants. Arch Dis
erythropoietin treatment: Investigational 144. Brown, MS, et al: Decreased response Child Fetal Neonatal Ed 88:F41, 2003.
new drug protocol for the anemia of the of plasma immunoreactive erythropoi- 161. Avent, M, et al: A comparison of high
acquired immunodeficiency syndrome. etin to “available oxygen” in anemia of versus low dose recombinant human
Overall results. Arch Intern Med prematurity. J Pediatr 105:793, 1984. erythropoietin versus blood transfusion
153326695 1993: 145. Stockman, JA, et al: Anemia of prema- in the management of anemia of prema-
Wie Semba, RD, et al: Highly active anti- turity: Determinants of the erythropoi- turity in a developing country. J Trop
retroviral therapy associated with etin response. J Pediatr 105:786, 1984. Pediatr 48:227, 2002.
improved anemia among HIV-infected 146. Stockman, JA: Anemia of prematurity. 162. Franz, AR, and Pohlandt, F: Red blood
women. AIDS Patient Care STDS Current concepts in the issue of when cell transfusions in very and extremely
15:473, 2001. to transfuse. Pediatr Clin North Am low birthweight infants under restrictive
123: Moore, RD, and Forney, D: Brief Soe MEOS6: transfusions guidelines: Is exogenous
Report: Anemia in HIV-infected 147. Stockman, JA: Anemia of prematurity. erythropoietin necessary? Arch Dis
patients receiving highly active anti- Clin Perinatol 4:239, 1977. Child Fetal Neonatal Ed 84:F96, 2001.
retroviral therapy. J Acquir Immune 148. Lachance, C, et al: Myocardial, erythro- 3. Amin, AA, and Alzahrani, DM: Effi-
Defic Syndr 29:54, 2002. poietic, and metabolic adaptations to cacy of erythropoietin in premature
129. Semba, RD, et al: Improvement of ane- anemia of prematurity. J Pediatr infants. Saudi Med J 23:287, 2002.
mia among HIV-infected injection drug 125:278, 1994. 164. Vamvakas, EC, and Strauss, RG: Meta-
users receiving highly active antiretro- 149. Strauss, RG: Controversies in the man- analysis of controlled clinical trials
viral therapy. J Acquir Immune Defic agement of the anemia of prematurity studying the efficacy of rHuEPO in
Syndr 26:315, 2001. using single-donor red blood cell trans- reducing blood transfusions in the ane-
. Buskin, SE, and Sullivan, PS: Anemia fusions and/or recombinant human ery- mia of prematurity. Transfusion 41:406,
and its treatment and outcomes in per- thropoietin. Trans Med Rev 20:34, 2006. 2001.
sons infected with human immunodefi- 150. Lenes, BA, and Sacher, RA: Blood 165. Strauss, RG: Managing the anemia of
ciency virus. Transfusion 44:826, 2004. component therapy in neonatal medi- prematurity: Red blood cell transfusions
13 — . Sullivan, PS, et al: Epidemiology of cine. Clin Lab Med 1:285, 1981. versus recombinant erythropoietin.
anemia in human immunodeficiency . Strauss, RG, et al: Commentary on Trans Med Rev 15:213, 2001.
virus (HIV)-infected persons: Results small-volume red cell transfusions for 166. Doyle, JJ: The role of erythropoietin in
from the multistate adult and adolescent neonatal patients. Transfusion 30:565, the anemia of prematurity. Semin Peri-
spectrum of HIV disease surveillance 1990. natol 21:20, 1997.
project. Blood 91:301, 1998. Ik, Meyer, MP, et al: Recombinant human 167. Widness, JA, and Strauss, RG: Recom-
. Dallman, PR: Anemia of prematurity. erythropoietin in the treatment of the binant erythropoietin in treatment of the
Annu Rev Med 32:143, 1981. anemia of prematurity: Results of a premature newborn. Semin Neonatol
3. Strauss, RG: Current issues in neonatal double-blind, placebo-controlled study. 3:163, 1998.
transfusions. Vox Sang 51:1, 1986. Pediatrics 93:918, 1994. 168. Brown, MS, et al: Postnatal changes in
. Widness, JA, et al: Erythropoietin Il). Frenck, RW, and Shannon, KM: erythropoietin levels in untransfused pre-
transplacental passage: Review of ani- Hematopoietic growth factors in pedi- mature infants. J Pediatr 103:612, 1983.
mal studies. J Perinat Med 23:61, 1995. atrics. Curr Opin Pediatrics 5:94, 1993. 169. Schaefer, RM, and Schaefer, L: The
135 . Schneider, H, and Malek, A: Lack of per- 154. Ohls, RK, and Christensen, RD: hypochromic red cell: A new parameter
meability of the human placenta for ery- Recombinant erythropoietin compared for monitoring of iron supplementation
thropoietin. J Perinat Med 23:71, 1995. with erythrocyte transfusion in the during rhEPO therapy. J Perinatol Med
136. Malek, A, et al: Lack of transport of treatment of anemia of prematurity. 232835 1995.
erythropoietin across the human pla- J Pediatr 119:781, 1991. 170. Kumar, P, et al: Recombinant human
centa as studied by an in vitro perfusion IIS). Shannon, KM, et al: Circulating ery- erythropoietin therapy for treatment of
system. Pflugers Arch 427:157, 1994. throid progenitors in the anemia of pre- anemia of prematurity in very low birth
. Zanjani, ED, et al: Studies in the liver maturity. N Engl J Med 317:728, 1987. weight infants: A randomized, double-
to kidney switch of erythropoietin pro- 156. Shannon, KM, et al: Recombinant blind, placebo-controlled trial. J Perina-
duction. J Clin Invest 67:1183, 1981. human erythropoietin stimulates ery- tol 18:173, 1998.
. Strauss, RG: Erythropoietin in the thropoiesis and reduces erythrocyte 17 ion. van Straaten HLM, et al: Evaluation of
pathogenesis and treatment of neonatal transfusions in very low birth weight a strategy to limit blood donor exposure
anemia. Transfusion 35:68, 1995. preterm infants. Pediatrics 95:1, 1995. in high risk premature newborns based
. Strauss, RG: Erythropoietin and neona- . Ohls, RK, et al: The effect of erythro- on clinical estimation of transfusion
tal anemia. N Engl J Med 330:1227, poietin on the transfusion requirements need. J Perinatol Med 28:122, 2000.
1994. of preterm infants weighing 750 grams 172. Strauss, RG: How I transfuse red blood
140 . Bauer, C: Erythropoietin: From gene or less: A randomized, double-blind, cells and platelets to infants with the
structure to therapeutic applications. J placebo-controlled study. J Pediatr anemia and thrombocytopenia of pre-
Perinat Med 23:77, 1995. 131:661, 1997. maturity. Transfusion 48:209, 2008.
PART 3
WHITE BLOOD CELL DISORDERS

Chapter

Cell Biology, Disorders

of Neutrophils, Infectious
Mononucleosis, and
Reactive Lymphocytosis
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
joe Marty, MS, MT(ASCP)
Ronald G. Strauss, MD

Neutrophils OBJECTIVES
Neutrophil Function
At the end of this chapter, the learner should be able to:
Disorders of Neutrophils
Eosinophils tie Characterize the changes in neutrophil count and morphology that develop in
response to bacterial infection.
Basophils
. Define the three modes of neutrophilic migration.
Monocytes
Absolute Monocytosis: Oo . Describe the changes in migration pattern and appearance that occur in a neutrophil
Reactive versus Malignant as a result of chemoattractant stimulation.
Causes
. Characterize the sequence of events that occur during phagocytosis.
Lymphocytes
Definition of Lymphocytosis . Define neutropenia.
Lymphocyte Morphology . Identify three causes of acquired neutropenia.
Causes of Reactive
Lymphocytosis OO}
=
a . Describe the etiology and classic clinical features of Chédiak—Higashi syndrome,
Laboratory Examination May-—Hegglin anomaly, Alder—Reilly anomaly, and Pelger—Huét anomaly including
Lymphocytopenia the associated changes in neutrophil morphology.

Case Study 1 . Describe the inheritance of and molecular basis for chronic granulomatous disease.

Case Study 2 . Compare and contrast three white cell anomalies in regard to morphology.

. Define lymphocytosis.

. List several disorders that present with lymphocytosis.

. Distinguish between absolute and relative lymphocytosis.

. Distinguish between benign and malignant lymphocytosis.

. Recognize morphological features of infectious mononucleosis and other reactive

lymphocytoses.

305
306 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

15. List clinical features of infectious mononucleosis.

16. Describe how the Epstein-Barr virus affects the B- and T-lymphocyte populations.
17. Utilize laboratory results for distinguishing infectious mononucleosis from other
lymphocytoses.

Neutrophils Variations in the number and morphology of leukocytes

in the bloodstream and bone marrow have long been used as
Granulocytes may be divided into three subsets—neutrophils, clinical guides for the diagnosis of many diseases. These
eosinophils, and basophils—based on cellular morphology variations reflect the response of normal leukocytes to an
(by light and electron microscopy) and on the staining char- underlying disease, or indicate a primary disorder intrinsic to
acteristics and contents of cytoplasmic granules. Neutrophils leukocytes, such as leukemia. A thorough knowledge of
are the most numerous leukocytes found in the peripheral established normal values and morphological characteristics
blood, accounting for 50% to 70% of all circulating white of all cellular elements is important for recognizing and
blood cells (WBCs) in the adult. Similar to monocytes, neu- interpreting unexpected findings.
trophils function as phagocytes that are capable of ameboid
movement into the tissues to engulf and destroy bacteria or
fungus; they are the first phagocytic cells to mobilize at a site Neutrophil Function
of infection. Neutrophils also play a role in mediating inflam-
The main function of the neutrophil is the internalization of
matory processes.
microorganisms for destruction, a process referred to as
Mature neutrophils (or polymorphonuclear leukocytes)
phagocytosis. Once bacteria infiltrate the tissues, neu-
are easily recognized on Wright’s-stained peripheral blood
trophils are stimulated for immediate action. For the pur-
smears by their dense multilobed nucleus and pinkish-tan
poses of discussion, phagocytosis is described as occurring
cytoplasm, which is peppered with pinkish-purple granules.
in three distinct phases: migration and diapedesis; opsoniza-
They are smaller than their myeloid precursors, and thus
tion and recognition; and ingestion, killing, and digestion
more mobile and deformable. Two types of cytoplasmic
(Fig. 15-1).
granules, primary (azurophilic or nonspecific) and sec-
ondary (specific), are present in the mature neutrophil;
MIGRATION AND DIAPEDESIS
however, only the secondary granules are visible with light
Neutrophils in the marginating pool roll along vessel endothe-
microscopy. A third type of granule (tertiary) has been iden-
lium in a random and nondirectional pattern called locomotion,
tified using electron microscopy.'? The contents of these
until a site of injury or infection is encountered. Bacteria and
primary, secondary, and tertiary granules are enzymes, most
sites of inflammation in the host send out signals in the form of
of which are involved in the killing and digestion of bacte-
chemoattractants (Table 15—2), which stimulate changes in
ria and fungi (Table 15-1).
morphology and migration pattern.* The chemoattractant forms
Neutrophils are considered to be one of the most mobile
cell lines in humans.' Once in the bloodstream, mature neu-
trophils are equally divided into marginating and circulating
pools between which there is a constant exchange of cells.
The marginating pool consists of cells adhering to vessel
endothelium within the vascular spaces. Marginating cells
are described as rolling along vessel endothelium in search of Primary Granules
an area of injury or inflammation, where, by diapedesis, they Lysozyme
enter the tissues for action. Neutrophils in the circulating Myeloperoxidase
Acid phosphatase
pool leave the blood by random migration after a half-life Elastase
of approximately 7 hours and do not return to the blood-
stream from tissues. Little is known of the kinetics of Secondary Granules
neutrophils after having entered the tissues; they are believed Lysozyme
NADPH oxidase
to remain in tissues for 2 to 5 days, where, if not used in an
Cytochrome b
inflammatory process, they die or are destroyed by other Lactoferrin
phagocytic cells. The bone marrow quickly replaces the neu-
trophils that have exited the bloodstream with cells from the Tertiary Granules
bone marrow storage pool. The daily production of neu- Plasminogen activator
Alkaline phosphatase
trophils is approximately 1 X 10"/L in adults, with 20% Gelatinase
remaining in circulation. !
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 307

, Blood vessel
with PMN
Activation
Diapedesis
_ Neutrophil Migration
Chemotaxis
= —> pyioge ofMigration _ Characteristics
{Ee ) Endothelial
naMN fe \ ceils Locomotion Cot Nondirectional
Chemokinesis Nondirectional acceleration
> d
322 NS
2 i
&
| of migration speed
ee i PMN Opsonization Chemotaxis Directional
Bacteria
(mS 7 »)
fira |’ Pa aia Att achment
ANNA, =

% y as y een © a Ingestion OPSONIZATION AND RECOGNITION

we ff lias Killing Migrating neutrophils cannot efficiently recognize and attach
\ Digestion to most microorganisms. A mechanism referred to as
opsonization facilitates recognition and attachment by mark-
Figure 15-1 m@ Illustration of the phases of neutrophilic phagocy- ing the organism for ingestion. The term opsonin 1s of Greek
tosis, which include activation, diapedesis, chemotaxis, opsoniza- origin and literally means “to prepare for dining.” Although
tion, ingestion, killing, and digestion. PMN = polymorphonuclear chemotaxis is taking place, circulating immunoglobulin and
neutrophil. (From Abramson, JS, and Wheeler, JG: The Natural
activated complement components coat the surface of the
Immune System—The Neutrophil. Oxford University Press, Oxford,
1993, p 110, with permission.) bacteria. The marked bacteria, referred to as an opsonin, is
readily recognized and ingested by the neutrophil. Ingestion
will not take place without the presence of membrane-bound
a concentration gradient, whereby the neutrophil migrates immunoglobulin. The plasma membrane of the neutrophil
toward the area of highest concentration. A re-engineering of carries receptors for the Fc fragment of immunoglobulin G
the plasma cell membrane occurs that transforms the neu- (igG) molecules and activated complement only. Proteins that
trophilic shape from smooth and round to ruffled and flat, with most effectively mark bacteria for recognition and attachment
pseudopods and a tail (Fig. 15-2). Under the stimulus of are IgG1, IgG3, C3b, and C3bi.°
chemoattractants, the activated neutrophil migrates in a repeti-
tive, wavelike motion to the site of infection. The process of PHAGOCYTOSIS: INGESTION, KILLING,
directional migration under the guidance of chemoattractants is AND DIGESTION
known as chemotaxis. The neutrophil adheres to endothelial The ingestion of the opsonized microbe begins as soon as the
receptors and, by diapedesis, penetrates through narrow junc- membrane surface receptor of the neutrophil and microbe
tions between endothelial cells into the tissues. In diapedesis, bind together. Membrane pseudopods extend around and
the neutrophil is briefly retained by the vascular basement envelop the microbe, forming an isolated vacuole within the
membrane, but then enters the tissues by passing through small neutrophil cytoplasm known as a phagosome. Simultane-
openings in this membrane. Chemoattractants further acceler- ously, cytoplasmic granules migrate to and fuse with
ate the rate of neutrophil migration by a process known as the membrane of the phagosome, forming a phagolysosome
chemokinesis.* With neutrophils being the first phagocytes to (Fig. 15-3). Once fusion is complete, the cytoplasmic gran-
migrate to the site of infection, the lag time between microor- ules undergo degranulation, whereby their contents are
ganism invasion and timely neutrophilic migration is crucial released into the phagolysosome. In vitro studies suggest an
for limiting the spread of (or even preventing) infection.’ All orderly sequence of events for phagolysosome formation,
three modes of neutrophilic migration contribute to the efficient with the degranulation of secondary granules preceding that
mobilization of neutrophils to the site of injury (Table 15-3). of the primary granules.° The ingested organism is exposed to

Round cell Elongated cell

1 Chemical Factors that

; Table 15-2
Signal Neutrophil -
_ Activation:

Chemontiractans LER

N-formyl Nicoeeoudes, cena

C5a, C3b, and C3bi factors Complement Figure 15-2 ™ The shape change of the neutrophil, from smooth
Interleukin-8 Monocyte o and round to ruffled and flat with pseudopods, is a result of stimu-
Leukotriene B, Membrane phospholipid lation by chemoattractants. (Adapted from Kaplan, S, and Brams, CA:
Platelet-activating factor Endothelium Testing for Neutrophil Function. Advance, 7[1], 1995, p 8, with
permission.)
308 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

opsonized particle.' A deficiency or dysfunction in any one or

a combination of these processes modifies neutrophil function.

QUANTITATIVE DISORDERS
NEUTROPHILIA Neutrophils play a central role in removing
infectious and inflammatory agents that challenge host immu-
nity. Within minutes, neutrophils are stimulated to migrate
from circulation through junctions between endothelial cells
to the site of infection or inflammation. The classic response
to infectious and inflammatory processes is an increase in the
relative number of neutrophils, termed neutrophilia. The
accelerated release of neutrophils from the bone marrow
reserve is accompanied by a “shift to the left” that is defined
as an increased number of metamyelocytes and band forms in
Figure 15-3 m Electron microscopy of phagolysosome formation.
(A) Staphylococci lie within phagocytic vesicles limited by sacs formed the circulating pool.? An increase in circulating neutrophils
from inverted pieces of the neutrophil membrane. Cytoplasmic gran- and immaturity is similarly observed in the early stages of
ules are approaching the phagocytic vesicles. (B) Higher magnifica- neoplastic conditions, such as chronic myeloid leukemia
tion shows degranulation with the discharge of granule contents (CML), and other chronic myeloproliferative disorders (see
into the vicinity of the staphylococcus.
Chaps. 17 and 18). The major distinction is that the myeloid
precursors released during the infectious response are more
limited to the metamyelocyte and band stages; whereas, in
the lytic activity of granular enzymes within the phagolyso- neoplastic processes, earlier precursor cells such as myelo-
some, which leads to eventual killing and digestion. cytes, promyelocytes, and blasts are also present. Table 15—4
Microorganism destruction (killing) is accomplished by lists the causes of secondary reactive neutrophilia. Leukocyte
oxygen-dependent and non-oxygen-dependent mechanisms.
alkaline phosphatase (LAP), by cytochemical staining, is use-
The non-oxygen-dependent mode of killing the internalized ful for differentiating neutrophilic response to infection
particle is represented by an alteration in pH and the release
(leukemoid reaction) from chronic myeloid leukemia, where
of the lysosomal and proteolytic enzymes into the phagolyso-
the LAP is increased in leukemoid reaction and decreased in
some.' The lytic enzymes possess bacteriocidal activity that
cleaves segments of the bacterial cell wall. Alternatively,
oxygen-dependent killing involves the release of nictinomide
adenine dinucleotide phosphatase (NADPH) oxidase, which
mediates the production of the active oxygen metabolites
superoxide and hydrogen peroxide during a process known as
the respiratory burst.° These active oxygen metabolites are
capable of microorganism injury. To enhance the antibacterial
activity further, superoxide and hydrogen peroxide react with
other products of phagocytosis such as myeloperoxidase
(in primary granules of neutrophils and monocytes) to pro- ¢ Infections
duce highly toxic agents such as hypochlorous acid (i.e., 1. Bacterial (most common)
household bleach).’* 2. Toxoplasma, mycobacterium, leptospiral, and viral (less
common)
¢ Inflammatory Disorders
¢ Tissue Necrosis
Disorders of Neutrophils
1. Burns
2. Trauma
Disorders of the neutrophilic cell line may predispose an
3. Infarct
individual to recurrent bacterial infections that are resistant to 4. Acute gout
treatment. Neutrophilic disorders are classified as quantitative ° Metabolic
and qualitative, reflecting changes in neutrophil number and 1. Uremia
function, respectively. Quantitative disorders may present a 2. Ketoacidosis
3. Eclampsia
significant decrease (or increase) in the absolute neutrophil
* Other Causes Unrelated to Infection or Inflammatory
number and are referred to as neutropenia and neutrophilia, Process
respectively. Neutropenia limits the potential for effective 1. Stress
mobilization and killing at the site of microbial invasion.? 2. Rigorous exercise
Alternately, qualitative disorders are marked by neutrophil 3. Smoking
dysfunction as a result of impaired migration or altered 4. Pregnancy
5. Trauma
bacteriocidal activity. As noted, phagocytosis occurs in a com-
6. Acute hemorrhage or hemolysis
plex sequence of events ranging from activation, migration, 7. Postsplenectomy
and diapedesis to the recognition, ingestion, and killing of an
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 309

CML (see Chap. 17). The kinetics of circulating neutrophils

Say
varies greatly depending on the type, duration, and intensity AS
)
%
of the infection. The immediate response to infection is tran- 4
*
Yas
sient neutropenia, resulting from increased margination and
accelerated delivery of neutrophils to the infected site. Within
an hour, neutrophils are released from the bone marrow
reserve into the bloodstream. In the early phases of infection,
the circulating half-life of neutrophils is shortened and cell
turnover is accelerated.'* Later, the circulating half-life
returns to normal.
In addition to changes in neutrophil number, reactive
changes in neutrophilic morphology may be observed in
infections, which is termed leukemoid reaction. Leukemoid
reaction is characterized by the presence of toxic granulation,
Dohle bodies, and cytoplasmic vacuolization in the neutrophils
(Figs. 154 to 15-6). Toxic granulation is frequently associated Figure 15-5 @ Dohle bodies (arrows). Note the large bluish bodies
with severe infection in which the cytoplasmic granules enlarge in the periphery of the cytoplasm.
and take on darker staining properties than normal. The toxic
granules have been identified as peroxidase-positive primary pregnancy, massive trauma, inflammatory processes, drug reac-
granules. Toxic granulation may also be accompanied by the tions, and other toxic states."
presence of Dohle bodies (1.e., pale blue inclusions at the periph- The function of circulating neutrophils may also be
ery of the cytoplasm). They consist of a few strands of rough affected by infection.'° Both enhanced and impaired functions
endoplasmic reticulum that have aggregated. Dohle bodies are have been reported when compared with normal values in stud-
similar, but not identical, to the inclusions found in the ies performed during infections.'* Despite the fact that excep-
May—Hegglin anomaly (a hereditary leukocyte and platelet dis- tions exist, itis generally accepted that mild infections enhance
order; discussed later in this chapter). Lastly, in response to neutrophil function, whereas severe infections impair neu-
infection, the cytoplasm may become vacuolated and, occasion- trophil function.'* The role that these acquired functional
ally, contain ingested microorganisms. All three of these abnormalities play in the course of infections is unknown.
morphological features are commonly observed, with toxic
granulation being reported as the most common morphological NEUTROPENIA
change in response to bacterial infection, followed by Dohle Neutropenia is defined as an absolute decrease in the number
bodies and cytoplasmic vacuolization.'' In addition, the pres- of circulating neutrophils and is suspected when patients pre-
ence of one or more of these reactive changes suggests progres- sent with absolute neutrophil counts of less than 1.5 xX
sion toward sepsis.’ The characteristics of leukemoid reaction 10°/L."' Absolute counts can be obtained by multiplying the
are listed in Table 15-5. total WBC count by the percentage of neutrophils (and bands)
Although these morphological features are viewed to be a seen in the differential cell count. The low neutrophil count is
clinically sensitive tool for predicting severe infection, they are not the sole indicator of disease and should be correlated with
not entirely specific to infection.’ All of these reactive changes patient history as well as clinical and laboratory findings. The
have occasionally also been observed during uncomplicated normal level of circulating neutrophils varies with age and

Figure 15-4 m Toxic granulation (peripheral blood). Note the Figure 15-6 ®@ Vacuolated neutrophils suggesting the presence of
prominent dark-staining granules. infection or a severe inflammation.
310 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Table 15-5 Characteristics of “Table 15-7 Pathgoenriaof

Leukemoid Reaction - _ Neutropenia —

¢ Toxic granulation of neutrophil * Increased Destruction or Removal ofNone enile

¢ Vacuolization of neutrophil 1. Infections
¢ Dohle bodies in the cytoplasm of neutrophils 2. Immune disorders
¢ “Shift to the left” increase in bands and metamyelocytes ¢ Maturation Defect
most often; myelocytes and other younger forms may also 1. Megaloblastic anemia
be seen Proliferation Defect
* Leukocytosis usually greater than 30,000/mm* (>30 10°/L); 1. Aplastic anemia
may be higher in young children; may be greater than 2. BM replacement disorders
50,000/mm* (0 x 10°) i in patients Teceiving CSF therapy _ 3. Idiosyncratic drug reactions
4. Myeloablative therapy
CSF = poleny-cammlatine Opto 5. BM fibrosis
¢ Abnormal Distribution
1. Hypersplenism

race, and refers to mature polymorphonuclear and band

forms, only. Recurrent bacterial infections are the hallmark
of persistent neutropenia, with the clinical severity being counseling family members in congenital cases. The patho-
reflected by the absolute neutrophil count as well as the fre- genesis of neutropenia is listed in Table 15-7.
quency and duration of neutropenic episodes.''! Neutropenia
can range from mild, with absolute counts from 1.0 to 1.5 < ACQUIRED NEUTROPENIA Most acquired neutropenias
10°/L, to moderate, with counts from 0.5 to 1.0 x 10°/L, to occur as transient conditions caused by factors extrinsic to the
severe, with counts less than 0.5 < 10°/L.° Life-threatening bone marrow. In order of frequency, concomitant viral infec-
infections are not generally observed until blood counts fall tion, the ingestion of certain medications, and alloantibody or
below 0.2 X 10°/L (Table 15-6)." autoantibody activity are all reported to be causes. (The drugs
Infections in the neutropenic patient are most commonly associated with causing neutropenia are listed in Table 15-8.)
caused by endogenous normal flora such as Staphylococcus Neutropenia may also be acquired as a secondary condition to
aureus, Streptococcus viridans, and gram-negative enteric processes such as aplastic anemia, malignancy of the bone
organisms.'? Although neutropenia increases host susceptibil- marrow, and dietary B,, or folate deficiency.'' The causes of
ity to bacterial infection, the risk of viral, fungal, and parasitic acquired neutropenia are listed in Table 15-9.
infection is not increased. Most infections in the neutropenic The patient’s age and race must both be considered in
individual occur in the cutaneous and soft tissues. The major diagnosing neutropenia. Newborns and infants have a higher
concern for these infections is the spread of the organism absolute neutrophil count; therefore neutropenia is defined as an
from the localized site into the bloodstream, resulting in a absolute count of less than 2500/mm}? or 2.5 10°/L. In addi-
superinfection such as septicemia. tion, approximately 25% of black children and black adults have
Neutropenic disorders are described as acquired or con- an absolute neutrophil count of 1.0 to 1.5 X 10°/L."
genital. Persistent cases of neutropenia are generally attrib-
uted to an intrinsic problem of the hematopoietic system, Infection
whereas transient conditions are linked to factors extrinsic to Viral infections are recognized to be the most common cause
the bone marrow. The absolute reduction in the circulating of acquired neutropenia, especially in children younger than
number of neutrophils may be attributed to increased periph- the age of 6. The neutropenia appears during the first few days
eral destruction, decreased production (proliferation defect), of illness, when the peak level of virus is reached and lympho-
impaired bone marrow release (maturation defect), or abnor- cytosis develops. Viruses implicated in inducing neutropenia
mal distribution. Identification of the cause is important for
selecting appropriate therapy, monitoring prognosis, and

‘Table,15-6 Classification of
_ Neutropenia — Drug Class Drug Prototype

Seyerty” Count Antibiotics Penicillin

Chloramphenicol
Mild 1000—1500/mm? Anti-Inflammatory Ibuprofen
(1.0-1.5 X 107/L) Anticonvulsants Phenytoin
Moderate 500-999/mm? Antithyroid Propylthiouracil
(0.5-1.0 X 10°/L) Cardiovascular Procainamide
Severe < 500/mm°* Hypoglycemic Chlorpropamide
(< 0.5 X 10°/L) Tranquilizer Phenothiazine
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 31]

IgG, IgM, or IgA immunoglobulin class, with the NA1 speci-

ficity being most commonly observed. Autoimmune neutrope-
nia has been identified in both adults and children, although it
is observed more frequently as the cause of chronic benign neu-
tropenia of childhood.'® Although no standard treatment has
been established, antibiotics are used to treat specific bacterial
¢ Infections
infections, and prednisone to limit the autoimmune response.
* Idiosyncratic drug reactions
¢ Autoimmune disorders
* Immunodeficiency disorders Congenital Neutropenia
¢ Hypersplensism Congenital or chronic neutropenias occur as persistent or
¢ Aplastic anemia intermittent disorders arising from an inherited abnormality in
¢ Megaloblastic anemia
cells of the myeloid line or those involving hematopoietic
* Malignant disorders replacing the BM
regulation'® (Table 15-10). The defective or deficient produc-
tion is intrinsic to the bone marrow microenvironment. Con-
sequences of these defects result in decreased or arrested cell
include influenza A and B, rubella, rubeola, herpes simplex, production, or impaired release by the bone marrow. These
hepatitis A and B, and respiratory syncytial virus. The accom- disorders are not caused by factors extrinsic to the bone mar-
panying neutropenia appears within 24 to 48 hours of the row such as infection, drug ingestion, or antibodies. Most are
onset of the viral infection and may persist during the acute extremely rare and genetically heterogeneous.'® Clinical pre-
viremic period.’ sentation varies from a mild, if any, increased risk of infection
(as seen in chronic benign neutropenia) to the overwhelming,
Immune-Mediated Neutropenia recurrent infections in severe congenital neutropenia that
Neutropenia may result from the activity of anti-neutrophil result in death within the first year of life. In severe congeni-
antibodies that mediate the removal of opsonized neutrophils tal cases, clinical benefits have been seen with the admuinistra-
from circulation. Immune-mediated neutropenia can be tion of granulocyte-colony-stimulating factor (G-CSF) in
caused by either alloantibody or autoantibody when patients terms of neutrophilic increment and infection prophylaxis."
present with absolute neutrophil counts less than 0.5 * 10°/L
and recurrent infections of mild to moderate severity.'° QUALITATIVE DISORDERS OF NEUTROPHILS
Alloimmune neonatal neutropenia has been described as DISORDERS OF NEUTROPHIL FUNCTION Qualitative dis-
being analogous to red cell hemolytic disease of the newborn. orders of neutrophil function are characterized by bacterial
The neutropenia develops after maternal sensitization to fetal infections that are caused by hereditary abnormalities in func-
neutrophilic antigens. The antigens are shared by the fetus tion. The occurrence of most qualitative conditions is
and the father, but are absent in the mother. During pregnancy, extremely rare; many are familial and stem from a general
anti-neutrophil antibody production is stimulated in the metabolic defect. The exact pathophysiology of nearly all
maternal immune system, and these IgG antibodies cross the qualitative neutrophilic disorders is unknown. Neutropenia
placenta, destroying fetal neutrophils. The anti-neutrophil commonly accompanies these disorders. The qualitative dis-
antibodies are directed against neutrophil-specific antigens, orders are classified according to the inajor defect expressed
such as NA1I, NA2, and NB1, or HLA antigens that are shared (1.e., cytoplasmic granules, disturbances of the respiratory
by neutrophils and other nucleated cells. Alloimmune neu- burst, or chemotaxis); however, multiple abnormalities
tropenia is encountered in approximately 3% of all live (including neutropenia) may be observed in some patients
births?; the condition is more common in firstborn children. (Table 15-11).
Severe neutropenia may persist for an average of 7 weeks or Functional defects of neutrophils, which can be acquired
until the anti-neutrophil antibodies are cleared from neonatal or inherited, are classified by the general type of defect:
circulation.” After this time, the neutrophil count returns to the (1) phagocytic/killing defects, (2) motility/chemotaxis defects,
normal range expected for this age group. Alloimmune (3) granule function and structure defects, and (4) adhesion
neonatal neutropenia may be accompanied by cutaneous
infections and infrequently progresses into respiratory infec-
tion and life-threatening sepsis.* Therapy is usually support-
ive and consists of antibiotics when infections occur.
Autoantibodies directed against neutrophils have also
been detected in the serum of some neutropenic patients.'* Chronic benign neutropenia
Survival studies utilizing radiolabeled techniques demonstrate Severe congenital neutropenia (Kostman’s)
that neutrophils coated by autoantibody exhibit a shortened Myelokathexis
Cyclic neutropenia
half-life in circulation.! Clinically, the condition is classified as Reticular dysgenesis
primary autoimmune neutropenia of idiopathic origin, or as Fanconi’s anemia
secondary to disease states such as Felty’s syndrome, rheuma- Dyskeratosis congenita
toid arthritis, systemic lupus erythematosus, and chronic Shwachman—Diamond syndrome
hepatitis.'* The anti-neutrophil autoantibodies may be of the
312 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Table 15-11 Classes of Qualitative Table 15-12 Classification of ==

Neutrophil Disorders Disorders of Neutrophil —
and Related Conditions Functions that CanBe
~ Inherited or Acquired
Cytoplasmic Granules
Chediak—Higashi syndrome ¢ Phagocytic/killing defects
Myeloperoxidase deficiency ¢ Chemotaxis/motility defects
Specific granule deficiency e Granule function and structure defects
¢ Adhesion defects
Biochemical Disturbance of Respiratory Burst
Chronic granulomatous disease
Glucose-6-phosphate dehydrogenase deficiency
Glutathione deficiency

Chemotaxis Two special laboratory tests are generally ordered to

Lazy leukocyte syndrome
Monosomy
evaluate neutrophil function: the nitroblue tetrazolium dye test
(NBT) and the myeloperoxidase stain. The NBT test is used to
indirectly detect the production of superoxide by neutrophils.
When neutrophils contain the blue-black precipitate, a positive
defects'' (Table 15-12). The acquired defects in each category reaction is recorded. Each laboratory should establish its own
are listed in Table 15-13. The inherited disorders of neutrophil normal ranges. It should be noted that ethylenediamine
function are listed in Table 15—14. WBC counts are variable in tetraacetic acid (EDTA) and heparin samples cannot be used to
neutrophil function defects depending on the nature of the dis- perform the NBT test. Although the NBT test is frequently
orders and whether the patient is actively infected. Examination ordered, the flow cytometry method that employs fluorescence
of the peripheral blood smear is generally unremarkable except detection methods to measure oxidant production is rapidly
for the very large granules that characterize Chediak-Higashi replacing this test.'' For the review of the myeloperoxidase
syndrome, and the bilobed nuclei with abnormal nuclear mem- stain, refer to Chapter 36.
branes and absent secondary granules in neutrophil-specific For practical purposes, the most clearly understood qual-
granule-deficiency disorders. itative disorders are addressed here.

Table 15-13 Acquired Disorders of Neutrophil Function —

Phagocytic/Killing Defects Chemotaxis and Motility Defects
. Chronic infections .
Autoimmune disorders
. Autoimmune disorders .
Numerous drugs and anti-inflammatory medications
. Diabetes .
Diabetes
. Cirrhosis .
Cirrhosis
. Splenectomy Renal failure
GVHD . Chronic infections
. Malnutritution GVHD
. Sickle cell anemia Thermal injury
. BM transplant CHNIAMWAWN>
Malnutrition
-—_ . Thermal injury 10. PNH
11. BM transplant
Granule Function and Structure Defects 12. Hematologic malignancies
1. Myeloid malignancies (CMPD, AML, 13. Recombinant interleukin-2 therapy
myelodysplastic syndrome)
2. Pregnancy Adhesion Defect
3. Severe thermal injury . Aging
4. Trauma and surgery . Drugs (1.e., aspirin, corticosteroids, epinephrine)
. Chronic infections
. Renal disorders
. Alcoholism
. Sickle cell anemia
. Paraproteinemia
. Diabetes
CONDNHDP
WN

GVHD = graft-versus-host disease; BM = bone marrow; CMPD = chronic myeloproliferative disorders; AML
= acute
myelocytic leukemia; PNH = paroxysmal nocturnal hemoglobinuria.
 fects Motility/Chemotaxis Defects
1. Chronic granulomatous disease 1. Lipid storage diseases (1.e., Gaucher’s)
2. Immunodeficiency disorders with 2. Complement disorders
decreased immunoglobulins 3. Chediak—Higashi syndrome
3. Complement disorders 4. Specific granule deficiency
4. Myeloperoxidase 5. Actin and microtubular defects
5. Specific granule deficiency 6. Hyperimmunoglobulin E syndrome

Granule Structure and Function Adhesion Defects

1. Chediak—Higashi syndrome 1. Type 1, 2, and 3 leukocyte adhesion
2. Myeloperoxidase deficiency deficiencies
3. Specific granule deficiency

Chediak—Higashi Syndrome phagocytic activity. The release of lysosomal enzymes is

Chediak—Higashi syndrome (CHS) is a rave autosomal-reces- impaired by the abnormal granule membrane fusion that results
sive multisystem disease featuring recurrent bacterial infections, in the characteristic giant lysosomal granules. These abnormal
oculocutaneous albinism, and the presence of giant lysosomal granules develop during early myelopoiesis because of an initial
granules in cells such as granulocytes, monocytes, lymphocytes, aggregation of primary granules followed by fusion with the
melanocytes, tissue macrophages, and platelets.'' Mild bleeding secondary granules (see Fig. 15-8). The giant lysosomal gran-
tendencies are frequently observed related to platelet granule ules are more evident in cells of the bone marrow than in periph-
defects (Figs. 15—7 and 15-8).'* Progressive neurologic compli- eral white cells. In circulation, the most striking granules may be
cations develop during childhood, with moderate neutropenia seen in natural killer cells (NK subset of lymphocytes). The
and thrombocytopenia accompanying nearly all cases.'' Many granules stain from dark red to deep purple with Wright’s stain
cells die within the bone marrow due to the defective cytoplas- and are strongly positive for peroxidase.
mic granulation. Defective chemotaxis (impaired motility) and Management of CHS, before the onset of the accelerated
defective degranulation are all characteristic of the abnormal phase, includes prophylactic antimicrobial therapy and high
cytoplasmic granulation found in granulocytes and monocytes daily doses of ascorbic acid.’ In the case of infection, aggres-
in Chediak—Higashi syndrome.’ Many of these patients die sive intravenous treatment is required. The Epstein-Barr virus
as a result of infection during early childhood. Those who (EBV) is believed to trigger the accelerated phase of CHS, so
survive into early childhood enter an accelerated phase that immunization against EBV may be beneficial.’ Bone marrow
progresses through pancytopenia, lymphoma-like cell infiltra- transplantation (BMT) holds the only hope for cure, with suc-
tion, organomegaly, systemic infections, and eventually death. cess being reported in a small number of patients. Optimally,
Staphylococcus aureus accounts for the majority of infections.’ BMT should be performed before the onset of the accelerated
In addition to the neutropenia, inefficient and prolonged phase.'®
bacterial killing has been identified as a key dysfunction in

Figure 15-8 @ Neutrophil from a patient with Chediak—Higashi

Figure 15-7 a Peripheral blood from a patient with Chediak-Higashi syndrome. The cytoplasm is filled with strikingly large primary
syndrome. (Right) Lymphocyte. (Left) Neutrophil. (From Dutcher, T: (azurophilic) granules. (From Dutcher, T: Hematology. In Listen, Look,
Hematology. In Listen, Look, and Learn, Health and Education and Learn. Health and Education Resources, Inc., Bethesda, MD, with
Resources, Inc., Bethesda, MD, with permission.)
permission.)
314. Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Chronic Granulomatous Disease present with deficiencies in one of the cytosol subunits (see
Chronic granulomatous disease (CGD) is a familial, heteroge- Table 15-15). Regardless of the subunit defect, superoxide
neous disorder of the neutrophil that may be chronic or inter- cannot be produced, and, in turn, bacterial killing is ineffec-
mittent. The mode of inheritance of most cases is X-linked tive.2* Ingestion of bacteria, degranulation, and phagolyso-
recessive; however, autosomal recessive cases have been some formation are normal.
described. CGD is the best-understood disorder of neutrophils. Diagnosis is established by demonstrating a bacterio-
It is seen in | out of every 500,000 individuals who exhibit cidal defect resulting from the absence of the oxidative
recurrent bacterial and fungal infections, generally during the burst on the nitroblue tetrazolium test (NBT), a luminol-
first year of life.!? The congenital abnormality is attributable to enhanced assay, or by measuring respiratory burst activity
a failure in the activation of the respiratory burst that results in with flow cytometry.*! The X-linked-recessive inheritance
little or no superoxide production and is the result of mutations may be confirmed by studying the family history and
in the genes encoding the subunits of the enzyme, NADPH performing molecular gene analysis.” Indicators of CGD
oxidase.”’ The characteristic clinical picture of these patients is include the presence of disease in male members of
of lymphadenitis, deep tissue infections such as osteomyelitis, the maternal family and intermediate to low neutrophil
and visceral and hepatic abscesses, recurrent pulmonary infec- activity in the mothers and sisters of affected boys. In most
tions, organomegaly, and infected eczematoid rash.*'** CGD cases, the female relatives are clinically well, but occasion-
patients exhibit neutrophilia rather than neutropenia. The hall- ally they may present with an increased susceptibility to
mark of CGD is the formation of granulomas during chronic infections or a syndrome resembling systemic lupus
inflammatory reactions that keep the organisms localized. erythematosus.
Life-threatening infections are often caused by Aspergillus, S. Progress has been made in modalities of treatment and
aureus, and enteric organisms.” the prognosis of CGD is improving. Aggressive prophylac-
The first cases of CGD were observed in boys presenting tic antibiotic therapy should be initiated as soon as a diag-
with severe bacterial infections by the first year of life, and were nosis is made. Gamma (y) interferon has been effective in
believed to be inherited as a sex-linked recessive disorder. Most limiting the frequency of infections, and granulocyte trans-
of these patients died of septicemia or chronic pulmonary disease fusions are useful in patients who respond poorly.*? Bone
in early childhood. Additional cases of CGD were identified in marrow and cord blood stem cell transplantation has been
girls who exhibited separate but genetically similar defects. It is proven to be successful when performed in children, even
now recognized that CGD may be inherited in a sex-linked- during ongoing infection; however, the rate of success is
recessive or autosomal-recessive pattern, depending on the sub- dependent on the HLA-match and supportive therapy with
unit of NADPH oxidase affected* (Table 15—15). both G-CSF and granulocyte infusions.*° In vitro studies of
Chronic granulomatous disease results from one of four gene correction for CGD have been carried out using
single gene mutations in NADPH oxidase, which is composed peripheral blood progenitor cells, making the prospect of a
of oxidase components known as phagocytic oxidase subunits, cure most encouraging.”!
or phox.*® The interaction of these subunits results in the for-
mation of superoxide during the respiratory burst. Each phox DISORDERS OF ABNORMAL NEUTROPHIL MORPHOLOGY
subunit is identified as a glycoprotein (gp) or a protein (p). Granulocytes with abnormal morphology can be acquired or
Two of the subunits, gp91-phox and p22-phox, are located in inherited. The acquired disorders of neutrophil morphology
the plasma membrane and together form the enzyme are listed in Table 15-16. Although rare, several hereditary
cytochrome b. The other subunits, p47-phox, p67-phox, and abnormalities in neutrophil morphology may be observed in
p40-phox, reside in the cytosol. Stimulation of NADPH oxi- patients without an association with infection, neutrophilic
dase leads to the migration of the cytosol subunits to the dysfunction, or altered neutrophil number. Such abnormali-
plasma membrane.”’ With the help of secondary mediators, the ties in neutrophilic morphology are collectively referred to as
phox subunits assemble into the active oxidant, superoxide.?’ “white blood cell anomalies”. The inherited disorders of
The classic X-linked form demonstrates a total absence of neutrophil morphology are listed and their characteristics
cytochrome b; whereas, most autosomal-recessive cases summarized in Table 15-17.
Hypersegmentation and hyposegmentation of the
nucleus are WBC anomalies that reflect the number of
segmented lobes demonstrated in the mature neutrophil.
Table 15-15 Molecular Basis of Hypersegmentation describes larger-than-normal neutrophils
~ Chronic Granulomatous with six or more nuclear lobes present (see Chaps. 5 and 7).
Disease A similar hypersegmentation of eosinophils has been
reported, with five or more nuclear lobes present. Hyperseg-
Cytochrome b Mode of Neutrophil Structure mentation in the granulocytes may be an indicator of an
Subunit Affected Inheritance Inveae acquired anemia such as megaloblastic anemia, or of a
p47-phox
benign autosomal-dominant condition known as hereditary
Autosomal Cytosol
p67-phox Autosomal Cytosol
hypersegmentation of neutrophils.*?
gp9 1-phox X-linked Plasma membrane Hyposegmentation of the nucleus is characteristic of
p22-phox Autosomal Plasma membrane Pelger—Huét anomaly, a condition in which the nucleus is
found to be bilobed or to have no lobulation whatsoever."!
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis SS

¢ Hypersegmentation
1. Megaloblastic anemia
2. Myeloid malignancies (i.e., AML, myelodysplastic
syndromes)
¢ Hypogranularity
1. Myeloid malignancies (i.e., AML, myelodysplastic
syndromes)
¢ Pseudo-Pelger—Huét
1. Myeloid malignancies (i.e., AML, myelodysplastic
syndromes)
2. Drugs (sulfonamides, colchicines, etc.)
¢ Cytoplasmic Inclusions Figure 15-9 @ Pelger-Huét anomaly (peripheral blood). (From
1. Cryoglobulinemia Hyun, BH, et al: Practical Hematology. A Laboratory Guide with
* Giant Neutrophils Accompanying Filmstrip. WB Saunders, Philadelphia, 1975, with
1. AIDS infection permission.)
2. G-CSF therapy

G-CSF = granulocyte colony stimulating factor.

Care must be taken to distinguish Pelger—Huét cells from a
“shift to the left,” in which an increase in metamyelocytes and
bands is observed during severe infection. Generally the
Moreover, nuclear chromatin is exceptionally coarse and nuclear chromatin is more condensed and coarse in
condensed. In the heterozygous state, predominantly bilobed Pelger—Huét neutrophils than in bands and metamyelocytes.
neutrophilic forms are present that are described as having a Further, in Pelger-Huét anomaly, more than 70% to 90% of
“dumbbell” or “pince-nez” appearance with two symmetric all neutrophils are affected.?
lobes being joined by a filament. In the homozygous state, no Morphological changes may also be noted in the neu-
segmentation is evident, and the nucleus takes on a round or trophilic cytoplasm. The presence of prominent, dark-staining,
oval appearance (Fig. 15-9). “True” Pelger—Huét anomaly is coarse cytoplasmic granules in neutrophils, eosinophils,
inherited in an autosomal-dominant pattern and is reported to basophils, monocytes, and occasionally lymphocytes is known
be a benign familial condition that is observed in | out of as Alder’s anomaly or Alder—Reilly inclusions" (Fig. 15—10).
6000 individuals.’ Acquired Pelger—Huét may be induced by In some patients, only one cell line may be affected.
drug ingestion or occur secondary to conditions such as These cytoplasmic inclusions are composed of precipitated
leukemia. Acquired forms, in which 10% of the neutrophils mucopolysaccharide and are seen in association with inherited
are trilobed, are often referred to as pseudo-Pelger—Huét.’ disorders of mucopolysaccharidosis, such as Hunter’s and
Hurler’s syndromes (see Chap. 23). These prominent granules
are similar to those in toxic granulation, with the exceptions
15-17 Inherited that they are larger, stain positive with metachromatic stains,
Neutrophil I and are a permanent morphological characteristic of the
neutrophils.
¢ Pelger—Huét Anomaly
¢ Autosomal dominant
* Bilobed or nonsegmented nuclei
¢ Normal cytoplasm
¢ May—Hegglin Anomaly
¢ Autosomal dominant
* Large blue cytoplasmic inclusions found in neutrophils,
eosinophils, basophils and monocytes
* Chediak—Higashi Syndrome
¢ Autosomal recessive
¢ Giant cytoplasmic granules in granulocytes and
lymphocytes
¢ Alder—Reilly Anomaly
* Autosomal recessive
* Cytoplasm of neutrophils contain dark azurophilic granules
* Eosinophils and basophils also affected
* Lymphocytes may also be abnormally granulated and
vacuolated
¢ Specific Granule Deficiency Figure 15-10 @ Alder-Reilly anomaly. (Left and middle) Note
* Autosomal recessive azurophilic granulation in cells from peripheral blood. (Right)
Bone marrow. (From Hyun, BH, et al: Practical Hematology. A Labo-
¢ Absence of secondary granules, neutrophils appear pale
ratory Guide with Accompanying Filmstrips. WB Saunders,
and hyposegmented
Philadelphia, 1975, with permission.)
* Abnormal granules in platelets and eosinophils
316 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Severity-

Mild 700-1400/mm*
(0.7-1.4 X 10°/L)
Moderate 1500-4900/mm?
(1.5-4.9 x 10°/L)
Severe >5000/mm*
(>5.0 X 10%/L)

reactive eosinophilia. The causes of secondary reactive

eosinophilia are listed in Table 15-19. Some common char-
Figure 15-11 ™ May—Hegglin anomaly. Note the Dohle body acteristics of secondary reactive eosinophilia are listed in
present in each neutrophil (arrows). Not shown in the slide but
Table 15-20. It should be noted that many times, a mild to
associated with May—Hegglin anomaly is the presence of giant
platelets. (From Dutcher, T: Hematology. In Listen, Look, and Learn.
moderate eosinophilia detected by a routine complete blood
Health and Education Resources, Inc., Bethesda, MD, with permission.) count (CBC), occurs without any known cause in most
outpatients.**
In malignant hematopoietic disorders such as CMPD and
As discussed previously, toxic granulation is an acquired
acute leukemias, eosinophilia may be seen because they are
transient change in morphology caused by infectious or toxic
part of the clone, and often demonstrate morphologic abnor-
agents. Granulocytes and monocytes in the hereditary
malities (see Chaps. 16, 17, and 18). The World Health Orga-
May-Hegglin anomaly demonstrate larger, blue-staining
nization now classifies the hypereosinophilic syndrome and
cytoplasmic inclusions that resemble Dohle bodies, except that
eosinophilic leukemia under the category of chronic myelo-
they are larger? (Fig. 15-11). Under electron microscopic
proliferative disorders.*°
examination, these neutrophils are shown to have large,
granule-free areas in the cytoplasm that contain fibrils of

=a,
RNA." This anomaly is also associated with thrombocytope-
nia and giant platelets, and variable neutropenia. Many of
these patients may be clinically asymptomatic (see Chap. 25).
Chediak—Higashi, previously discussed under neu-
trophils with abnormal function, is also classified as a neu- ae
ti pat

trophil disorder with abnormal morphology. The very large

cytoplasmic granules characteristic of this disorder are found
in all granulated cells within the bone marrow, blood, and
solid tissues.'' It should be noted that these large fused lyso-
somal granules are also observed in lymphocytes."! ¢ Allergic Disorders
1. Asthma
2. Urticaria
Eosinophils 3. Allergic rhinitis
¢ Parasitic Infections
The normal percent of eosinophils found upon examination 1. Helminthic (must invade tissue to produce eosinophilia)
on the peripheral blood smear is approximately 2% to ¢ Skin Disorders
4%. Eosinophils function to release their secondary granules 1. Bullous pemphigoid
2. Pemphigus
to destroy parasites and function in immediate hypersensi- 3. Atopic dermatitis
tivity reactions upon migration to the tissues. Eosinophilia 4. Eczema
occurs when the absolute eosinophil count is greater than ¢ Pulmonary Disorders
600 cells/mm? or 0.6 X 10° cells/L.** There are no age-related 1. Loeffler syndrome
variations in the absolute counts. Eosinophilia can be arbitrar- 2. Bronchiectasis
3. Pneumonia
ily classified using the absolute count. The absolute counts 4. Cystic fibrosis
for mild, moderate, and severe eosinophilia are listed in 5. Churg—Strauss syndrome
Table 15-18.** ¢ Other Inflammatory Disorders
The most common cause of eosinophilia worldwide is 1. Celiac disease
parasitic infections. In order to produce eosinophilia the par- 2. Vasculitides
asites must invade the tissues. Helminthic infection is the 3. Inflammatory bowel disease
4. Collagen vascular disorder
most common parasite causing eosinophilia. In developed 5. Sarcoidosis
countries, allergic disorders are the most common causes of
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis Bile

to 900 monocytes/mm* or 0.1 to 0.9 X 10°/L.'' In newborns and

young infants, the number of monocytes is higher (S00 to
1200 monocytes/mm*). Beyond infancy, however, there are
no age-related variations. Monocytes, on migration to the
tissues, function in phagocytic and antimicrobial activities,
* Mild to moderate eosinophilia
* Represents a compensatory bone marrow response to an tissue repair, and various other functions in cellular and humoral
increased tissue demand for eosinophils immunity. Monocytes also contain numerous very fine granules
* Eosinophilia disappears with the resolution of the causative in their cytoplasm which play a major role in destruction of
disease microbes. The enzymes found in the granules of monocytes are
listed in Table 15-22. Some of the functions of the many pro-
teins secreted by monocytes are listed in Table 15—23.
Basophils Monocytosis occurs when the absolute monocyte count
exceeds 1000 monocytes/mm* or 1.0 X 10°/L.'' Monocytosis is
The normal percentage of basophils present upon examina- generally associated with a chronic infection as opposed to an
tion of the peripheral blood smear is 1% to 2%. Basophilia acute infection. The causes of secondary reactive monocytosis
occurs when the absolute count is greater than 200 cells/mm? are listed in Table 15-24. Recovery from cyclic neutropenia, a
or 0.2 x 10° cells/L.'' A moderately increased absolute form of agranulocytosis, is preceded by a transient monocyto-
basophil count may occasionally occur secondary to allergic sis.'' Absolute monocytosis can also be found in malignant
disorders, hypersensitivity reactions and inflammatory disor- hematopoietic disorders such as chronic myelomonocytic
ders; however, this is an uncommon finding.'! The secondary leukemia (CMML), chronic myelogenous leukemia (CML),
reactive causes of basophilia are listed in Table 15—21. acute monocytic leukemia and acute myelomonocytic leukemia
More common causes of basophilia, with absolute counts (AMML) (see Chaps. 16, 17, 18, and 19).
that are much higher, are malignant hematological disorders
such as CMPD. Chronic myelogenous leukemia (CML) is the Absolute Monocytosis: Reactive versus
most common cause of basophilia even though the percentage Malignant Causes
of basophilia in the differential count is less than 10%.*’ The
absolute basophil count is very high due to the strikingly ele- Reactive monocytosis secondary to chronic infection may be
vated WBC count in patients with CML, which is a consistent accompanied by a neutrophilia, and a mild to moderate ane-
feature (see Chap. 17). mia is usually present. The WBC count is usually elevated
and the platelet count is highly variable. Patients with mono-
cytosis, as a result of a malignant clone, have a moderate to
Monocytes severe anemia, a highly variable WBC count (which is
usually increased), and a severe thrombocytopenia.'! The
The normal percentage of monocytes present on examination of
morphology of monocytes in reactive monocytosis shows rel-
the peripheral smear is 2% to 9% with an absolute count of 100
atively mature monocytes with clumped nuclear chromatin
which is present in a folded, indented, or unusually shaped
Table15-21 Causes of Secondary — nuclei. The cytoplasm is usually spread out and frequently
~ Reactive alee
(Absolute Count ©
ee 200mm ! ey Enzymes Found iinthe | :
Soro 107) _ Granules ofMon 0 cytes %
¢ Infections ¢ Lipozyme
1. Chicken pox ° Collagenase
2. Smallpox ¢ Acid phosphatase
3. Influenza ¢ Elastase
¢ Inflammatory Disorders
1. Rheumatoid arthritis
2. Ulcerative colitis
3. Collagen vascular disease ts Table15-23 Functions of Proteins Ka
¢ Allergic Reactions
1. Urticaria : Secreted by Monocytes eae
2. Allergies to food and drugs
3. Erythroderma ¢ Regulation of hematopoiesis
¢ Chronic Renal Disease ¢ Stimulation of inflammatory reactions
¢ Endocrine Disorders e Removal of infectious organisms by phagocytosis
1. Diabetes ¢ Removal of senescent blood cells
2. Hypothyroidism ¢ Modulation of the immune function
¢ Exposures to Radiation ¢ Stimulation of host defense against tumor cells
318 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

The term reactive lymphocytes is used to describe

Table 15-24 Causes of Secondary transformed or benign lymphocytes. The term atypical
Reactive Absolute should not be used interchangeably with reactive because, in
Monocytosis (Adults: pathology, atypical may imply malignant-appearing cells.
Absolute Counts Other terms that have been used to describe the spectrum of
reactive lymphocytes include immunocytes, transformed
>1.0 < 10°/L; Newborns: plasmacytoid lymphocytes,
lymphocytes, immunoblasts,
>1.2 < 10°/L) Turk cells, and Downey cells. Reactive lymphocytes occur in
normal patients, but usually account for less than 10% of the
Chronic Infection Chronic Neutropenia
total lymphocytes. To maintain consistency with laboratory
¢ Fungal ¢ Acquired neutropenia reporting, laboratories should have criteria and normal ranges
¢ Bacterial ¢ Inherited cyclic
for reactive lymphocytes in their procedure manual. Control
¢ Protozoal neutropenia
¢ Rickettsial
slides with reactive lymphocytes should be reviewed on a reg-
ular basis with staff to ensure uniformity in the reporting of
Inflammatory Conditions Malignant Hematological reactive lymphocytes.
Disorders
¢ Sarcoidosis
* Collagen vascular disorders ¢ Hodgkin lymphoma
¢ Inflammatory bowel diseases ¢ Non-Hodgkin lymphoma
Lymphocyte Morphology
¢ Sprue ¢ Multiple myeloma
Most lymphocytes are small; however, intermediate and some
Other large lymphoctes may be observed. Normal lymphocytes
¢ Immune thrombocytopenic have a high nuclear-to-cytoplasmic ratio, with the nucleus
purpura (ITP) being oval or round and the chromatin clumped and staining
¢ Hemolytic anemia dark purple. The cytoplasm is blue, with varying intensity
¢ Postsplenectomy from light to dark in different cells. Although most lympho-
cytes do not have granules, large lymphocytes may have a
few well defined purplish-red granules which can be easily
counted at the periphery of the cytoplasm. Large granular
lymphocytes (LGLs) are characterized by a few cytoplasmic
vacuolated. Numerous dust-like fine granules, which are nor- granules (Fig. 15-12). These granules are azurophilic, appear
mally hard to see, may be very prominent in these reactive red or purple, and are larger than the fine, pink, secondary
monocytes. In monocytes associated with a hematopoietic granules present in neutrophils. LGLs may be of T-cell or true
malignancy, more immature forms with fine nuclear chro- natural-killer cell origin.” They normally represent 10% to
matin and nucleoli are observed, usually without the reactive 15% of the peripheral blood mononuclear cells and their
morphological features previously described. absolute number in the peripheral blood is normally less than
0.6 X 10°/L.° They normally represent fewer than 1% of the
bone marrow lymphocytes. In contrast, the cytoplasm of
Lymphocytes monocytes contains many small granules and has a “ground-
Definition of Lymphocytosis glass” cloudy appearance.
Reactive lymphocytes and normal lymphocytes vary in
Lymphocytosis refers to an increase in lymphocytes in the size, shape, and immunophenotypic markers (polyclonal)
peripheral blood. The normal percentage of lymphocytes pre-
sent on examination of the peripheral smear is 20% to 44%
with an absolute count of 1200 to 3400 lymphocytes/mm+* or
1.2 to 3.4 X 10°/L.** The absolute lymphocyte count is deter-
mined by multiplying the percentage of lymphocytes
(obtained from the peripheral blood differential) by the total
leukocyte count (obtained from the CBC).
Absolute lymphocyte counts decrease with age. In
infants and young children, lymphocyte counts greater than
10.0 X 10°/L represent lymphocytosis. In adults, a finding of
more than 4.0 < 10°/L lymphocytes is defined as an absolute
lymphocytosis.**
Relative lymphocytosis refers to an increase in the per-
centage of lymphocytes when the absolute lymphocyte count
is within the normal range. This situation may occur when
other hematopoietic elements are decreased; that is, in neu-
tropenia, in which lymphocytes are not affected, or when the Figure 15-12 mA large, mature-appearing lymphocyte with
patient is dehydrated. azurophilic granules.
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 319

The ratio of the nuclear area to the visible cytoplasmic rim

(i.e., the N:C ratio) varies with reactive lymphocytes. One of
the most distinguishing features of reactive lymphocytes is
the abundant cytoplasm, when compared with smaller resting
Reactive Resting Small lymphocytes. Resting small lymphocytes have relatively little
Eymphocyte SSE
cytoplasm, whereas NK (null-killer) lymphocytes may have
Size Large (9-30 Hee Small (8-12 ae moderate amounts of cytoplasm, resulting in the terminology of
N:C Ratio* Low to moderate High to moderate “large granular lymphocytes” for these cells. In Figure 15-13,
Cytoplasm Abundant Scant three lymphocytes are present. In the center of the field is a
Colorless to dark blue Colorless to
reactive lymphocyte, specifically a large granular lymphocyte
light blue
Nucleus Round to irregular Round
with abundant cytoplasm (large arrow). The cytoplasm is usu-
Chromatin Coarse to moderately Coarse ally pale blue, with occasional azurophilic granules. A signifi-
fine* cant morphological feature of reactive lymphocytes is the
Nucleoli Absent to distinct Absent uneven staining of the cytoplasm. In reactive lymphocytes,
_Typing Polyclonal _ Polyclonal _
peripheral portions of the cytoplasm often stain darker blue than
*Norsal Pol blood may contain a i medium-sized areas of the cytoplasm closer to the nucleus. Also, the cytoplas-
natural killer cells that are larger than resting small mic border is usually round with an occasional indentation.
lymphocytes but smaller than large reactive lymphocyies.
‘The N:C ratio is the ratio of the nuclear area to the visible The nucleus in reactive lymphocytes may be round,
cytoplasmic rim. indented, or lobulated (Fig. 15—14). In reactive conditions, the
*The chromatin is not as fine as that seen in blast cells. appearance of the nuclear chromatin in the lymphocyte popu-
lation is variable and not monotonous. Generally, the nuclear
chromatin is coarse or clumped, and prominent clumping or
coarseness of the chromatin similar to plasma cells may
(Table 15—25). The cells do not originate from one precursor cell occur. A small “plasmacytoid” lymphocyte with prominent
or clone. In contrast, lymphocytes in malignant disorders are chromatin clumping, perinuclear halo, and an _ eccentric
similar with regard to size and shape (monomorphic), and nucleus similar in appearance to a plasma cell is illustrated in
immunophenotype, because they originate from the same malig- Figure 15-15. The nucleus in resting small lymphocytes is
nant clone (monoclonal). Malignant lymphocytes may vary in round and less variable. The chromatin may vary in coarse-
size depending on the particular malignancy. However, for any ness but not to the extent seen with reactive lymphocytes.
one malignancy, the size and appearance tend to be constant. Nucleoli may be present in reactive lymphocytes. Such
Reactive lymphocytes range in size from 9 to 30 um.’ reactive lymphocytes may be differentiated from lymphoid or
The size variation that may be seen with reactive lymphocytes myeloid blasts by having more abundant cytoplasm (i.e., a
are illustrated in Figure 15-13. Resting small lymphocytes lower N:C ratio) and a clumped chromatin pattern. A reactive
tend to be much smaller than reactive lymphocytes, ranging lymphocyte with a prominent nucleolus, often referred to as an
in size from 8 to 12 um, and are similar in size to the top right “immunoblast,” is illustrated in Figure 15—16. Immunoblast
lymphocyte in Figure 15—13 (right arrow). is a descriptive morphological term and has no bearing on the
lymphocyte type. Nucleoli are not apparent in resting small
lymphocytes. Reactive lymphocytes are compared with small
resting lymphocytes in Table 15—25.

sth

Figure 15-15 m Three lymphocytes are present, one with abundant

cytoplasm (low N:C ratio) and two with moderate amounts of
cytoplasm, from a patient with infectious mononucleosis. Note the
variation in the chromatin coarseness. The larger or reactive-appearing Figure 15-14 @ Lymphocyte with nuclear indentation.
lymphocyte has prominent cytoplasm indentations (center arrow).
320 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Adenovirus
Chickenpox
Cytomegalovirus
EBV (infectious mononucleosis)
Hepatitis
Herpes simplex
Herpes zoster
Human immunodeficiency virus (HIV)
Influenza
Paramyxovirus (mumps)
Rubella (measles)

Bacterial
Figure 15-15 @A small plasmacytoid lymphocyte with coarse Brucellosis
chromatin and an eccentric nucleus. Paratyphoid fever
Pertussis (whooping cough)
Tuberculosis
Morphological criteria alone usually enable one to dis- Typhoid fever
tinguish reactive lymphocytoses from malignant disorders.
Drug Reactions
When there is ambiguity, clinical history, cell typing, and During recovery from acute infections (especially in children)
serologic findings may help in distinguishing these entities. If
necessary, a bone marrow or lymph node biopsy may lead to Miscellaneous
the correct diagnosis. The most common causes of a reactive Acute infectious lymphocytosis
Allergic reactions
lymphocytosis are shown in Table 15-26. Malignant condi- Autoimmune diseases
tions that may be confused with a reactive lymphocytosis are Hyperthyroidism
listed in Table 15-27. Malnutrition
Rickets
Syphilis
Causes of Reactive Lymphocytosis Toxoplasmosis

INFECTIOUS MONONUCLEOSIS
HISTORY In 1907, Turk described clinical symptoms in sev-
eral patients who probably suffered from infectious mononu- terminology of morphology is seldom used today. Infectious
cleosis (IM) caused by EBV infection.*° Reactive lymphocyte mononucleosis is a historical term. We now know that the
morphology and clinical symptoms were correlated in 1920 large mononuclear cells originally described in IM are lym-
by Sprunt, who named the syndrome “infectious mononucle- phocytes and not monocytes.
osis.”*? Downey, in 1923, described in further detail the Later, in 1932, Paul noticed that serum from patients with
unusual lymphocyte morphology associated with IM.*° IM contained antibodies against sheep erythrocytes.*' This dis-
Although morphology is still important, Downey’s descriptive covery was the basis for the Monospot test (Ortho Pharmaceu-
ticals). This contemporary test is the most commonly used and
the simplest method available for measuring IgM heterophile
antibodies. Heterophile antibodies are antibodies that also react
with cells of other species. In particular, the IgM antibodies
react with sheep and horse erythrocytes. The antibodies are not
absorbed by guinea pig kidney in the Monospot test and, thus,
if present, agglutinate horse erythrocytes.
The DNA virus responsible for IM was first observed in
lymphoblasts cultured from patients with Burkitt’s lym-
phoma**** (see Chap. 21). This virus is now known as the
Epstein-Barr virus (EBV). In addition to Burkitt’s lymphoma,
EBV has been associated with other malignancies, including
B-cell lymphoproliferative syndromes, nasopharyngeal carci-
noma, Hodgkin lymphoma, and gastric carcinoma.*! The
malignancies appear to occur after the virus has been dormant
in the host for years.
Figure 15-16 @A large reactive lymphocyte with prominent In regard to acute viral illness, EBV was first linked as
nucleolus (immunoblast). the causative agent for IM by Henle in 1967.4 He was able to
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive lymphocytosis 321

of the sore throat pain leads to difficulty in swallowing and

anorexia. Other commonly observed symptoms include nau-
sea, headache, myalgia, sweats, and chills. Infrequently, indi-
viduals present with mild autoimmune hemolytic anemia
because of cold-reacting antibodies (agglutinins) specific for
the “i” red cell antigen (see Chap. 13), and immune thrombo-
Acute Lymphocytic Leukemias
cytopenia caused by increased splenic activity.
Acute Myelocytic Leukemias Multiple organ involvement with related symptoms may
M3m be present in IM. Enlargement of the anterior cervical lymph
M5a nodes is another physical finding during the first week. Follow-
Chronic Leukemias
ing a week or two, the swelling usually subsides. The nodes are
Chronic lymphocytic leukemia (CLL) firm but not tender or warm. At least one-half of patients
Hairy-cell leukemia demonstrate a palpable spleen during the course of infection
Lymphocytosis of large granular lymphocytes and, rarely, splenic rupture may occur. Hypersplenism may
Polymorphocytic leukemia contribute to mild anemia or immune thrombocytopenia, or
Leukemic Phase of Lymphoma both. Hepatomegaly may be detected in up to 25% of patients
Follicular lymphoma (typically small cleaved cell) with IM. Liver enzymes and bilirubin levels may be elevated
Mantle cell lymphoma because of liver involvement.
Other non-Hodgkin’s lymphomas (e.g., large cell) Infectious mononucleosis is uncommon in older adults
Miscellaneous and, therefore, the differential diagnosis of lymphocytosis is
Adult T-cell leukemia/lymphoma (ATL/L) influenced by the age of the patient. For example, in a 50-year-
Mycosis fungoides old patient with increasing numbers of benign-appearing lym-
Sézary syndrome phocytes, the diagnosis of chronic lymphocytic leukemia (CLL)
Plasma cell leukemia
is more likely than IM; however, in a child or young adult, CLL
is extremely rare. It should be noted that many children contract
IM in early childhood which is misdiagnosed as a bad cold or
flu. In most cases, the development of antibodies results in long-
establish a lymphocyte cell line from the blood of a laboratory term immunity.
worker who was infected with EBV and managed to show by
serologic studies that EBV was responsible for IM. Additional DIFFERENTIAL DIAGNOSIS The differential diagnosis for
evidence supporting the role of EBV in IM has been provided IM includes a variety of entities listed in Tables 15-26 and
by the detection of EBV nucleic acid sequences using poly- 15-27. The differential may be narrowed considerably
merase chain reaction on lymphoid tissue from patients with by evaluation of a peripheral blood smear and _ serologic
IM.***° Unlike EBV-associated malignant conditions, IM findings. The skilled morphologist should be able to readily
results from primary infection. differentiate the reactive lymphocytes seen in IM from malig-
Humans can be infected by one of two strains of EBV, nant lymphoid cells seen in leukemia or lymphoma.
type A or B, which are widespread in North America and Cytomegalovirus, rubella, hepatitis, and other viral illnesses
may coinfect the same individual.*’? EBV primarily infects may have reactive lymphocytes and may require additional
B lymphocytes and epithelial cells of the pharynx and cervi- serologic findings to distinguish them from EBV infection.
cal lymph nodes, which all share the CD21 receptor.” Inter- Acute streptococcal pharyngitis, diphtheria, and other bacterial
estingly, the majority of atypical lymphocytes associated infections are identifiable by culture, and the lymphocytes usu-
with IM have been identified to be T lymphocytes of the ally do not have the reactive morphology seen in viral infec-
cytotoxic-suppressor subset, although T helper cells and NK tions. Classical reactive lymphocytes and definitive serologic
cells are also present.*” Initial stimulation of the T cells findings may not always be present and, therefore, it may be dif-
appears to be nonspecific, later followed by direct stimula- ficult in some instances to distinguish between IM and other
tion from EBV-infected B lymphocytes.** The direct stimu- processes. In those rare cases, additional studies, such as lymph
lation results in such rapid T-lymphocyte proliferation that node biopsy may be indicated.
the lymph nodes enlarge and the cells released are reactive
in appearance. TREATMENT, CLINICAL COURSE, AND PROGNOSIS Treat-
ment of IM is mostly symptomatic, with some patients requir-
CLINICAL MANIFESTATIONS Infectious mononucleosis is ing bed rest. Acetaminophen or ibuprofen products are often
more commonly present in young individuals, with the peak taken to relieve muscle pain and headache. Little progress has
incidence between 17 and 25 years of age. The virus enters been made in decreasing the duration of illness, with antiviral
the body orally through the lymphoid tissue in the pharynx therapies showing few benefits.*’ In the few patients with severe
and, in turn, infects B lymphocytes. The onset is generally or persistent cases, corticosteroids have been indicated to treat
abrupt, the most consistent initial symptoms being sore throat, associated hemolytic anemia and progressive neurologic prob-
lymphadenopathy, fever, dysphagia, general malaise, and lems. Intravenous gamma (‘y)-globulin is frequently adminis-
excessive fatigue that may persist for 2 to 3 weeks. The severity tered to treat [M-associated immune thrombocytopenia,” and
322 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

limited success has been seen using y-interferon to treat compli- be seen in 4% of patients.°* Morphologically, these cells
appear similar to those seen in CLL/small lymphocytic lym-
cations such as pneumonia or encephalitis.°°
phoma (SLL). In pertussis, immunologic marker studies
Antibiotics are not useful unless there are complications
reveal that the lymphocytes are predominantly helper T
such as streptococcal pharyngitis. The fatality rate of IM is
cells.°° Prominent lymphadenopathy is notably unusual in
approximately | in 3000 cases.*° In acute cases, complete
children with pertussis or infectious lymphocytosis.
recovery usually occurs within 2 months, and recurrences are
extremely rare. It should be noted that not all children who
MALIGNANT CONDITIONS
have acquired immunity had a prior history or clinical symp-
Malignant disorders that may be confused with reactive lym-
toms of IM. EBV infections in immunocompromised patients
phocytoses are listed in Table 15—27. In general, the malig-
may be life threatening. In view of the increasing incidence of
nant cells in leukemia and lymphomas are monotonous in
patients with immunodeficencies, the morbidity rate is likely
appearance and, therefore, are morphologically different from
to increase.
the reactive lymphocytes seen in benign conditions. Malig-
nant lymphoid cells in leukemias and lymphomas are mono-
CYTOMEGALOVIRUS INFECTION
Cytomegalovirus (CMV) belongs to the herpes virus family clonal when immunophenotypically analyzed.
and is endemic worldwide. The virus may be transmitted by The morphology of lymphoblasts seen in acute lym-
oral, respiratory, and sexual means or by blood transfusion phoblastic leukemia (ALL) is different from that of reactive
and organ transplantation. Patients with CMV infection may lymphocytes. ALL is morphologically classified as L1, L2, or
have recurrent infection or reactivation of a latent infection, L3, depending on the morphology of the cells (see Chap. 16). In
as seen in herpes simplex infection, or may be reinfected with contrast to reactive lymphocytes, blasts of L2 leukemia have
fine chromatin and prominent nucleoli (Fig. 15-17). The blasts
a different CMV strain. CMV infection is the most common
cause of heterophile-negative IM. The diagnosis is made by of L1 leukemia are monotonous in appearance and do not have
demonstrating the presence of IgM antibodies to CMV or by as much cytoplasm as do reactive lymphocytes (Fig. 15-18).
shell vial tissue cultures. The N:C ratio is much higher in LI blasts than in reactive
lymphocytes. In L3 blasts (Fig. 15-19), there are abundant
CLINICAL MANIFESTATIONS In the majority of immuno- cytoplasmic vacuoles that are lacking in reactive lymphocytes.
competent individuals, CMV infection is usually asympto- The lymphoid cells present in prolymphocytic leukemia (PLL)
matic. The clinical manifestations reflect the involved may also be confused with reactive lymphocytes. The promi-
organ(s). When CMV infection occurs in previously healthy nent, usually single, nucleoli that are present in PLL are shown
individuals, the symptoms mimic EBV infection. Symptoms in Figure 15-20. Reactive lymphocytes have more abundant
include fever, sore throat, splenomegaly, lymphadenopathy, cytoplasm and less prominent nucleoli than prolymphocytes.
and myalgia. Morphological changes in lymphocytes are Severe cytopenias that occur in acute leukemias are
indistinguishable from those seen in EBV infection. Because infrequent in benign lymphocytosis. With anemia or severe
of liver involvement, mild to moderate elevations of liver thrombocytopenia, the diagnosis of leukemia must be
function tests are common. considered (see Chap. 16).
In immunocompromised individuals, CMV infection may Patients with lymphoma may have a leukemic phase
be life threatening. Because CMV infection is endemic, par- with circulating malignant cells. Except for the large-cell
enterally acquired infection from transfusions is a concern, lymphomas, circulating lymphoma cells may be easily
especially with the increase in the numbers of individuals who distinguished from reactive lymphocytes. The nuclear
are immunocompromised from chemotherapy, immunosuppres-
sive drugs, or human immunodeficiency virus (HIV) infection.

OTHER VIRAL INFECTIONS

Any viral infection has the potential to elicit an immune
response, resulting in an absolute lymphocytosis. Lymphocytes
that appear “stimulated” or reactive may be seen in CMV and
infectious hepatitis, in addition to IM. With a negative
Monospot test, other viral infections (e.g., rubella, HIV,°'*? her-
pesvirus-6,°° and the adenoviruses) should be considered.

BACTERIAL INFECTIONS
Lymphocytosis in bacterial infections occurs more commonly
in chronic infections and during the recovery period follow-
ing acute infections. The organisms most often responsible
include Brucella, Mycobacterium tuberculosis, and spiro-
chetes. Marked lymphocytosis with mature-appearing lym- Figure 15-17 mL2 leukemia. The L2 lymphoblasts have fine chro-
phocytes is seen in children with pertussis (whooping cough), matin with a moderate amount of cytoplasm and nucleoli. Note the
and absolute lymphocyte counts exceeding 50 X 10°/L may prominent nucleolus in one of the lymphoblasts (arrow).
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis eS)i)Ww

iL

Figure 15-18 @L1 leukemia. These L1 lymphoblasts have a high Figure 15-20 @ Prolymphocytes with moderate amounts of cyto-
N:C ratio (scant cytoplasm), fine chromatin, and indistinct nucleoli. plasm and prominent nucleoli.
Except for the fine chromatin, note the similarity to mature
lymphocytes. These cells should not be confused with reactive
lymphocytes. Note the large “smudge cells” (arrows). observer. A reactive lymphocyte with abundant cytoplasm (low
N:C ratio) and indented cytoplasmic borders (large arrow) is
shown in Figure 15-13. The less experienced observer may
clefting seen in the leukemic phase of follicular small
mistake these unusual-appearing reactive lymphocytes for
cleaved-cell lymphoma is shown in Figure 15-21. Fortu-
monocytes or blasts. However, as seen in Figure 15—22, mono-
nately, in large-cell lymphoma, malignant cells in the periph-
cytes typically have linear condensation of chromatin, whereas
eral blood are rare. Prominent generalized lymphadenopathy
blasts (see Fig. 15-17) have chromatin strands that are finer and
is unusual in IM, but may occur in non-Hodgkin’s or evenly dispersed.
Hodgkin lymphomas (see Chap. 21).
The lymphoblasts in L2 leukemia or the monoblasts in
MS5a ieukemia (a category of acute myeloid leukemia) may also
Laboratory Examination be mistaken for reactive lymphocytes. An MSa, monoblastic
leukemia 1s illustrated in Figure 15-23. Monoblasts have a low
A variety of laboratory procedures may help in the correct N:C ratio but fine chromatin is present as well as prominent
diagnosis of patients with an absolute lymphocytosis. CBC, nucleoh. The fine chromatin and prominent nucleoli (arrow)
serology, and microbiologic culture are often performed. Of present in an L2 lymphoblast is shown in Figure 15—17. With
these procedures, a CBC with differential and serologic stud- reactive lymphocytes, a careful review of the cell morphology
ies are the most useful. will reveal that overall, the chromatin is not fine enough for the
Proper evaluation of the peripheral blood smear is crucial cells to be classified as blasts and that the overall morphology
for the correct differential diagnosis in patients with an absolute is variable and not monotonous as seen in leukemias.
lymphocytosis. Especially in IM, reactive lymphocytes are Serologic tests play a critical role in establishing the
prominent and should easily be identified by the experienced diagnosis in patients with an absolute lymphocytosis. In the

1 Ss a

Se
4

Beane %
Figure 15-19 m L3 leukemia. Note the abundant cytoplasmic vac- Figure 15-21 ™ Lymphocytes from a patient with follicular small
uoles and clumped chromatin. cleaved-cell lymphoma. Note the prominent nuclear clefting.
324 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Figure 15-22 ™ This monocyte has very fine granular cytoplasm, Figure 15-25 @ MSa leukemia. These monoblasts have abundant
cerebriform nucleus, linear condensation of chromatin, and no cytoplasm (low N:C ratio), fine chromatin with some chromatin
nucleolus. clumping, and prominent nucleoli.

proper clinical setting, a positive Monospot (heterophile anti- The Monospot test is most frequently used for diagnos-
body) test with reactive lymphocytes in the peripheral blood ing IM, because the rise in titer of heterophile antibodies
(see Fig. 15-13) is diagnostic of IM. A variety of antibodies parallels the rise in titer of the more specific EBV antibod-
formed by humoral responses may be measured when it is ies. The Monospot test is much quicker, easier to use, and
necessary to elucidate further the etiology of lymphocytosis less expensive than measuring viral-specific antibodies. If
in difficult cases or in cases in which the Monospot test is the Monospot test is initially negative, it should be repeated
negative” (Table 15—28). in | week, if IM is suspected. Serum may be analyzed for
In the first week of viral infections, IgM antibodies are antibodies to specific EBV antigens; antibodies to CMV and
formed against viral capsid antigens. During the second hepatitis may be measured, and viral cultures may be per-
week, as the immunologic response matures, IgG antibod- formed. Some of the possible testing strategies are dia-
ies are formed. The pattern of the immunologic response grammed in Figure 15—25.
and lymphocytosis that is expected in patients with IM In cases where malignancy is being considered, lympho-
is illustrated in Figure 15-24. A rise in titer should be cytes may be further characterized by flow cytometric
demonstrated by comparing serum obtained during acute immunophenotypic analysis. With leukemias and lym-
and convalescent phases. Also, by using enzyme-linked phomas, flow cytometric immunophenotyping usually reveals
immunosorbent assay (ELISA) techniques, antibodies to a monoclonal cell population.°’ As previously described in
CMV and hepatitis may be measured. Finally, viral cultures IM, B cells are infected by EBV with most of the reactive
may be performed. lymphocytes representing polyclonal T cells.

Antigen Test Used Description

Pech Monospot Antibodies to a variety appears hes in re first Week

of antigens detected with Monospot
test; transient
EBV-VCM (IgM) ELISA IgM antibody to viral Detectable in first week of
capsid antigen infection; earliest detectable
antibody; declines rapidly
after second week
EBV-VCA (IgG) IgG antibody to viral Detectable approximately
capsid antigen 7 days after exposure; levels
persist for life; responsible
for immunity
Antibody to EBV nuclear Appears late in first month of
antigen infection and persists for life;
may indicate past infection
Antibody to EBV early Seen in < 5% of normal, healthy
antigen complex subjects; may indicate
EBV-carrier state
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 3:
i)Wn

10,000 INFECTIOUS MONONUCLEOSIS SYMPTOMS

- fan WITH LYMPHOCYTOSIS
= ny \ Mean total
-| 6000 N lymphocyte
4 4000 ~. counts Monospot Test
(Heterophile Antibody)
2000 ——__ eee ae
0 s— Mean reactive
lymphocyte
©
ocea counts
Infectious Repeat Monospot
FE 300 IgG anti-VCA see ra ee ae (1 week later)
5 & vs
fa
9 & 200 IgG anti-VCA
Or ¥ Y
5 5 150 IgG anti-EA (D)
Infectious EBV-IgM
==<& 400 ;
IgG anti-EBNA Mononucleosis Antibody Test

©
rsZ 50
oO = Se ee Be ee OOF
Op |
0 1 5 3 4 5 6 > g antibodies
Heterophile Negative Y
TIME IN WEEKS FROM ONSET OF ILLNESS (0) Infectious Mononucleosis Sa

Figure 15-24 m Time-course relationship between heterophile

antibodies, various anti-Epstein—Barr antibodies, and the mean total
y Y
and reactive lymphocyte counts. VCA = viral capsid antigen. EBNA =
CMV Hepatitis Tests
Epstein-Barr nuclear antigen, EA = early antigen, D = diffuse compo- Mononucleosis Toxoplasma Titer
nent. (From Lee, RG, et al: Wintrobe’s Clinical Hematology, ed 9. Viral Culture etc.
Lea & Febiger, Philadelphia, 1993, p 1658, with permission.)
Figure 15-25 Testing strategies for infectious mononucleosis
differential diagnosis.
Lymphocytopenia
As mentioned previously, the normal percentage of lympho- 2.0 X 10° cells/L in children.°* The disorders associated with
cytes present on examination of the peripheral smear is 20% to lymphocytopenia, which usually presents with a normocytic
44%, with an absolute count of 1200 to 3400 lymphocytes/mm* normochromic anemia, are listed in Table 15—29; granulocy-
or 1.2 to 3.4 x 10°/L.°8 Lymphocytopenia occurs when the topenia may also be present.°* It should be noted that most
absolute count is less than 1000 lymphocytes/mm* or 1.0 X patients with this disorder have a decrease in T lymphocytes,
10° cells/L in adults and less than 2,000 lymphocytes/inm* or primarily CD4+ (helper) T cells.

Tel

‘Table 15-29 Causes of Secondary Reactive Lymphocytopenia (Absolute Counts: srt <
OX 10°/L in Adults; < 2.0 x 10°9/L in Children)

Infections Systemic Diseases

HIV Sarcoidosis
TB Renal disease
Hepatitis Burns
Influenza Celiac disease
Typhoid fever
Babesiosis
Pneumonia
Sepsis

Autoimmune Disorders Malignant Disorders

SLE Hodgkin lymphoma
Rheumatoid arthritis Carcinoma
Myasthenia gravis

Medical Treatment Complication Other

Chemotherapy Congenital immunodeficiency disorders
Radiation therapy Nutritional deficiencies
Glucocorticoid Idiopathic CD4+ T lymphopenia
Anesthesia and surgery
Antilymphocyte globulin
Thoracic duct drainage
326 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

Case Study I Case Study 2

HISTORY OF PRESENT ILLNESS HISTORY OF PRESENT ILLNESS
A 3-year-old boy was admitted with a diagnosis of liver A 13-year-old girl went to her physician because of sore
abscess after ultrasound examination. He had been ill with throat, a 1-week history of general malaise, and fever. She
many infections since the age of 1 month. Types of infections also complained of some nausea and difficulty in drinking
included recurrent episodes of pneumonia and ear infection, fluids. Physical examination revealed bilateral, enlarged,
perirectal abscess, osteomyelitis of the metacarpal bones, firm cervical lymph nodes, mild splenomegaly, and
suppurative lymphadenitis, and mastoiditis. Organisms that hepatomegaly.
were recovered from these sites of infection included Staphy-
lococcus aureus and Aspergillus. Despite these episodes of LABORATORY DATA
infection, he experienced normal growth and development.
The family medical history was remarkable in that an older The laboratory data revealed a WBC count of 15.0 x 10°/L;
brother and a cousin (a son of a maternal aunt) had suffered a hematocrit, 42%; a platelet count, 4.5 x 10°/L; and a retic-
similar chronic infections, and one had died of chronic pneu- ulocyte count, 2.0%. A differential count of the peripheral
monia caused by a gram-positive organism. On physical blood revealed 65% lymphocytes, 25% granulocytes, and
examination, the boy appeared to be generally well. The skin 10% monocytes. The differential report noted that 36% of
was covered with scattered areas of crusted scabs described the lymphocytes were reactive (see Fig. 15—13). Chemistry
as an infected eczematoid rash. A Gram siain of the rash results showed that liver enzymes and bilirubin levels were
revealed gram-positive cocci in clusters (Staphylococcus). slightly increased. No other abnormalities were noted.
Lymph nodes and liver were enlarged. After physical examination, additional laboratory work
was ordered. The throat culture for streptococci was nega-
LABORATORY DATA tive. A Monospot test was positive. No further laboratory
work was ordered. After a few days of rest at home, the
The hemoglobin level was 9 g/dL; the erythrocyte morphol- patient was allowed to return to school. No further problems
ogy was normal with slight hypochromia. The total WBC were noted.
count was 33.0 * 10°/L (28.0 X 10°/L neutrophils). The neu-
trophils contained moderate toxic granulation and vacuoles. DISCUSSION
The platelet count was 427 x 10°/L. Concentrations of serum
immunoglobulins and complement components were all The differential diagnosis in this case includes EBV IM,
moderately increased. Neutrophilia was consistently found on viral pharyngitis, diphtheria, and streptococcal pharyngitis.
several occasions on retrospective review of previous labora- The clinical history and clinical presentation are not consis-
tory data. Neutropenia was never documented. Studies of tent with other infections or malignancy. If the Monospot
neutrophilic migration and phagocytosis were normal. Oxida- test had been negative, the differential diagnosis would need
tive metabolism in response to neutrophilic stimulation was to be expanded.
completely absent. Specifically, there was no post-phagocytic With a negative Monospot test, further testing should first
increase in Oxygen consumption, and superoxide anion and include an ELISA for EBV-VCA (IgM) and possibly a
hydrogen peroxide were not formed. Finally, neutrophils were repeat Monospot in a week. If a diagnosis of IM is excluded
unable to oxidize and kill Staphylococcus that had been based on such testing, then other laboratory procedures
phagocytized. Neutrophils from the mother were analyzed should be performed. These would include further cultures,
and performed at about 50% of normal capacity. serologic tests, and possibly lymph node and bone marrow
biopsies. In this case, the liver enzymes were elevated; up to
DISCUSSION 25% of individuals with IM have liver involvement.

This patient exhibits characteristic features of the X-linked

recessive form of chronic granulomatous disease of child-
hood, with severe and persistent infections caused by Staphy-
lococcus organisms. Anemia and persistent neutrophilia are
seen even when these patients are relatively free of infection.
Neutrophils from these children are numerous; they migrate
normally, are capable of phagocytosis; and form phagocytic
vesicles. The neutrophils of these patients, however, are
unable to kill the phagocytized microorganisms, since they
are not able to generate active oxygen metabolites (such as
superoxide anion and hydrogen peroxide). In some families,
the X-linked nature of the inheritance pattern may be estab-
lished by finding disease in male members of the maternal
family and by finding moderate defects in the mother.
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 327

1, The enz matic contents of primary (azurophilic) gran- i, (Defective bacterial killing
ules include: 4 . d. Abnormal phagolysosome formation
a. NADPH oxidase and hydrogen peroxide 9. Identify the disease characteristic(s) associated with
b. Cytochrome b and collagenase
Chediak—Higashi syndrome.
ee ea lysozyme artial albinism, recurrent infections, and mild bleed-
. Alkaline phosphatase
and gelatinase
ing tendencies
2. Directional migration toward a gradient stimulated by a b. Granulomas, osteomyelitis, and hepatic abscesses
emoattractant is referred to as: c. Hypopigmentation of skin and chronic, swollen lymph
ee nodes :
b. Random mobility d. Periodic pneumonia that may result in lesions called
c. Opsonization pneumatoceles
d. Chemokinesis
10. Pelger—Huét anomaly may be described by:
3. The marking of an invading microbe with IgG and com- a. Large neutrophils and hypersegmentation of the
plement to facilitate recognition is referred to as: nucleus with greater than six lobes
Chemokinesis b. Dark-staining, coarse granules in cytoplasm of neu-
Soin ona trophils, eosinophils, basophils, and monocytes
c. Phagolysosome fusion c. Pale blue inclusions of the cytoplasm of neutrophils
d. Signal transduction and giant platelets
4. Which sequence reflects the correct order for phago- Hyposegmentation of the nucleus with the majority of
cytosis? neutrophils being bilobed or monolobed ,
a. Release of cytoplasmic granules; binding of particle; 11. Reactive lymphocytes may best be distinguished from
ingestion; fusion of phagolysosome blasts by the presence of which of the following mor-
b. Ingestion; binding of particles; fusion of phagolyso- phological features?
some; release of cytoplasmic granules a. Prominent nucleoli
Binding of particle; ingestion; fusion of phagolyso- Fine chromatin
some; release of cytoplasmic granules Heterogeneous cell population
d. Fusion of phagolysosome; binding of particle; release . High N:C ratio
of cytoplasmic granules; ingestion 12. Which of the following antigens is detectable first by
5. In oxygen-dependent killing, the enzyme responsible for ELISA?
meditating the production of active oxygen metabolites a. EBNA
during the respiratory burst is: BV-VCA (IgM)
a. Myeloperoxidase c. EBV-VCA (IgG)
b. Lysozyme d. Heterophile
c. Lactoferrin 13. The Epstein-Barr virus infects which of the following cells?
@ NADPH oxidase a. Helper T lymphocytes
6. The two most important biochemical products of the res- b.)\Cytotoxic T lymphocytes
piratory burst that are involved with particle digestion c. B lymphocytes
during active phagocytosis are: d. NK cells
a. Lactoferrin and gelatinase 14. What is the most frequent cause of a heterophile
b. Superoxide dismutase and catalase (Monospot) negative mononucleosis-like syndrome?
. Glutathione peroxidase and copper-zinc enzymes _ HIV
Superoxide anion and hydrogen peroxide (som
7. The morphological characteristic(s) associated with c. Hepatitis C
hediak—Higashi syndrome is (are): d. Toxoplasma gondii
a lysosomal granules 15. In which of the following conditions are reactive lym-
b. Hypersegmented agranular neutrophils with vacuolization phocytes found?
c. Prominent dark-staining granules and pyknotic nuclei a. Infectious mononucleosis
d. Pale blue inclusions in cytoplasm of neutrophils and b. CMV infection
giant platelets c. Rubella
8. The defect in chronic granulomatous disease is attributed to: (4 All of the above
a. Delayed degranulation
b. Inefficient ingestion of microbes
328 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis

16. Absolute lymphocytosis is best described as: c. Fatigue

a. Greater than 70% lymphocytes on differential d. Fever
b. Presence of nucleoli in lymphocytes 19. Which of the following features best differentiates
c. Monoclonal population of lymphocytes malignant lymphomas from infectious mononucleosis?
d. Greater than 4.0 < 10° lymphocytes per liter in an a. Clonality .
adult b. Monotony
17. Which of the following features are seen in reactive lym- c. Pattern of lymphadenopathy
phocytes? d. All of the above
a. Low N:C ratio 20. Which of the following viral agents causes infectious
b. Blue cytoplasm mononucleosis?
c. Indented cytoplasmic borders a. Heterophile virus
All of the above b. Human herpes-6 virus
18. Which of the following clinical manifestations would be c BBY
unexpected in infectious mononucleosis? d. HIV
a. Skin rash
b. Sore throat See answers at the back of this book.

min Alder’s anomaly, prominent, dark-staining, coarse

~’}] SUMMARY CHA cytoplasmic granules are observed in neutrophils.
g Individuals with May—Hegglin anomaly demonstrate
g Neutrophils are capable of amoeboid movement into the
large, dark blue-staining cytoplasmic inclusions in granu-
tissues to engulf and destroy bacteria and fungus.
locytes; thrombocytopenia; and giant platelets.
m Phagocytosis occurs in three distinct phases: migration
gw Lymphocytosis is known as an increase in the number of
and diapedesis; opsonization and recognition; and inges-
circulating lymphocytes.
tion, killing, and digestion.
m@ The term reactive lymphocytes is used to describe trans-
m The three modes of migration that contribute to efficient
formed or benign lymphocytes, which usually account for
neutrophil mobilization to a site of injury are random
less than 10% of the total lymphocytes present.
locomotion, directional chemotaxis, and accelerated
chemokinesis. # An absolute lymphocyte count is determined by multiply-
ing the percentage of lymphocytes (from the peripheral
BA shift to the left is defined as the early release of bands
blood differential) by the total leukocyte count (from the
and metamyelocytes from the bone marrow into circula-
complete blood count).
tion in response to infection or inflammation.
Reactive changes in lymphocyte morphology are hetero-
w Neutrophilia is known as an increase in the number of cir-
geneous and include a low N:C ratio; round, indented, or
culating neutrophils.
lobulated nucleus; abundant, uneven staining cytoplasm
a In response to bacterial infection, reactive changes in neu- with a round or indented cytoplasmic border; and the pos-
trophil morphology can be observed in the forms of toxic sible presence of nucleoli.
granulation, Dohle bodies, and vacuolization.
Malignant cells in leukemia and lymphoma can be distin-
w Neutropenia, which can be acquired or congenital, is guished morphologically from reactive lymphocytes in as
defined as an absolute decrease in the number of circulat- much as malignant cells are monotonous in appearance,
ing neutrophils below 1.5 X 10°/L. similar in size and shape, as they originate from a single
mwChediak—Higashi syndrome is a rare disorder of neu- clone.
trophil function that is characterized by recurrent bacter- @ Reactive changes in lymphocytes commonly accompany
ial infections, partial albinism, and the presence of giant infectious mononucleosis, cytomegalovirus (CMV), rubella,
lysosomal granules in nucleated cells. hepatitis, and other viral infections.
m Chronic granulomatous disease, the best understood dis- m Caused by the Epstein-Barr virus (EBV), infectious
order of neutrophil function, is a defect that is attributed mononucleosis is characterized by sore throat, fatigue,
to one of four mutations in NADPH oxidase, resulting in fever, headache, difficulty swallowing, and generalized
ineffective bacterial killing. malaise in teenagers and young adults.
m In Pelger—Huét anomaly, more than 70% to 90% of neu-
trophils have a bilobed nucleus or no nuclear segmenta-
tion at all.
 Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis 329

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 Chapter

Introduction to Leukemia
and the Acute LeuKemias
Ken Gatter, MD, JD
Frank Cruz Ill, MD
Rita Braziel, MD

Introduction OBJECTIVES
Definition and Overview
Comparison of Acute At the end of this chapter, the learner should be able to:
and Chronic Leukemia L. Define leukemia.
Historical Perspectives
Etiology and Risk Factors 2 . Compare and contrast acute versus chronic leukemia, and leukemia versus lymphoma.
Classifications 3 . Compare and contrast acute myeloid and acute lymphoblastic leukemia.
Introduction to Acute 4. Describe the FAB classification and list the characteristics of each type.
Leukemia
Incidence 5 . List and describe each of the WHO subtypes of acute lymphoblastic and acute
Clinical Features myeloid leukemia, paying particular attention to immunophenotypic and cytogenetic
Laboratory Evaluation of features.
Acute Leukemia . Describe characteristic morphology and cytochemical staining patterns for each of
Acute Myeloid Leukemia the subtypes of acute myeloid leukemia.
FAB Classification of AML
. List the WHO categories of acute myeloid leukemias with recurrent cytogenetic
WHO Classification of AML
abnormalities.
Acute Lymphoblastic
. List the criteria for immunologic classification of acute lymphoblastic leukemia,
Leukemia
including early precursor B- and T-cell acute lymphoblastic leukemias.
Classification of ALL and
Introduction . Describe the role of genetic analysis of newly diagnosed leukemia and know some of
Review of Lymphocyte the prognostic implications of the more common genetic alterations seen in acute
Ontogeny leukemias.
FAB Classification of ALL
. Describe the morphologic and cytogenetic abnormalities associated with acute
WHO Classification of ALL
promyelocytic leukemia (FAB M3) and understand some of the clinical implications.
Burkitt’s Leukemia/
Lymphoma (Mature Lt Correctly identify a blast on a peripheral blood smear or bone marrow aspirate, be
B-Cell ALL) able to distinguish between the typical morphologic features of a lymphoblast and a
Childhood versus Adult ALL myeloblast and list some of the flow cytometry antibodies useful for distinguishing
Treatment of Acute between myeloid and lymphoid.
Leukemia
Treatment of ALL
Treatment of AML
Measurement of Response
to Therapy for Acute
Leukemia
Case Study 1
Case Study 2
Case Study 3

oS)Lo —
332. Chapter 16 Introduction to Leukemia and the Acute Leukemias

Introduction
Definition and Overview
Leukemia is a malignant disease of hematopoietic tissue,
characterized by replacement of normal bone marrow ele-
ments with abnormal (neoplastic) blood cells. These Type of Leukemia % of Cases
leukemic cells are frequently (but not always) present in the
peripheral blood and commonly invade reticuloendothelial Acute myelogenous (AML)
Acute lymphocytic (ALL)
tissue, including the spleen, liver, and lymph nodes. They
Chronic myelogenous (CML)
may also invade other tissues, infiltrating any organ of the Chronic lymphocytic (CLL)
body. If left untreated, leukemia eventually causes death.
Leukemia can be a rapidly progressive disease character- Source: Cancer Facts and Figures 2006. American
Cancer Society.
ized by an abnormal expansion of immature cells or blasts
(acute leukemia), or a slowly progressive disorder charac-
terized by an abnormal expansion of mature cells (chronic
leukemia). Acute and chronic leukemias are compared in 60 years of age or older, with the most prevalent incidence in
Table 16-1. the 70- to 90-year-old range! (see Chap. 17).
Leukemia can be divided into two major cell types: Leukemia can strike anyone of either sex. The frequency
myelogenous and lymphocytic. Each has an acute and chronic of all types of leukemia is slightly higher among males
form, which leads to four major types of leukemias: than females, with approximately 57% of new cases of
leukemia diagnosed in males in 2006.'In terms of race and
¢ Acute myeloid leukemia ethnicity, leukemia rates are higher among Americans of
¢ Chronic myeloid leukemia European descent than among African Americans, and the
e Acute lymphoblastic leukemia lowest among American Indians and Alaskan natives.' In the
¢ Chronic lymphocytic leukemia
United States, an estimated 208,080 individuals are living
The overall incidence of leukemia in the United States is 8 to with leukemia. !
10 new cases per 100,000 individuals per year. In 2006, an
estimated 35,070 new cases of leukemia were diagnosed in
the United States.' The percentages of new cases in 2006 for
Comparison of Acute and Chronic
each type of leukemia are estimated in Table 16—2.' Leukemia
Most cases of leukemia occur in older adults, with acute
The clinical and laboratory features of acute and chronic
myeloid leukemia (AML) and chronic lymphocytic leukemia
leukemia differ in a number of respects, as summarized in
(CLL) being the most common types. As the elderly popula-
Table 16—1. Patients with acute leukemia usually present with
tion continues to increase, leukemia will strike many more
a sudden onset of symptoms, and if left untreated, the disease
adults than children. Currently, the ratio of adult cases to child-
runs a rapidly fatal course of 6 months or less. In contrast,
hood cases is 10:1.' More than 50% of all cases of leukemia
patients with chronic leukemia tend to have an insidious
occur after the age of 64.' In children, the most common form
onset and a more indolent clinical course, usually lasting 2 to
of leukemia is acute lymphoblastic leukemia (ALL). Chronic
6 years. Mean survivals in chronic leukemia also increase as
leukemia, whether of lymphoid or myeloid lineage, is gener-
diagnostic sensitivities increase and patients are diagnosed at
ally considered to be a disease of adults. CLL is extremely rare
an earlier stage in their disease.
in children and is unusual before the age of 40 (see Chap. 20).
Patients with acute and chronic leukemia also show dif-
CML increases dramatically among individuals who are
ferences in their hematologic parameters. In general, bone
marrow failure and its sequelae are much more prominent in
the initial presentation of acute leukemia than in that of the
chronic form. In acute leukemia, there is a loss of normal
bone marrow function. Anemia is consistently observed in the
acute leukemic patient, although its severity is variable.
Thrombocytopenia is common. The white blood cell count
Chronic varies; it may be markedly elevated with numerous blasts, or
Adults it may be normal, or in some cases, even decreased. Typically
Clinical onset Insidious the various normal circulating white blood cells are dimin-
Course (untreated) 2-6 yr ished in number, resulting in a neutropenia. In chronic
Leukemic cells Immature Mature leukemic patients, anemia is often mild at presentation. The
Anemia Mild to severe Mild
Thrombocytopenia Mild to severe platelet count is usually normal, but it may actually be
Mild
White blood cell count Variable Increased increased in CML. Chronic leukemias almost always present
Organomegaly Mild Prominent with elevated white blood cell counts, which are often very
high (> 50,000/uL).!
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 333

Both acute and chronic leukemia patients can display Bone marrow and stem cell transplantation have improved the
enlargement of the spleen, liver, or lymph nodes, but this is survival of many patients with leukemia, especially those
more consistently seen in patients with chronic leukemia, in with acute forms. The effectiveness of treatment is most
whom organomegaly tends to be more prominent and can notable for children with acute lymphoblastic leukemia
occasionally be massive. (ALL). Before the 1960s, childhood ALL was universally
fatal, but with modern treatment regimens the survival rates
Historical Perspectives have dramatically improved over the past 30 years. The
majority of children with ALL younger than 15 years of age
The initial description of leukemia as a clinical entity was are now cured.' The survival rates, however, are not as
made by John Bennett in Scotland and Rudolf Virchow in impressive for adults with ALL and AML. For example, ALL
Germany, who independently published their findings in patients older than 60 years of age have five year survival
1845. They described a series of autopsy studies of victims of rates below 20%.° Similarly, AML patients older than
a progressive chronic disorder of unknown origin, in which 60 show dismal survival rates in most instances.? In addition,
enlarged spleens and purulent-appearing blood were found. survival rates among adults with leukemia show differences
The blood, when examined microscopically, revealed an according to race. Whites have slightly higher rates than
astounding increase in “coloriess” corpuscles. Bennett ini- many minority groups.'
tially suggested that the marked increase in white blood cells
was the result of an inflammatory process. Virchow chose the
term Weisses Blut (white blood), which was later translated Etiology and Risk Factors
into Greek as leukemia. He proposed that leukemia was
The origin of leukemia at the genetic level appears, in most
caused by a neoplastic proliferation of white blood cells. The
cases, to be related to mutation and altered expression of onco-
ensuing debate between Bennett and Virchow continued for
genes and tumor suppressor genes. Most oncogenes regulate
several years, but eventually even Bennett rejected inflamma-
cell proliferation and differentiation. Abnormal oncogene or
tion as the etiology of leukemia.
tumor suppressor gene expression induced by translocation
Virchow continued his study of leukemia, and defined
and genetic fusion or mutation often results in unregulated cel-
two groups characterized predominantly by either splenic or
lular proliferation. Although the events that lead to this are not
nodal involvement. Today these are recognized as chronic
entirely understood, a number of host and environmental fac-
myelogenous leukemia and chronic lymphocytic leukemia,
tors have been identified that are associated with increased risk
respectively (see Chaps. 17 and 20).
of leukemia transformation. The epidemiologic aspects of
In 1857 Friedreich gave a classic account of a rapidly
acute leukemia have been reviewed* and are summarized in
progressive form of leukemia, to which Epstein applied the
Table 16-3.
term acute in 1889. Further classification was made possible
in 1877 by Paul Ehrlich’s discovery of a tri-acid stain that per-
mitted the morphological characterization of blood cells. Classifications
Examination of the morphology enabled investigators to
show that acute leukemia was associated with immature cells Leukemia is classified according to cell type—with regard to
whereas chronic leukemia was associated with mature, well both cell maturity and cell lineage. Cell maturity is used to dis-
differentiated cells. At the turn of the century, Naegeli tinguish between acute and chronic forms of leukemia. When
described the myeloblast and divided the acute leukemias into the malignant cells are immature (stem cells, blasts, or other
myeloblastic and lymphoblastic forms. A decade later, immature precursors), the leukemia is classified as acute; when
Shilling described a monoblastic (blasts with the morphologic the cells are predominantly mature, it is described as chronic. In
features of immature monocytes) variant of the myeloblastic general these two groups correspond to a rapid (acute) or slow
form. Thus, the main morphological variants of acute and (chronic) natural history. Leukemias are further defined accord-
chronic leukemia were well established by 1930. Since that ing to cell lineage as lymphoid or myeloid. The term myeloid
time classification of leukemia has been refined, and clini- (from myelo, Greek for marrow, and eidos, form) encompasses
cally distinct subgroups have been characterized. granulocytic, monocytic, megakaryocytic, and erythrocytic
Today the clinical laboratory plays an important role in the leukemias. Today, there are two major classifications: the
diagnosis and classification of leukemia. The morphological French-American-British (FAB) and the World Health Organi-
analysis of leukemia is now augmented by cytochemical, cyto- zation (WHO), which are presented later in this chapter.
genetic, immunologic, and molecular techniques. Together The FAB and WHO classification schemes broadly divide
these methods are used to delineate specific categories of acute leukemia into acute lymphoblastic leukemia (ALL) and
leukemia for which distinct treatment protocols are used. acute myeloid leukemia (AML). Alternate names for AML
Certainly the most significant application of the biologic include “acute nonlymphoblastic leukemia (ANLL),” “acute
understanding of leukemia has been the improvement in out- myelogenous leukemia,” “‘acute myeloblastic leukemia,” and
come as a result of continued advances in treatment. Complex “acute granulocytic leukemia.”
therapeutic protocols with multiple cytotoxic drugs and radi- The types of acute leukemias, their common abbrevia-
ation, as well as more “targeted,” less toxic approaches such tions, the FAB classification, and their alternate names are
as tyrosine kinase inhibitors and protease inhibitors are used. listed in Table 16-4.
334 Chapter 16 Introduction to Leukemia and the Acute Leukemias

7 thine ae
taple 16-3 Host and Environmental Factors Associated with an Increased Risk
of Leukemia

Host Factors Environmental Factors

Hereditary Ionizing radiation

Congenital chromosomal abnormalities Chemicals (e.g., benzene)
Chromosomal fragility
Bloom’s syndrome
Fanconi’s anemia
Abnormal chromosomal number Drugs (e.g., chloramphenicol, phenylbutazone
Down syndrome (10- to 20-fold increase) alkylating drugs)
Klinefelter’s syndrome
Turner’s syndrome
Immunodeficiency Viruses
Ataxia-telangiectasia Human T-cell leukemia/lymphoma
Sex-linked agammaglobulinemia virus | (HTLV-1)
Chronic marrow dysfunction HTLV-2
Myelodysplastic syndrome Epstein—Barr
Myeloproliferative disorders
Aplastic anemia
Paroxysmal nocturnal hemoglobinuria

Table 16-4 Classification of Leukemia

ae ege e °

Type of Leukemia Abbreviation Alternate Names

Acute Myeloid Acute nonlymphoblastic

(ANLL)
Acute myeloblastic leukemia
without cytologic maturation
With minimal maturation
With maturation
Acute promyelocytic leukemia APL Hypergranular promyelocytic
Acute myelomonocytic leukemia AMML Naegeli-type leukemia
Acute monocytic leukemia AMoL Schilling-type leukemia
Erythroleukemia AEL Di Guglielmo’s syndrome,
erythremic myelosis
Acute megakaryoblastic leukemia AMegL

“French—American—British classification of acute leukemia.

Introduction to Acute Leukemia Clinical Features

Incidence The majority of patients with acute leukemia display clinically

abrupt onset of signs and symptoms of only a few weeks’ dura-
Acute leukemia occurs in individuals of all ages, but ALL is tion. Patients often seek medical attention because of weakness,
more common in children and AML is more common in bleeding abnormalities, or flu-like symptoms. These abnormali-
adults. Seventy-five percent of childhood leukemias are clas- ties reflect the failure of the bone marrow to produce adequate
sified as ALL whereas nearly 80% of AML cases occur in numbers of normal cells and are caused by the proliferation and
adults.! accumulation of leukemic cells in the marrow. Leukemic
The incidence of acute leukemia increases exponentially replacement eventually results in marrow failure and the resul-
with age, and the median age at diagnosis of acute myeloid tant life-threatening complications of anemia, thrombocytope-
leukemia (AML) in the United States is currently 63 years.’ nia, granulocytopenia, and their sequelae. Anemia, the most
Males have a slightly increased incidence compared to consistent presenting feature, is associated with fatigue, malaise,
females. In 2008, there were a total estimated number of and pallor. Hemorrhagic complications related to thrombocy-
13,290 new cases of AML and 5430 new cases of ALL in the topenia and, in some cases, to disseminated intravascular coag-
United States. ! ulation (DIC), are also common. These may be mild and
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 335

restricted to easy bruising, petechiae, and mucosal bleeding; or

they may be more severe, involving gastrointestinal tract, geni- Table 16-5,
5 Clinical Features of
tourinary tract, or central nervous system hemorrhage. Infec- de Acute Leukemia
tions result from severe granulocytopenia. Bacterial infections
are common (e.g., Staphylococcus, Pseudomonas, Escherichia Pathogenesis) Clinical Manifestations
coli, and Klebsiella) but fungal infections also occur (e.g.,
Bone Marrag Failure
|
Candida and Aspergillus). Viral infections are less frequent. Anemia Fatigue, malaise, pallor
Infiltration of other tissues, especially organs that Thrombocytopenia Bruising, bleeding
play a role in fetal hematopoiesis, is often manifested by Granulocytopenia Fever, infections
hepatosplenomegaly or lymphadenopathy, particularly in ALL Organ Infiltration
and the acute monoblastic leukemia, a subtype of AML. A Marrow expansion Bone or joint pain
myeloid or granulocytic sarcoma is defined as a tumor mass of Spleen Splenomegaly
myeloid cells outside of the marrow and may be the first Liver Hepatomegaly
evidence of AML. Gingival hypertrophy and oral lesions are Lymph nodes Lymphadenopathy
Central nervous system Neurologic symptoms
primarily seen in acute monoblastic leukemia. A mediastinal
Gums, mouth Gingival hypertrophy, oral
mass resulting from thymic involvement is a hallmark of pre- lesions
cursor T-ALL. Bone or joint pain, caused by pressure of the
expanding leukemic cell population in the marrow cavity, com-
monly accompanies the acute leukemias. Leukemic infiltration
of the central nervous system, an ominous feature infrequently Laboratory Evaluation of Acute Leukemia
observed at initial presentation, is associated with signs and
symptoms of increased intracranial pressure (nausea, vomiting, When acute leukemia is suspected, a series of laboratory tests
headache, papilledema) or cranial nerve palsies. These clinical confirms the diagnosis and helps classify the disease. Infor-
features and their relationship to pathophysiology are summa- mation from the laboratory will help determine prognosis and
rized in Table 16-5. guide therapy. The distinction between AML and ALL 1s par-
Between 5% and 10% of cases of AML are preceded ticularly important; major features of this distinction are out-
by a recognizable myelodysplastic or “‘preleukemic” syn- lined in Table 16-6.
drome. The myelodysplastic syndromes (MDS) are more Preliminary evaluation includes a complete blood count
common in patients older than 50 years of age and are (CBC), platelet count, white cell differential, and peripheral
associated with unexplained and _ persistent anemia, blood smear examination. Anemia, which may be mild to
leukopenia, thrombocytopenia, and monocytosis, alone or severe and is usually normocytic normochromic, is a consis-
in combination.® These peripheral cytopenias are typically tent finding. Platelet counts are typically decreased. The white
associated with a hypercellular bone marrow. Although blood cell count is variable, ranging from significantly
myelodysplastic syndromes often progress with increasing decreased to markedly elevated. The blood smear usually
blast counts, not every person with MDS will progress to reveals blasts or other immature cells, but they may be rare or
acute leukemia. The myelodysplastic syndromes are dis- absent (“aleukemic” leukemia). Circulating nucleated red
cussed in more detail in Chapter 19. blood cells are occasionally seen. Myelodysplastic features

Table 16-6. Comparison of Acute Myeloblastic. and Acute Lymphoblastic Leukemia ee

Factor AML” ALT

Age Goren in one rare inPehildren Connon innila rare inaddi

Blood Anemia, neutropenia, thrombocytopenia Anemia, neutropenia, thrombocytopenia;
myeloblasts and promyelocytes lymphoblasts and prolymphocytes
Morphology Medium-to-large biasts, more cytoplasm Small or medium blasts, scarce
than lymphoblasts, cytoplasmic cytoplasm, no granules; fine nuclear
granules, Auer rods; fine nuclear chromatin and indistinct nucleoli
chromatin and distinct nucleoli
Cytochemistry Positive peroxidase and Sudan Negative peroxidase and Sudan black;
black; negative TdT positive TdT
Extramedullary and Common in spleen and liver; less Common in lymph nodes, spleen, liver,
_focal disease common in ier nodes and CN CNS, and ‘gonads _

eae ae = acuteSlimehe
AML= acute Ferelsbiasticle leukemia; TdT = ne Rope teens transferase;
CNS= central nervous system.
Source: From Kjeldsberg, CR (Ed): Practical Diagnosis of Hematologic Disorders. ASCP Press, Chicago, 1989, p 349,
with permission.
336 Chapter 16 Introduction to Leukemia and the Acute Leukemias

are sometimes present, including pseudo-Pelger—Huét cells SPECIMENS

and hypogranular neutrophils. These are more common in At the outset, care must be taken to ensure that an adequate
elderly patients with AML. specimen is obtained and that it is properly handled. Lack
Once the diagnosis of leukemia is suggested by the clin- of technical excellence may obscure or complicate an
ical history, physical examination, CBC, and peripheral blood otherwise straightforward diagnosis; an inadequate or
smear findings, a bone marrow aspirate and biopsy is usually improperly handled specimen is a common cause of diag-
obtained. Morphological examination of the bone marrow is nostic error.
usually required to establish the diagnosis. The WHO classi- Evaluation of the peripheral blood cell morphology is
fication system for acute leukemias requires a minimum blast routinely performed by making a smear from blood drawn in
count of 20% in the peripheral blood or bone marrow for con- ethylene diaminetetraacetic acid (EDTA) (purple top) tube.
firmation of the diagnosis of acute leukemia in comparison to The anticoagulant EDTA causes subtle morphological arti-
the FAB classification which requires a blast count of 30%.° facts of nucleated cells and platelets. A specimen left in
Although a diagnosis of acute leukemia may be established EDTA for longer than 30 minutes may show artifactual vac-
by a peripheral blood examination, well-prepared smears of uolation of monocytes and neutrophils, nuclear shape changes
bone marrow aspirate material are still the specimen of choice and swelling, as well as degranulation of platelets.? These
for classification of leukemia by morphological and cyto- possible alterations should be kept in mind if only a routine
chemical criteria. EDTA-anticoagulated peripheral blood smear is available for
A systematic approach to the classification of leukemia review. A smear made from a nonanticoagulated fingerstick
begins with a review of cell morphology, which provides will not show these abnormalities.
important clues about cell lineage and guides further studies Before a bone marrow specimen is collected, arrangements
required to make a definitive diagnosis. Morphological eval- for any special studies, including cytochemistry and flow cyto-
uation should be followed by immunologic cell marker stud- metric immunophenotyping, should be made, and any special
ies and in some cases cytochemical staining. Cytochemical handling procedures should be noted; such procedures are also
stains help define granulocytic and monocytic differentiation important to cytogenetic and ultrastructural studies. During the
and continue to be useful to distinguish AML from ALL. bone marrow procedure, the aspirate is collected first and
When positive, cytochemical studies permit subclassification smears are made immediately to avoid clotting. As the aspirate
of most cases of AML and exclude ALL. Immunologic analy- smears are pulled, the presence of bone marrow spicules should
sis is routinely done on new acute leukemias using flow be confirmed. If spicules are not present, another aspiration may
cytometry, which allows determination of blast immunophe- be necessary. For immunophenotyping or cytogenetic studies,
notype. There are many useful flow markers, one of which is the marrow aspirate is anticoagulated by aspirating directly into
the nuclear enzyme terminal deoxynucleotidyl transferase a syringe coated with heparin. After sufficient aspirate material
(TdT), present in most cases of ALL and rarely expressed in is collected, the biopsy is obtained. Care should be taken to
AML. Other immunologic cell marker studies are routinely ensure that the biopsy has an adequate amount of bone marrow,
used to help determine myeloid or lymphoid lineage and for because a biopsy consisting only of periosteum and cortical
further subclassification.’ bone is useless and inadequate. If adequate, the biopsy should
Increasingly important are laboratory studies that help be used, before fixation, to make touch preparations by gently
identify genetic abnormalities in the malignant blasts, touching or rolling the biopsy along a glass slide. The biopsy
particularly as pharmacologic agents are developed to should be blotted before making the touch preparations, because
specifically target the effects of a particular genetic abnor- excess blood will obscure morphological detail. Touch prepara-
mality. Such laboratory studies include conventional kary- tions are especially important when the aspirate produces a
otyping, molecular genetic studies, DNA flow cytometry, “dry” tap. Further details of the bone marrow procedure are
and rarely, electron microscopy. The molecular genetic discussed in Chapter 2.
studies commonly use fluorescent in situ hybridization
(FISH) and polymerase chain reaction (PCR) analysis.® EVALUATION OF MORPHOLOGY
Chromosome analysis is a valuable tool in identifying prog- Cellular morphology is evaluated on a Romanowsky (Wright-
nostically important subgroups of patients, and providing Giemsa) stained blood or bone marrow smear in carefully
baseline data that may be useful in monitoring patients. chosen areas in which cells are not distorted by overcrowding.
Molecular diagnostic studies play an important and expand- The experienced morphologist will often be able to identify
ing role in primary diagnosis and detection of minimal blasts and classify the leukemic cell type as myeloid or lym-
residual disease. DNA flow cytometry yields prognostic phoid; however, additional testing is always necessary to con-
information in patients with ALL, with hypodiploidy asso- firm the diagnosis.
ciated with poor prognosis and hyperdiploidy of greater Several morphological features (outlined in Table 16-7)
than 50 chromosomes associated with a favorable progno- are helpful in distinguishing lymphoblasts from myeloblasts.
sis. Electron microscopy is now rarely utilized, except in These include the size of the blast, amount of cytoplasm,
evaluation of poorly differentiated leukemia or acute nuclear chromatin pattern, and the presence of nucleoli. The
megakaryoblastic leukemia. typical myeloblast (Fig. 16-1) is a large cell (15 to 20 um in
The purpose and principles of these laboratory methods diameter) with a moderate amount of cytoplasm. Its nucleus
and detailed procedures are outlined in Chapters 34 and 35. has a fine, reticulated chromatin pattern, and multiple distinct
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 337

Feature

Blast size Prvean Fras nee Variable, aah to ieee size

Nuclear chromatin Usually finely dispersed Coarse to fine
Nucleoli 1-4, often prominent Absent or | or 2, often indistinct
Cytoplasm Moderately abundant, fine granules often Usually scant, coarse granules
present sometimes present (~7%)
Auer rods Present in 60%-—70% of cases Not present
Other cell types Often dysplastic changes in maturing Usually not dysplastic
myeloid cells —

nee From Kickster CR2 (Ed): FPractical

Hernan of foaanee Terie ee
ed 2. ASCP ee Gis 1995, p5 381,
with permission.

nucleoli are often present. The typical lymphoblast (Fig. 16—2) important aid in the classification of acute leukemia because
is a smaller cell with scant cytoplasm. The nuclear chromatin they identify cellular components that are associated with
often appears denser than in the myeloblast, and nucleoli are specific cell lines. For example, a positive myeloperoxidase
usually indistinct when present. For a review of morphologi- or Sudan black B stain indicates myeloid differentiation, and
cal descriptions of the blast stage, see Chapter 1. a positive nonspecific esterase stain indicates monocytic dif-
Granulocytic differentiation is suggested by the presence ferentiation. When any of these stains are positive even in a
of azurophilic granules. A very helpful morphological feature is relatively small percentage of blasts, lymphoid origin is ruled
the Auer rod, the presence of which excludes ALL. Auer rods out, with rare exceptions. Thus, the cytochemical stains help
are cytoplasmic inclusions that result from an abnormal fusion distinguish between ALL and AML. They are also used to
of primary granules and are pathognomonic for a myeloprolif- subclassify AML.
erative process, particularly AML. On Romanowsky-stained The cytochemical reactions are performed by applying
smears, Auer rods appear as pink- or purple-staining rods or staining techniques to peripheral blood smears, bone mar-
splinter-shaped inclusions (see Fig. 16-1). They are present in row smears, or touch preparations. Fresh preparations are
up to 60% of patients with AML,” but it may take a long, care- preferred, especially for enzyme reactions. Control smears
ful review of the blood or marrow smear to find them; given can be fixed in the appropriate fixative, allowed to air-dry,
their diagnostic importance, this search is well worth the effort. and stored at —20°C for future staining. Caution should be
In acute promyelocytic leukemia, Auer rods are easy io find, taken when interpreting cytochemical stains. It is the
some cells having “bundles” of cigar-shaped rods and are leukemic cell population whose identity (cell lineage) is in
termed “faggot cells.” question; therefore, a positive reaction is determined by
finding positive staining in the leukemic blasts rather than in
CYTOCHEMISTRY mature cells. For a description of all the cytochemical stains
Special stains are used to identify chemical components of cells including principles of reactions, methods and interpreta-
such as enzymes or lipids. These cytochemical stains are an tions, refer to Chapter 36. The cytochemical reactions that

Figure 16-1 m™ Myeloblast (note the Auer rod). Figure 16-2 ™Lymphoblasts (peripheral blood).
338 Chapter 16 Introduction to Leukemia and the Acute Leukemias

Special Stain Site of Action Cells Stained . Comment

Myeloperoxidase Mainly primary Late myeloblasts, granulocytes; Valuable in that the primary
granules; Auer rods monocytes less intensely granules are not always
visible; separates AML
(+ blasts) from ALL (— blasts)
Sudan black B Phospholipids: Late myeloblasts, granulocytes; Parallels peroxidase, but
sterols, neutral fats monocytes less intensely smears do not need to be
fresh
Specific esterase (Naphthol Cytoplasm Neutrophilic granulocytes; Parallels peroxidase, but
AS-D chloroacetate) mast cells less sensitive; useful on
paraffin-embedded tissues
Nonspecific esterase Cytoplasm Monocytes; focal staining in Useful for determining
Alpha-naphthy! acetate T cells; ANAE also + degree of monocytic
(ANAE) and butyrate in megakaryocytes differentiation; separates
mono (+) from myelo
(—) blasts
Periodic acid—Schiff Glycogen and related Lymphocytes, granulocytes, Helpful in supporting
substances megakaryocytes diagnosis of
erythroleukemia

are useful in the classification of acute leukemia are summa- granulocytic cells and, to a lesser extent, in monocytic lyso-
rized in Table 16-8. somal granules.
The SBB is the most sensitive stain for granulocytic pre-
MYELOPEROXIDASE (MPO) Peroxidase is present in the cursors, with a staining pattern that generally parallels the
primary granules of myeloid cells (Fig. 16-3). These granules myeloperoxidase stain. As with the peroxidase stain, the SBB
first appear in the early promyelocyte (late blast) and persist is used to differentiate AML from ALL. Positivity seldom
through subsequent stages of cell maturation. Monocytes occurs in lymphoid cells; SBB is less specific than MPO. The
have variable staining with peroxidase and are most often SBB stain, whose reactivity does not diminish with time, is
only weakly positive. This enzyme is not present in lympho- particularly useful for specimens that are not fresh.
cytes or their precursors and is, therefore, useful in differenti-
ating AML from ALL. It is more specific for granulocytic SPECIFIC ESTERASE (NAPHTHOL AS-D CHLOROACETATE)
differentiation than the Sudan black B stain. The specific esterase stain (Fig. 16-5), commonly referred to
as chloroacetate esterase (CAE), roughly parallels the perox-
SUDAN BLACK B (SBB) Phospholipids, neutral fats, and idase and SBB stains, although it is not as sensitive. It is
sterols are stained by Sudan black B (SBB) (Fig. 16-4). Phos- negative in eosinophils and monocytes, but positive in neu-
pholipids occur both in primary and secondary granules of trophils, basophils, mast cells and their precursors. Its most

Figure 16-3 ™ Myeloperoxidase positivity in acute promyelocytic Figure 16-4 @ Sudan black B positivity in acute myeloblastic
leukemia. leukemia (AML), M2.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 339

diffuse staining pattern and lymphocytes have a granular pat-

tern. Normal erythroid precursors are negative.
The PAS reaction is not very useful for characterizing
acute leukemia and it is no longer commonly used for classi-
fication. The typical block positivity (Fig. 16-7) associated
with lymphoblastic leukemia may also occur in acute myeloid
leukemia, especially the acute monocytic type and ery-
throleukemia. Strong PAS positivity may be present in ery-
throblasts in erythroleukemia, a potentially helpful feature for
differentiating erythroleukemia from pernicious anemia, in
which the PAS reaction is negative except in rare cases. Nor-
moblasts may also be positive in iron deficiency, thalassemia,
severe hemolytic anemias, and some of the myelodysplastic
syndromes. The PAS stain, however, does not reliably distin-
guish AML from ALL, and typically other methods are
Figure 16-5 @ Specific esterase (naphthol AS-D chloroacetate) employed to make clinically relevant distinctions.
positivity in AML, M2.
IMMUNOLOGIC MARKER STUDIES
important use is in demonstrating myeloid differentiation in A number of immunologic methods are indispensable to the
paraffin-embedded tissue sections. diagnosis and classification of acute leukemia. Antibodies
detect markers associated with cell lineage (lymphoid vs.
NONSPECIFIC ESTERASE (ALPHA-NAPHTHYL ACETATE myeloid) and maturation stage. Occasionally, leukemic blasts
OR BUTYRATE) The nonspecific esterase (NSE) stain have an aberrant immunophenotype that facilitates detection
(Fig. 16-6) is used to identify monocytic cells. It is diffusely of a relatively low level of an abnormal blast population. For
positive in these cells and negative in granulocytic cells. Lym- example, myeloid blasts at diagnosis of an AML may aber-
phoid cells are negative, except for T lymphocytes, which can rantly express CD7, which is an antigen commonly seen on
demonstrate a focal dotlike cytoplasmic staining pattern. T and NK lymphocytes but not on myeloid cells. After treat-
Different substrates are available for use in the NSE ment, flow cytometric analysis may specifically look for these
stain. Alpha (a)-naphthyl butyrate is the most specific for immunophenotypically aberrant CD7 expressing myeloid
monocyte differentiation, whereas a-naphthyl acetate is more blasts to help identify low levels of remaining leukemic
sensitive. Both are positive in monocytes and their precursors blasts.
as well as in macrophages. The e-naphthyl acetate, but not the In clinical practice, there are essentially two techniques
butyrate, is also positive in megakaryocytes and platelets. used to detect antigens either on the blast cell surface or
within the blast cytoplasm: flow cytometry and immunohisto-
PERIODIC ACID-SCHIFF The periodic acid—Schiff (PAS) chemistry. Flow cytometry requires a cell suspension and is
reaction stains for glycogen and related compounds, includ- best done on peripheral blood or bone marrow aspirate.
ing mucoproteins, glycoproteins, glycolipids, and polysac- Immunohistochemistry is typically done on paraffin sections
charides. Stains of lymphocytes, granulocytes, monocytes, of the core biopsy, clot section made from the aspirate, or
and megakaryocytes may be positive. Granulocytes have a on paraffin sections of other biopsy material. The following

Be ea

Figure 16-6 m™ Nonspecific esterase (alpha-naphthy! butyrate) pos- Figure 16-7 ® Periodic acid-Schiff positivity in acute lymphoblastic
itivity in acute monocytic leukemia (M5). leukemia (ALL). Note the “block” staining pattern.
340 Chapter 16 Introduction to Leukemia and the Acute Leukemias

paragraphs briefly introduce the immunologic cell marker The availability of myeloid-specific monoclonal anti-
procedures. Their utility in the evaluation of acute leukemia is bodies has permitted surface marker analysis of AML. The
discussed in the individual sections on AML and ALL. rationale for this is based on the notion that AML is a clonal
disorder derived from a myeloid stem line, which displays the
CELL SURFACE MARKERS Cell surface markers are pro- surface membrane antigens expressed in normal myeloid dif-
teins on the cell membrane that can be detected using flow ferentiation pathways. A schematic representation of surface
cytometry and immunohistochemistry. Different stages of antigen expression in normal myeloid differentiation is shown
maturation express different proteins. Some proteins are pre- in Figure 16-8.
sent early in development whereas others do not appear until Surface marker analysis has begun to replace conven-
much later. Still other proteins may appear, disappear, and tional cytochemical methods for lineage determination in
then reappear at a later stage of development. This unique acute leukemia. Surface marker studies are used to distin-
expression of proteins enables the diagnostician to use these guish AML from ALL, particularly when the leukemic cells
proteins as markers of both cell lineage and maturation stage. are poorly differentiated or negative and indistinct with cyto-
Although some surface markers can be detected with chemical stains, such as in the case of AML, minimally
polyclonal antisera, many can be detected only with mono- differentiated.
clonal antibodies. Some antibodies that are commonly used to When surface marker analysis is performed to distinguish
evaluate leukemias and other lymphoproliferative and myelo- AML from ALL, it is important to choose a panel of markers
proliferative disorders are listed in Table 16-9. that includes antibodies to several myeloid-associated antigens

od
table 16-9 Monoclonal Antibodies Used for Study of Leukemia _
and Lymphoma 4

Cluster Designation Specific Antibodies Major Hematopoietic Reactivity

CDia T6, Leu-6 Thymic and Langerhans’ cells

CD2 Tie eu=5 E-Rosette-forming T cells
CD3 T3, Leu-4 Mature T cells
CD4 T4, Leu-3 Helper-inducer T-cell subset
CD5 Tl, Leu-1 Pan-T and some B cells
Diz Leu-9 Pan-T, early thymocytes
CD8 T8, Leu-2 Suppressor-cytotoxic T-cell subset
CD10 Jon GAMEA B-cell pre, some thymocytes, grans and ALL
CD11b Mol, Leu-15 Monos and grans, C3bi receptor
CDI 1c Leu-M5 Monos, myeloid precursors, HCL
CD13 My 7 Most grans, minority of monos, and other
CD14 My 4, Leu-M3 Monos, minority of grans, and DRC
DIS Leu-M1 Myeloid cells, RS
CD19 B4, Leu-12 B cells, early B-cell precursors
CD20 Bl, Leu-16 B cells, midstage B-cell precursors
opyAl B2 C3d receptor on B cells and DRC
CD22 Leu-14 B cells, ALL, HCL
CD23 Bo B cells, EBV-transformed B lymphoblasts
CD25 IL-2R1, IL-2R IL-2 receptor on T cells and other, HCL, NHL
CD30 Ber-H2 Activated B and T cells, RS, ALCL
Cbss My 9 Myeloid progenitors
CD34 My 10, HPCA Hematopoietic progenitor-stem cells
CD38 T10, Leu-17 Plasma cells, ALL, AML
CD41 INS} Plts and megakaryocytes (GPIIb/IIa), AML M7
CD42b ANS1 Plts and megakaryocytes (GPIb), AML M7
CD43 DF-T1 T cells, myeloid cells
CD45 T200, HLe, LCA Leukocytes
CD56 Leu-19 NK cells, T-cell subset
CD57 Leu-7 T cells, NK cells
CD61 gplila Pits, AML (M7)
CD75 LN-1 B cells, follicular center cells
CD79a MB-1 B cells, ALL
CD103 HML-1 Intraepithelial lymphocytes, HCL
HLA-DR, la B cells, activated T cells, and monos
CD = cluster designation; B cells = B lymphocyte; T cells = T lymphocyte; Pan = reactivity with many leukocyte
populations; ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia; DRC = dendritic reticulum
cells;
grans = granulocytes; monos = monocytes; Plts = platelets; RS = Reed Sternberg cells; HCL = hairy
cell leukemia;
ALCL = anaplastic large cell lymphoma; NK = natural killer cells.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 341

Monocyte Promonocyte Monoblast Myeloblast Promyelocyte Myelocyte PMN

(CD14) My4 [ear
(CD11b) Mot |”

A
(CD33) Myo r ae r
(CD13) My7 |
HLA-
DR

Figure 16-8 ® Distribution of myeloid and monocytic surface antigens with bars depicting the strength of antigen reactivity. The wider the
bar, the stronger the reaction, the narrowing indicating a weaker reaction. PMN = polymorphonuclear neutrophil; CFU-GM = colony-forming
unit granulocyte macrophage/monocyte.

(e.g., CD33, CD13, and CD14), as well as B- and T-lymphoid Cytoplasmic markers are very useful in assessing cell
antigens.'' Multiple myeloid lineage-specific markers are nec- lineage in ALL. Cytoplasmic CD3 1s present early in T-cell
essary because no single marker defines all forms of AML. development, and strong expression is seen in most cases of
This topic will be addressed later in the chapter in the section precursor T-cell ALL. Likewise, the presence of cytoplasmic
on “acute leukemias of ambiguous lineage.” CD22 and CD79a aids in defining B-cell lineage in ALL.
The surface phenotypes expressed in cases of AML do not The presence of cytoplasmic IgM heavy chain (\) is a
correlate absolutely with the WHO classification or with spe- defining characteristic of pre-B cells; the earlier precursor
cific genetic alterations, although certain trends exist.° For B cells are cytoplasmic pt negative. Pre-B-cell ALL has tradi-
example, cases of AML with monocytic differentiation may tionally been associated with a worse prognosis than early-
express monocyte-related surface markers (CD14), and cases of pre-B-cell ALL. However, the poor prognosis is now known
acute promyelocytic leukemia tend to express a promyelocytic to be more closely linked to the frequently associated translo-
phenotype (CD13 and CD33 positive, HLA-DR and CD34 neg- cation, t(1;19) in pre-B-cell ALL, rather than to the pre-B-cell
ative). Other AML subgroups exhibit widely variable pheno- phenotype.'* Accordingly, cytoplasmic pu staining is no longer
types. Occasionally, mixed lineage phenotypes are encountered, routinely performed in many laboratories; instead, FISH is
with coexpression of myeloid and lymphoid markers. These now used to identify t(1;19) and other prognostically impor-
biphenotypic or bilineage leukemias may reflect origination of tant cytogenetic abnormalities.
the malignant cell line from an early progenitor capable of both Another helpful cytoplasmic marker used in immunophe-
myeloid and lymphoid differentiation. notyping acute leukemias is cytoplasmic myeloperoxidase.
Flow cytometric analysis requires a cell suspension, typi- This myeloid marker is often used as part of a flow cytometric
cally from bone marrow aspirates or peripheral blood. It is panel, in addition to the traditional myeloid markers, CD13
important to have fresh specimens with viable cclls; nonviable and CD33. Immunohistochemical stains for cytoplasmic
cells lead to nonspecific staining, which may make interpreta-
tion impossible. An immunofluorescent method (direct or
indirect) is used to stain the cells, and a flow cytometer is used
to analyze them. (See Chap. 34 for a review of flow cytometry.)
The surface-staining pattern, as observed with a fluorescent
microscope, that is typical of a strongly positive reaction is
shown in Figure 16-9.

CYTOPLASMIC MARKERS Cell marker studies can also be

directed at cytoplasmic antigens using either immunohisto-
chemistry or flow cytometry. When using flow cytometry, an
additional step must be taken to fix and permeabilize the cells
to allow antibody to enter through the cell membrane and into
the cytoplasmic space. Often the quantity of antigen on the
cell surface and in the cytoplasm varies, and some antigens
are exclusively cytoplasmic. Plasma cell neoplasms, for
example, often have very weak surface and strong cytoplas- Figure 16-9 @ Surface immunoglobulin (slg)-positive cells in B-cell
mic immunoglobulin staining intensity. ALL, L3.
342 Chapter 16 Introduction to Leukemia and the Acute Leukemias

myeloperoxidase can also been performed on formalin-fixed, Philadelphia chromosome t(9;22) associated with CML and
paraffin-embedded sections. However, immunohistochemical the translocation t(15;17) consistently observed in acute
staining with currently available antibodies does not seem to promyelocytic leukemia. The WHO classification for AML
be as specific for myeloid lineage as either the traditional includes six entities with recurrent cytogenetic abnormalities,
cytochemical method or flow cytometry. each with clinical significance. Cytogenetic abnormalities
also provide important prognostic information in ALLs. In
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE Anti- addition to cytogenetic evaluation, flow cytometric analysis
bodies can also be directed at nuclear antigens using the for DNA ploidy status provides useful prognostic information
same cell membrane permeabilization procedure. Terminal mA
deoxynucleotidyl! transferase (TdT) is a unique nuclear enzyme Cytogenetic analysis may be carried out in different
(DNA polymerase) present in stem cells and precursor B- and ways, including karyotyping, FISH, PCR, or sequence analy-
T-lymphoid cells.'* High levels are found in the majority (90%) sis of particular base pairs. Karyotyping evaluates stained
of the lymphoblastic leukemias, including both B- and T-lineage chromosome metaphase preparations to detect the numeric
ALL. TdT, although not lineage specific, provides information and structural karyotype (from karyon, Greek for nucleus,
useful in the distinction of ALL from AML. TdT is present in plus typos, meaning mark) abnormalities. Normal human
most cases of lymphoblastic lymphoma and in approximately cells have 46 chromosomes and are diploid (diplous, double,
one-third of cases of chronic myeloid leukemia in ALL blast plus eidos, form), having two alleles of each gene. That is,
crisis (CML-BC).'* Detectable levels of this enzyme are also in they have two haploid sets of 23 chromosomes. An aneuploid
5% to 10% of the cases of AML,'* although the level is usually population can be either hypodiploid or hyperdiploid, and can
significantly lower than in lymphoblasts.'* be identified as such by DNA flow cytometry as well as by
TdT is detected using a number of immunologic meth- cytogenetic examination.
ods, including flow cytometry and immunohistochemical Karyotyping is capable of detecting structural rearrange-
methods, that laboratories routinely use. It is important to note ments, including translocations, inversions, deletions, duplica-
that only nuclear staining 1s considered a_ positive tions, and isochromosomes. Current standard karyotyping,
reaction on immunohistochemical stains. TdT can also be however, will fail to detect chromosomal abnormalities in
detected by a variety of other immunologic techniques 40 to 50% of AMLs.'° Other methods, such as FISH, can
(ammunocytochemical or immunofluorescent), using fresh detect very small but significant genetic abnormalities that will
smears, touch preparations, or cytopreparations (Fig. 16—10). not be apparent by standard karyotyping. Some mutations
When heparinized specimens are studied, removing the (such as NPM1 and FLT3-ITD mutations), will look normal by
heparin by washing the sample is essential to reducing false- current standard cytogenetic techniques, including FISH.
negative results. Of particular importance to the study of acute leukemia are
the translocations that result from the movement of a DNA
CYTOGENETICS segment from one chromosome to another and that are often
Cytogenetic analysis of leukemic cells is an essential compo- associated with oncogene rearrangement, genetic fusion, and
nent in the evaluation of the newly diagnosed leukemic abnormal gene expression. Translocations result from a recipro-
patient. It plays a major role in diagnosis, subclassification, cal interchange of portions of two nonhomologous chromo-
prognosis, selection of appropriate therapy, and monitoring somes and are the most common structural chromosomal
the effects of'therapy. In the WHO classification a number of abnormalities. A number of these are associated with distinct
chromosomal abnormalities have been associated with dis- subgroups of AML or ALL and have prognostic significance.
tinct forms of leukemia. Classic examples of this are the For example, patients with acute myelomonocytic leukemia
who exhibit an inversion or deletion of the long arm of chromo-
some 16 (inv[16q] or del[16q]) have a longer median survival
than patients with other types of AML.
Chromosomal abnormalities (both numeric and struc-
tural) are found by routine clinical methods in the majority
of patients with acute leukemia. Importantly, just because
current techniques do not identify a genetic abnormality in
a patient’s acute leukemia does not mean that there is none.
As laboratory techniques improve, the percentages of
genetic abnormalities identified in both AML and ALL
will increase. The recent development of fluorescently
labeled chromosome painting probes and automated differ-
ential color analysis (automated spectral karyotyping)
should greatly enhance the sensitivity and accuracy of
karyotypic analysis. This technique holds great promise
for the identification of additional chromosomal abnormal-
Figure 16-10 ® TdT positivity in ALL using immunofluorescence ities in leukemic patients, who frequently have complex
method. karyotypes.'°
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 343

Table 16-10 lists common cytogenetic abnormalities assays, but are valuable at diagnosis and relapse. FISH has
associated with acute leukemia. several advantages, including the ability to use cells in either
metaphase or interphase, the ability to use paraffin-embedded
MOLECULAR GENETICS tissue, and the ability to detect some abnormalities not seen
The molecular genetic basis for many of the acute leukemias by conventional karyotyping.'* FISH and PCR are used
has been elucidated in recent years. Extensive studies of the primarily for confirmation of the presence of a suspected
immunoglobulin genes, T-cell antigen receptor genes, and chromosomal abnormality not detected by conventional cyto-
other genes involved in the many recurrent cytogenetic abnor- genetics, for the analysis of specimens that are not suitable for
malities described in the preceding section have been cloned conventional cytogenetics, for confirmation of relapse, and
and characterized.'* DNA FISH probes and polymerase chain for the monitoring of minimal residual disease following
reaction (PCR)-based primers are available for rapid and pre- therapy.'*
cise detection of many specific cytogenetic abnormalities at These methods permit detection of residual leukemic dis-
the molecular level.'° FISH assays are not as sensitive as PCR ease at extremely low levels, and clinical applications of these

alities and Mol cular Correlates ere

Chromosome Involved Genes on

Abuormality “Associated. Disorder Respective Comes: piece

(6:9) AML (M2) and AMML DEK-CAN RT-PCR

(M4) with marrow basophilia
(8:21) AML with myelocytic maturation ETO-AMLI Southern blot
(M2) (associated with a favorable RIERER
prognosis and good response
to therapy)
t(15;17) Unique to APL (M3 and M3m) PML-RARa RT-PCR or FISH
(associated with a favorable
prognosis)
t(11;17) APL variants; not ATRA responsive PLZF-RARa
t(5;17) NuMa—RARa
16q abnormality: AMML with abnormal eosinophilia CBFB-MYHI1 RT-PCR or FISH
inv(16) and del(16) (M4Eo) (associated with a favorable
prognosis)
(9322) Most common in CML; occasionally ABL-BCR RT-PCR or FISH
found in AML and ALL (early
pre-B, pre-B, cell, and T cell) (very
poor prognosis in ALL)
11q23 reciprocal Poor prognosis in pediatric ALL MLL RT-PCR or FISH
translocations and AML
t(9;11) AMOL, especially poorly differentiated AF9-MLL
(MS5a); other types of AML, e.g.,
AMML (M4)
t(4;11) Mixed lineage leukemia with AF4—MLL
lymphoblastic (early pre-B) or
monocytic features; common in
infants
t(11;19) Precursor B-cell ALL ENL-MLL
t(12;21) ALL, precursor B cell TEL-AMLI
(favorable prognosis)
t(1;19) ALL, pre-B-cell phenotype PBX1-E2A
(30% incidence in this group;
associated with a very poor
prognosis)
ALL, B-cell phenotype (Burkitt’s cMYC-IgH
t(8;14), t(2;8)
and t(8;22) lymphoma), c-myc translocated to
chromosome 1|4 heavy chain or one
of the light-chain genes on
chromosome 2 (kappa) or 22 (lambda)
t(1;14) ALL, T-cell phenotype TALI-TCRaé
co thymocyte) _

inv= inversion; del = deletion; RT-PCR= reverse transcriptase tee Rin

t= Perera
344 ~~ Chapter 16 Introduction to Leukemia and the Acute Leukemias

techniques are still being developed. The sensitivity of molecu- M3: Promyelocytic; M3m: promyelocytic microgranu-
lar techniques enables detection of leukemic subpopulations lar variant
that are not identifiable by conventional morphology or other M4: Myelomonocytic; M4Eo: myelomonocytic with
methods; therefore, patients who are in clinical remission may bone marrow eosinophilia
be observed to possess minimal residual disease when PCR- M5: Monocytic (a) poorly differentiated and (b) well
based techniques are employed.'*” The significance and clinical differentiated
applications of minimal residual disease need to be explored
Mo: Erythroid
further, because standard treatment regimens are designed to
address those levels of residual disease traditionally detected by M7: Megakaryoblastic
morphologic means. These groups are defined according to the predominant cell type
observed on Romanowsky and cytochemically stained blood
and bone marrow aspirate smears (Table 16-11). Additional
Acute Myeloid Leukemia specialized studies are required to confirm the diagnosis of MO
FAB Classification of AML (AML without cytologic maturation) and M7 (acute megakary-
ocytic leukemia). A summary of the cytochemical reactions in
In 1976 a group of French, American, and British hematol- each type of AML is found in Table 16-12.
ogists, prompted by the need for uniform nomenclature and
classification of acute leukemia, proposed a morphological AML FAB M0
classification scheme for leukemia.'* This scheme, the MO is AML without morphological or cytochemical matura-
French—American—British Classification, or better known tion (Fig. 16-11). It comprises approximately 5% of the adult
as the FAB Classification, proved to be useful in standard- cases of AML.!? AML MO have primitive leukemic blasts that
izing the morphological classification of both acute show no distinctive myeloid morphological features and lack
myeloid.and lymphoid leukemias. In the years since it was reactivity with the conventional battery of cytochemical
first introduced, the FAB cooperative group has made sev- stains (myeloperoxidase, Sudan black B, NSE). Accordingly,
eral modifications, striving to make the classification as flow cytometry or another form of immunophenotyping is
objective and unambiguous as possible.° Although there are required for the diagnosis. Typically, these undifferentiated
still some areas of ambiguity, the FAB system has gained AMLs exhibit immunologic reactivity for at least one
wide acceptance, and continues to be relevant even after the myeloid lineage-specific antigen (CD33, CD13, CD117) in
adoption of the most recent WHO Classification scheme. the absence of lymphoid antigens (particularly CD3, CD79a,
Importantly, the new WHO includes parts of the FAB Clas- CD22). Generally, undifferentiated AML is negative for
sification system. expression of antigens of myelomonocytic differentiation,
The FAB classification of AMLs are divided into the such as CD14 and CD15.'! The prognosis of MO appears to be
following groups: poor compared to other types of AML.
MO: Myeloid without cytologic maturation
AML FAB M1
M1: Myeloid with minimal maturation M1 is AML with minimal morphologic and cytochemical
M2: Myeloid with maturation maturation (Fig. 16-12). It comprises 15% of the adult cases

Myeloid without cytologic maturation: Morphologically undifferentiated leukemic blasts with myeloid immunophenotype
Myeloid without maturation: Marrow leukemia cells are primarily myeloblasts with no azurophilic granules
Myeloid with maturation: Leukemia cells show prominent maturation beyond myeloblast stage
Promyelocytic: Abnormal, hypergranular promyelocytes dominate; Auer rods easily found; increased incidence of DIC; strong
MPO staining
Microgranular variant of M3: Indistinct granules; rare to occasional Auer rods, nucleus often reniform or bilobed; increased
incidence of DIC; strong MPO staining
Myelomonocytic; Both monocytic (monocytes and promonocytes) and myeloid differentiation (maturation beyond myeloblast
Stage)
M4 with bone marrow eosinophilia: Similar to M4 with marrow eosinophilia (abnormal and immature); associated with
abnormal 16q karyotype
Monocytic, poorly differentiated: Monoblasts predominate, typically with abundant cytoplasm and single distinct nucleoli
Monocytic, well differentiated: Predominantly promonocytes in marrow and more pronounced maturation in blood
Erythroleukemia: Dysplastic erythroblasts with multinucleation, cytoplasmic budding, vacuolation, and megaloblastoid
changes
Megakaryoblastic: Wide range of morphology; cytoplasmic projections sometimes present; electron microscopy or
immunocytochemical stains necessary for diagnosis
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 345

AKoL

MPO
SBB
CAE
NSE
AcP
PAS hf /-

CrP “=

“Negative reaction is for erythroblasts only; myeloblasts present will stain positive with MPO and SBB.
‘Positive reaction is with a naphthyl acetate and negative reaction is with alpha naphthyl butyrate nonspecific esterases.
+ =blasts stain positive; — = blasts stain negative; +/— = blasts stain negative or weakly positive (< 3%).
MPO = myeloperoxidase; SBB = Sudan black B; CAE = chloroacetate esterase, NSE = nonspecific esterase; AcP = acid
phosphatase; PAS = periodic acid-Schiff; CrP = cyanide-resistant peroxidase; AML = acute myeloblastic leukemia:
APL = acute promyelocytic leukemia; AMML = acute myelomonocytic leukemia; AMoL = acute monocytic
leukemia; EL = erythroleukemia; MegL = megakaryoblastic leukemia; AEoL = acute eosinophilic leukemia.
«

of AML.’ M1 is characterized by poorly differentiated adult cases of AML and is, therefore, the most common
myeloblasts (type | blasts) representing greater than 90% of type.'’ M2 is characterized by myeloblasts (type | and type 2)
nonerythroid cells, with no evidence of maturation to more and greater than 10% of the immature cells are promyelocytes
mature myeloid cells. The nucleus of these type | myeloblasts or later stages of myeloid maturation. M2 is also character-
typically has loose open lacy chromatin, distinct nucleoli and ized by a specific translocation involving chromosome 8 and
immature cytoplasm without any granules. Fewer than 10% 21; t(8;21) in approximately 15% of the cases of M2 AML.
of the myeloblasts are type 2 blasts which have the same mor- Myeloblasts in M2 will stain positive for MPO, SBB, and
phology as type | but also have <15 azurophilic granules in NCAE cytochemical stains (Fig. 16-15). AML with matura-
the cytoplasm. Auer rods may be present in M1 myeloblasts. tion immunophenotypically resembles AML without matura-
M1 myeloblasts will demonstrate a positive reaction with tion, as evidenced by expression of CD13, CD33, and CD117.
MPO, SBB, and NCAE cytochemical stains, demonstrating In addition to these markers, the blasts may also express
myeloid lineage. Immunophenotypically, at least two myeloid CD19, CD34, and CD56."
antigens are expressed (CD13, CD33, CD117, and cytoplas-
mic MPO), but lymphoid antigens are generally absent.'' The AML FAB M3 AND M3m
course of this leukemia is generally described as aggressive. M3 is acute promyelocytic leukemia (APL; Fig. 16-16). It
comprises approximately 10% of the adult cases of AML." It
AML FAB M2 is characterized by abnormal promyelocytes (>30% of non-
M2 is AML with some maturation to or beyond the promye- erythroid cells) with heavy granulation, sometimes obscuring
locyte stage (Figs. 16-13 and 16-14). It comprises 25% of the the nucleus, and, often, abundant cytoplasm. Auer rods are

Figure 16-11 @ Acute myeloblastic leukemia (AML) without mat- Figure 16-12 AML, M1, peripheral blood.
uration, MO, bone marrow.
346 = Chapter 16 Introduction to Leukemia and the Acute Leukemias

Ss.

Figure 16-15 ® AML with maturation, M2, bone marrow. Figure 16-16 @ Acute promyelocytic leukemia (APL), M3, bone
marrow.

frequently seen, and some cells may contain bundles or stacks

of Auer rods (‘faggot cells”).© The nucleus varies in size
and shape and is often reniform (kidney-shaped) or bilobed.
These cells are strongly positive with the MPO and SBB stain
(Fig. 16-17), and are usually negative with the NSE stain.
Unique to M3 is a characteristic translocation t(15;17) that
leads to a fusion gene and oncogenesis of APL. M3 is associ-
ated clinically with a high incidence of Disseminated
Intravascular Coagulation (DIC). The abnormal promyelo-
cytes in M3 leukemia are rich in thromboplastic substances
that can trigger DIC when released. DIC is associated with
coagulation abnormalities, hemorrhagic manifestations and
the presence of schistocytes on the peripheral blood smear
(see Chap. 27). Thrombocytopenia is almost always present.
A second form of APL is the microgranular variant,
M3m (Fig. 16-18). The leukemic cells of APL, microgranu-
lar variant, have primary granules that are not readily visible
on Romanowsky-stained smears. These granules can, how-
Figure 16-14 @ AML, M2. ever, be demonstrated with strongly positive myeloperoxidase
and Sudan black staining (in contrast to the negative or weak
staining of typical monocytes).'* The disease also has a high
incidence of DIC, and it is, therefore, important to recognize

Figure 16-17 m APL, M3 (Sudan black B stain).

 Chapter 16 Introduction to Leukemia and the Acute Leukemias 347

Figure 16-18 ® Acute “microgranular” promyelocytic leukemia, Figure 16-19 ™ Acute myelomonocytic leukemia (AMML), M4,
M3m, peripheral blood. bone marrow.

it for therapeutic considerations. Patients with the microgran- vacuolated is also a feature that is often seen in promono-
ular variant tend to have a higher white blood cell count than cytes. Monocytes predominate in the peripheral blood with an
patients with typical APL and may have a shorter survival.'? absolute count greater than 5 * 10°/L (Fig. 16—20).'? Serum
Morphologically, microgranular APL can be mistaken for and urinary muramidase, a lysozyme, is increased in M4. The
acute myelomonocytic or monocytic leukemia. The leukemic myeloblasts must positively stain for myeloperoxidase or
cells appear monocytoid with prominent nuclear folding and SBB. Generally, monoblasts, promonocytes, and monocytes
abundant cytoplasm. The nucleus of most cells in the periph- will stain positive with NSE.
eral blood is reniform or bilobed. Granulation of these cells is AMML expresses myeloid antigens, as would be
scant or absent, although occasional cells with heavy granula- expected. In addition to these, antigens of monocytic differen-
tion are almost always present. The bone marrow aspirate tiation may also be expressed, including CDI1b, CDl1Ic,
may reveal a morphological pattern that more closely resem- CD14, CD64, and CD4.'!7° It should be noted that CD14,
bles typical APL.'? CD11b, CD13, CD15, CD33, and CD64 may be absent or at
The diagnosis of APL, microgranular variant, can be least partially diminished in M4 and M5." Therefore it is
confirmed with cytochemical studies, including myeloperoxi- important to correlate cytochemistry, morphology, and cyto-
dase or Sudan black stain, which are strongly positive. The genetics with flow cytometric immunophenotyping. Exclud-
NSE reaction is usually negative.'* Cytogenetic studies of ing the cases of inv(16) AMML, the cytogenetic features of
microgranular APL reveal the same abnormal karyotype AMML are diverse and not specific for this diagnosis. AMML
t(15;17) that is found in the hypergranular form. may respond to aggressive therapy, but this is varied. Again,
Immunophenotypic analysis of APL typically shows a AMML has a prognosis similar to that of other AMLs.
characteristic phenotype, featuring CD13 and CD33 antigen
expression with the lack of CD34 and HLA-DR expression. AML FAB M4Eo MA4Eo is AMML associated with bone mar-
Monocytic markers (CD14 and CD15) are generally not row eosinophilia in which abnormal and immature
expressed. Reverse transcriptase (RT) -PCR and FISH meth-
ods are also available for detection and rapid characterization
of this disease.'® The prognosis of this disease is comparable to
that of AML M2.

AML FAB M4
M4 is acute myelomonocytic leukemia (AMML,; Fig. 16-19).
M4 is one of the more commonly diagnosed forms of AML,
representing approximately 20% of the adult cases.'” The
leukemic cells of AMML are characterized by both granulo-
cytic and monocytic differentiation. Morphologically,
monoblasts have round nuclei, prominent nucleoli, and deli-
cate chromatin, surrounded by a basophilic cytoplasm that
may contain fine azurophilic granules. The distinction be-
tween monoblasts and promonocytes is often difficult, but
promonocytes generally have a more convoluted nucleus
and a lower nuclear:cytoplasmic ratio than do monoblasts. A Figure 16-20 ®AMML, M4, peripheral blood.
more granular cytoplasm that is less basophilic and more
348 Chapter 16 Introduction to Leukemia and the Acute Leukemias

ee

Figure 16-21 ™ Acute monocytic leukemia (AMoL), poorly differ- Figure 16-25 @ Gum hypertrophy: A clinical manifestation of
entiated, MSa, peripheral blood. acute leukemia (M5).

eosinophils are present. M4Eo has a longer median survival cells in monoblastic leukemia). In contrast, in acute mono-
than M4 and is associated with an inversion of chromosome cytic leukemia (FAB M5b) the majority of monocytic cells
16. Similar to M4, this type of AML shows myeloid and are promonocytes and monocytes; however, the predominant
monocytic differentiation and stains positive with MPO, cell in the peripheral blood is the promonocyte.
SBB, and NSE. MS5a and M5b leukemia both have distinctive clinical
manifestations associated with the monocyte’s propensity to
AML FAB MS5a AND MS5b migrate to extramedullary sites. Skin and gum involvement are
MSa is acute monocytic leukemia (AmoL), poorly differenti- particularly characteristic (Fig. 16-23). Lymphadenopathy fre-
ated and M5b is AmoL, well differentiated (Figs. 16—21 and quently occurs, and sometimes hepatosplenomegaly is promi-
16-22). Acute monoblastic and acute monocytic are some- nent. CNS involvement also has an increased incidence in these
times used as synonyms for MSa and MS5b leukemias, respec- patients. Serum and urinary muramidase is increased in M5a
tively. M5 leukemias comprise approximately 10% of the and M5b. Typically, monoblasts and promocytes are strongly
adult cases of AML.'? These leukemias are characterized by positive for NSE. Monoblasts are usually myeloperoxidase
an excess of cells of the monocytic cell line. In these cases, and SBB negative. In general, MSa and M5b leukemia follows
cells of monocytic lineage (monoblasts, promonocytes, and an aggressive course. MS usually expresses CD64, CD56,
monocytes) must comprise greater than 80% of the leukemic HLA-DR, CD4, CD13, and CD33.'' The monocytic antigen
cells (not overall marrow cells). In the FAB criteria, CD14 is often absent or frequently diminished in MS5."!
leukemias of the monocytic lineage are subdivided into a and
b based upon the relative proportion of monoblasts and AML FAB M6
promonocytes. In cases of acute monoblastic leukemia (FAB M6 is acute erythroleukemia and is characterized by an
MS5a), monoblasts comprise the majority of monocytic cells abnormal proliferation of erythroid and myeloid precursors
(typically monoblasts are greater than 80% of the monocytic (Figs. 16-24 and 16-25). M6 is uncommon, comprising

Figure 16-22 ™ AMoL, well-differentiated, M5b, peripheral blood. Pigure 16-24 m Erythroleukemia, M6, bone marrow.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 349

platelet peroxidase (PPO) ultrastructural studies using electron

microscopy. PPO, which is distinct from myeloperoxidase, is
specific for the megakaryocytic cell line.?! Acute megakaryoblas-
tic leukemia is defined as an acute leukemia in which greater
than or equal to 50% of the blasts are of megakaryocytic lineage.
The blasts observed in acute megakaryoblastic leukemia
display a wide range of morphology, from small cells with
scant cytoplasm and dense chromatin, to large cells with a
moderate amount of cytoplasm and a fine reticulated chro-
matin pattern. Cytoplasmic projections are sometimes pre-
sent, and in some cases, azurophilic granules resembling early
granular megakaryocytes can be seen. The presence of
megakaryocytic fragments and micromegakaryoblasts in the
peripheral blood is suggestive of acute megakaryoblastic
leukemia. Conventional cytochemistry can suggest the diag-
Figure 16-25 m@ Erythroleukemia, M6, peripheral blood. nosis of acute megakaryoblastic leukemia, but is not defini-
tive. The myeloperoxidase and Sudan black reactions are
negative, whereas PAS is usually positive. Nonspecific
approximately 5% of the adult cases of AML." Patients with esterase, a-naphthyl acetate, shows dot-like positivity.
M6 have a hypercellular BM with a predominance of erythroid Electron microscopy for platelet peroxidase may be per-
precursors 250% of all nucleated BM cells. Myeloblasts com- formed, but the diagnosis of acute megakaryoblastic leukemia
prise at least 30% of all nonerythroid cells and Auer rods may is usually made by using an immunophenotypic demonstra-
be seen. The erythroid hyperplasia is characterized by mega- tion of platelet-specific antigens. Megakaryoblasts will gener-
loblastic changes, dyserythropoiesis, and ineffective erythro- ally express at least one of the platelet associated antigens
poiesis. in the BM, ringed sideroblasts can be seen in M6 when CD41, CD42b, and CD61. CD36 and factor VIII antigen
stained with Prussian blue for iron. Abnormal erythroid features expression are typical.''!
characteristic of M6 include bizarre multinucleation of ery-
throblasts, and erythroblasts with vacuolated cytoplasm. The FAB REVISED CRITERIA
erythroblasts in M6 stain strongly positive with PAS stain. AML, especially M6 type (erythroleukemia), is sometimes dif-
Abnormal megakaryocytes may also be observed. Erythroblasts ficult to distinguish from certain myelodysplastic syndromes
will stain negative with MPO, SBB, and NSE; however, (see Chap. 19). To address this problem, the FAB cooperative
myeloblasts will be positive for MPO, SBB, and specific group proposed a revised criteria for the classification of AML,
esterase (CAE). including a stepwise evaluation of the bone marrow aspirate
(Fig. 16—27).” A differential count is done of all nucleated cells
AML FAB M7 (ANC) and of only nonerythroid cells (NEC). The ANC differ-
M7 is acute megakaryoblastic leukemia (Fig. 16-26) and is rela- ential is used to determine the percentage of erythroblasts (all
tively uncommon, comprising approximately 5% of the adult nucleated erythroid precursors). Cases with greater than
cases of AML.'° M7 is characterized by a neoplastic proliferation 50% erythroblasts in the ANC differential are further evaluated
of megakaryoblasts (>30%) and atypical megakaryocytes in the to differentiate between M6 and myelodysplastic syndrome
bone marrow. Recognition of this entity was aided by the use of (MDS). M6 cases have myeloblasts greater than 30% of NEC,
and MDS cases have blasts less than 30% of NEC. Those cases
with less than 50% erythroblasts are evaluated to differentiate
between AML, types M1 to M5 (blasts greater than 30% of
ANC), and MDS (blasts less than 30% of ANC). The final clas-
sification of AML (M1 to MS) is based on characterization of
the NEC fraction.
The FAB classification system has improved diagnostic
accuracy and uniformity. It is useful for the reader as an aid to
recognizing the wide morphological variations possible in
leukemic cells and providing diagnostic criteria for each mor-
phologic subclassification. However, the FAB classification
system has largely failed to define groups of patients with
AML that have distinct clinical outcomes. These patients gener-
ally have similar clinical courses, regardless of their FAB
subtype. Exceptions to this are patients having AML with mono-
cytic differentiation (M4 and M5), who tend to have a lower rate
of complete remission and a lower rate of survival, and those
Figure 16-26 m™ Acute megakaryoblastic leukemia (AMegL), M7. with acute promyelocytic leukemia (M3), which is associated
350. Chapter 16 Introduction to Leukemia and the Acute Leukemias

BM aspiration ——> Hypocellular BM on films the disease. Molecular techniques such as RT-PCR and FISH
Y are used to determine recurring genetic abnormalities in the
CELLULAR BM BM biopsy leukemic cells.
The WHO classification incorporates five major cate-
gories in its classification of acute myeloid leukemias:
ERYTHROBLASTS % OF ALL NUCLEATED BM CELLS (ANC)
1. Acute myeloid leukemias with recurrent genetic abnor-
malities
<50% Erythroblasts 250% Erythroblasts
2. Acute myeloid leukemia with multilineage dysplasia
3. Acute myeloid leukemia, and myelodysplastic syndromes
(MDS), therapy related (t-AML and t-MDS)

“non-erythroid c 4. Acute myeloid leukemia not otherwise categorized.

5. Acute leukemias of ambiguous lineage

AML WITH RECURRENT GENETIC ABNORMALITIES

BL 230% BL <30% BL <30% BL 230% According to the WHO classification, patients with any of the
following clonal, recurring cytogenetic abnormalities should
be considered to have AML regardless of the blast percentage.®

AML WITH t(8;21)(q22;q22);(ETO-AMLI) This translocation

NEC % (M1 to M5) occurs in up to 15% of cases of AML and may be the most com-
mon translocation. The ETO-AMLI translocation occurs
Figure 16-27 @ Suggested steps in the analysis of a bone marrow
(BM) aspirate to reach a diagnosis of acute myeloid leukemia (AML,
chiefly in younger patients and often in cases of acute
M1 to M6) or myelodysplastic syndrome (MDS). BL = blast cells; myeloblastic leukemia with maturation, FAB M2. The translo-
ANC = all nucleated bone marrow cells; NEC = nonerythroid cells, cation involves the fusion of the AVL/ gene on chromosome 21
bone marrow cells excluding erythroblasts. (From Bennett, JM, with the ETO gene on chromosome 8, resulting in the creation
et al: Proposed revised criteria for the classification of acute myeloid of a dominant inhibitory protein that prevents the transcription
leukemia. Ann Intern Med 103:626, 1985, with permission.)
of a number of important hematopoietic genes. Expression of
myeloid markers, such as CD13, CD33, and MPO, are typical,
with a longer average survival rate.° Because of the limitations but in addition, these leukemias express markers of the lym-
of morphological and cytochemical classification in prediction phoid lineage, most frequently CD19. FISH and RT-PCR assays
of biologic behavior in AML, the WHO classification has for this abnormality are quite reliable.* The t(8;21) is associated
included immunologic and cytogenetic techniques to augment with a favorable prognosis and good response to therapy.”
the FAB classification. These methods provide additional data
that are useful in determining appropriate patient management. AML WITH iny(16)(p13q22) OR (16;16)(p13;q22);(CBFB/
The new WHO classification essentially includes most of the MYH1I) Pericentric inversion of chromosome 16 is associ-
FAB MI through M7 AML leukemias under the category of ated with another type of AML that has a favorable prognosis,
“AML, not otherwise categorized.” However, it does exclude acute myelomonocytic leukemia with marrow eosinophilia,
M3, which is classified as AML with t(15;17) and some M2 M4eo under the FAB system. The inv(16) results in the fusion
cases which have recurrent cytogenetic abnormalities.° of the CBFG gene on 16q22 with the MYH// gene on 16p13.73
The inversion leads to the sequestration of much of the CBFB
WHO Classification of AML protein in the cytoplasm, preventing its function as a tran-
scription factor. This type of leukemia shows myeloid and fre-
Advances in the understanding of the molecular/genetic fea- quently monocytic antigen expression, but again as discussed
tures of hematopoietic malignancies have led to the new in the preceding text, none of these antigen patterns are spe-
WHO classification, which seeks to incorporate this new cific for AML inv(16). Similar to patients with AML t(8;21),
information. Most pathologists and hematologists/oncologists AML inv(16) patients achieve higher rates of remission with
have adopted the WHO terminology in their routine practice. therapy containing high-dose cytosine arabinoside.
Importantly, however, the FAB classification remains relevant
because it is incorporated into the WHO category of “AML ACUTE PROMYELOCYTIC LEUKEMIA WITH 1(15;17)
not otherwise categorized” and many clinicians and (q225;q12); (PML/RARa AND VARIANTS) Acute promyelo-
researchers continue to reference the FAB system. In addi- cytic leukemia (known as M3 under the FAB system) is
tion, archived material and several older databases use the composed of abnormal promyelocytes with heavy granula-
FAB criteria. Remember, in the WHO classification the blast tion, sometimes obscuring the nucleus, and abundant cyto-
count in the blood or bone marrow for diagnosis of AML is plasm. Auer rods are frequently seen, and some cells may
20%, and in the FAB classification the blast count is 30%.° In contain bundles or stacks of Auer rods (“faggot cells”). The
the WHO classification, genetic abnormalities of the leukemic nucleus varies in size and shape and is often reniform
(neoplastic) cells must be identified. Cytogenetic studies (kidney-shaped) or bilobed. These cells are strongly positive
should be initially performed and at regular intervals throughout with the peroxidase stain and are usually negative with the
 Chapter 16 Introduction to Leukemia and the Acute Leukemias = 35]

NSE stain, but cases with positive NSE activity have been reaction is usually negative, but can be positive.'* Cytogenetic
reported. In addition to characteristic morphology, acute studies of microgranular APL reveal the same abnormal kary-
promyelocytic leukemia (APL) contains a translocation that otype t(15;17) that is found in the hypergranular form, and
results in the fusion of a transcription factor called PML on rapid FISH analysis for the translocation is often valuable for
chromosome 15 with the alpha («)-retinoic acid receptor gene confirming the diagnosis.'’
(RARa) on chromosome 17, giving rise to one of the most Due to the hemorrhagic complications of DIC associated
striking instances of genotype-phenotype correlation in with APL, and the opportunity for a directed therapeutic inter-
pathology: the hybrid gene, PML-RARa.”> Because wild-type vention, namely ATRA, the recognition and characterization of
RARa acts as a transcriptional activator, when translocated, it this disease, when confronted with a new diagnosis of acute
acts as a repressor of transcription leading to a maturation leukemia, is of paramount importance. The wise diagnostician
arrest at the promyelocyte stage.’ In the normal state, RARa will consider APL whenever monoblastic or myelomonoblastic
interacts with transcriptional co-repressors and, the levels of leukemia enters the differential. Immunophenotypic analysis
retinoic acid constitutively present are sufficient for overcom- typically shows a characteristic phenotype, featuring variable
ing this repression. However, the fusion of RARa to PML CD13 and strong CD33 antigen expression with the lack
leads to the formation of a co-repressor complex that is of CD34 and HLA-DR expression. Lymphoid markers such as
markedly enhanced.** The formation of this enhanced CD2 and CD9 may be expressed as well. Monocytic markers
complex is involved in the oncogenesis of APL through and markers of maturity (CD15) are generally not expressed."!
mechanisms that are not entirely understood, but high doses The prognosis of this disease is comparable to that of
of all-trans-retinoic acid (ATRA) are effective as a differenti- AML t(8;21) or AML inv(16). Deaths in APL are typically
ating agent in APL.** ATRA in combination with an anthracy- early and due to hemorrhagic complications. Variant translo-
cline forms the basis for therapy. cations, including t(11;17) and t(5;17), may occur in cases
APL often presents with the signs and symptoms of that are morphologically and immunologically similar to APL
DIC. The majority (80%) of patients with APL present with with the classic t(15;17). Although these translocations also
hemorrhagic manifestations, including petechiae, small involve the RARa gene, t(11;17) is resistant to ATRA.'”
ecchymoses, hematuria, and bleeding from venipuncture and
bone marrow sites.'* The abnormal promyelocytes are rich in AML WITH 11q23 (MLL) ABNORMALITIES The mixed lin-
thromboplastic substances that, if released, trigger DIC. The eage leukemia or myeloid lymphoid leukemia (MLL) gene is
most consistent coagulation abnormalities include a pro- involved in at least 80 different translocations, many of which
longed prothrombin time and thrombin time, elevated fibrin are seen in AML, ALL, and MDS.* In AML, MLL gene
degradation products, and decreased amounts of plasma fib- translocations are associated with either AML with monoblas-
rinogen. Thrombocytopenia, which tends to be more severe tic differentiation or AML associated with previous topoiso-
than in other types of AML, is almost universally present. merase II inhibitor therapy.** Functionally, MLL gene appears
Schistocytes are sometimes evident on the peripheral blood to act to modulate the expression of HOX genes, but the pre-
smear. cise mechanism is not clear. MLL gene translocations typi-
A second form of APL is the microgranular variant cally occur in children or younger adults.* Clinically, patients
(known as M3m in the FAB classification).'* The leukemic may present with extramedullary monocytic sarcoma or
cells of the microgranular APL have primary granules that are DIC.* A global approach to the detection of MLL gene abnor-
not readily visible on Romanowsky-stained smears. These malities is prudent, given the heterogeneity of abnormalities
granules can, however, be demonstrated with strongly posi- that are associated with this gene. Southern blot, RT-PCR,
tive myeloperoxidase staining (in contrast to the negative conventional cytogenetic studies, and FISH analysis all have
staining typical for monoblasts and promonocytes), as well as a role in the characterization of AMLs with such abnormali-
by Sudan black staining.'? The disease also has a high inci- ties. Typically, MLL gene abnormalities denote a poor
dence of DIC, and it is, therefore, important to recognize it for outcome.**
therapeutic considerations. Patients with the microgranular It should be noted that the WHO maintains that a diag-
variant tend to have a higher white blood cell count than nosis of AML can be made in patients with a blast count of
patients with typical APL and may have a shorter survival.'* less than 20% and a clonal recurring genetic abnormalities of
Morphologically, microgranular APL can be mistaken t(8;22), inv(16), t(16;16), and t(15;17).° Notwithstanding, the
for acute myelomonocytic or monocytic leukemia. The WHO also emphasizes that the diagnosis of acute leukemia is
leukemic cells appear monocytoid with prominent nuclear not synonymous with a decision to treat as an acute
folding and abundant cytoplasm. The nucleus of most cells in leukemia.° Many clinical factors should be considered before
the peripheral blood is reniform or bilobed. Granulation of treating a patient with chemotherapy.
these cells is scant or absent, although occasional cells with
heavy granulation are almost always present. The bone mar- AML WITH MULTILINEAGE DYSPLASIA

row aspirate may reveal a morphological pattern that more AML with multilineage dysplasia is an acute leukemia with
closely resembles typical APL."° greater than 20% blasts and dysplasia in two or more cell
The diagnosis of microgranular APL can be confirmed lines.° This dysplasia must be significant, meaning that it must
with cytochemical studies, including myeloperoxidase or be present in greater than 50% of the cells in at least two lines,
and it must be in a pretreatment specimen.° Typically, this is a
Sudan black stain, which are strongly positive. The NSE
352 Chapter 16 Introduction to Leukemia and the Acute Leukemias

disease that occurs in elderly patients in a setting of MDS, battery of cytochemical stains (myeloperoxidase, Sudan black
although it may also occur de novo. Most often patients pre- B, NSE). Accordingly, flow cytometry or another form of
sent clinically with severe pancytopenia. This is a morpholog- immunophenotyping is required for the diagnosis. Typically,
ically heterogeneous disease, and these features are reflected these minimally differentiated AMLs exhibit immunologic
in the immunophenotype, which is highly variable, often reactivity for at least one myeloid lineage-specific antigen
showing aberrant and promiscuous antigen expression. Cyto- (CD33, CD13, CD117) in the absence of lymphoid antigens,
genetic alterations are often complex, but may involve the particularly cCD3, cCD79a, cCD22.'' Generally, minimally
loss of segments of chromosomes 5q and 7q. The prognosis is differentiated AML is negative for expression of antigens of
not favorable."’ myelomonocytic differentiation, such as CD14 and CD15."
Rarely, blasts may also express a lymphoid antigen, but the
AMLs AND MYELODYSPLASTIC SYNDROMES, expression is dimmer than that seen in lymphoid malignan-
THERAPY RELATED cies and there should be more myeloid antigens expressed. In
AML and myelodysplastic syndromes may arise as a result of the differential diagnosis for minimally differentiated AML is
chemotherapy or radiation. Presentation as a myelodysplastic ALL, acute megakaryoblastic leukemia, and acute leukemia
syndrome, presenting as a cytopenia or pancytopenia at some of ambiguous lineage. The prognosis appears to be poor com-
time after treatment, is more common than presenting with pared to other types of AML.°
outright AML. In some cases, the myelodysplastic syndrome
develops into an acute leukemia, but in other cases mortality ACUTE MYELOBLASTIC LEUKEMIA WITH MINIMAL
is due to the long term effects of MDS.” MATURATION Known as AMLM1 under the FAB classifica-
The WHO recognizes two major subtypes of therapy- tion, acute myeloblastic leukemia with minimal maturation is
related AMLs: (1) alkylating agent/radiation related, and composed of predominantly poorly differentiated myeloblasts,
(2) topoisomerase II inhibitor related.© The development of typically representing greater than 90% of nonerythroid cells.**
leukemiais more rapid after topoisomerase II inhibitor therapy, Importantly, myeloblasts predominate and there should be no
with a reported mean of about 33 to 34 months after beginning significant evidence of maturation to more mature myeloid
chemotherapy, compared with about 5 to 6 years after alkylating cells such as neutrophils. The nucleus of the myeloblast typi-
agent/radiation.® As is the case with AML with multilineage cally has a fine, lacy chromatin pattern and distinct nucleoli.'?
dysplasia, immunophenotypic analysis of the increased blast The quantity of cytoplasm is usually moderate, and azurophilic
population in therapy-related AML and MDS shows no specific granules or Auer rods may be seen, but there are fewer than
pattern, rather, the immunophenotype is heterogeneous.° In 10% of the blasts with azurophilic granules. The myeloperoxi-
addition to pan-myeloid antigen expression, the blasts may dase or Sudan black reactions must demonstrate at least 3%
aberrantly express CD56 and CD7."’ positivity in the blast population to document myeloid differen-
The rationale behind the further subdivision of therapy tiation.** Immunophenotypically, at least two myelomonocytic
related AML and MDS is based on distinct cytogenetic and antigens are expressed (CD13, CD33, CD117, and MPO), but
prognostic differences between the two main groups. Therapy- lymphoid antigens are generally absent. The course of this
related AML and MDS related to alkylating agents/radiation leukemia is generally described as aggressive.!!
therapy typically surface 5 to 6 years after therapy and show
clonal evolution from MDS, frequently harboring deletions or ACUTE MYELOBLASTIC LEUKEMIA WITH MATURATION
translocations involving 5q and 7q, in addition to other chro- Morphologically synonymous with the FAB M2, cases of AML
mosomal abnormalities.'’ These leukemias are generally asso- with maturation show a marrow infiltrate with greater than
ciated with a very poor outcome and short survival time. 20% blasts and evidence of maturation to or beyond the
In contrast, AML arising from topoisomerase inhibitor promyelocyte stage.** There should be more than 10% of cells
treatment have a shorter time to onset after treatment, typically at different stages of granulocyte maturation, and the percent-
about 3 years. Second, these therapy related AMLs have differ- age of maturing myeloid cells is typically higher than 10%.”* At
ent cytogenetic abnormalities, the most common of which is a least 3% of the leukemic cells are myeloperoxidase or Sudan
translocation involving 11q23 (the MLL gene).*° Other reported black positive, and this percentage is usually also much
genetic abnormalities in AML after topoisomerase treatment higher.** Monocytes comprise less than 20% of the bone mar-
include t(8;21), inv(16) and even APLs with t(15;17).° Lastly, row; thus NSE activity must not exceed 20% of marrow cells.°
it may be that these patients have a better outcome than those AML with maturation immunophenotypically resembles
with alkylating agent/radiation associated MDS or AML. AML without maturation, as evidenced by expression of CD13,
CD33, and CD117."' In addition to these markers, the blasts
ACUTE MYELOID LEUKEMIA NOT OTHERWISE may also express CD15, CD34, and HLA-DR."'! As described
CATEGORIZED in the preceding text, many cases of AML with these features
ACUTE MYELOBLASTIC LEUKEMIA WITHOUT harbor the t(8;21) and are best classified under AML with
MATURATION Also known as FAB MO, these cases of acute recurrent genetic abnormalities.'’ In the remaining cases, other
myeloid leukemia show no definitive myeloid differentiation translocations including t(6;9) and t(8;16) have been described.
by conventional morphological and cytochemical analysis. but have yet earned specific classifications as recurrent genetic
These AMLs have primitive leukemic blasts that show no dis- abnormalities under the 2001 WHO classification scheme.
tinctive myeloid morphological features and lack reactivity Survival rates are variable, and evidence shows that AML with
(less than 3% of blasts staining positive) with the conventional maturation responds to aggressive therapy.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 353

ACUTE MYELOMONOCYTIC LEUKEMIA Acute myelo- promonocytes are weakly to mildly positive, although not
monocytic leukemia (AMML), M4 in the FAB system, is one uniformly.
of the more commonly diagnosed forms of AML. The leukemic The immunophenotype of these leukemias expresses
cells of AMML are characterized by both neutrophilic and myeloid antigens (CD13, CD33, and CD117) and also expresses
monocytic differentiation. By WHO criteria, not only must markers of monocytic differentiation (CD14, CD64, CD4, and
blasts be greater than or exceed 20%, but neutrophils and pre- lysozyme).** Often, CD34 is not expressed on these cells. While
cursors, as well as monocytes and precursors, must each some differential expression of myeloperoxidase may be seen
comprise equal to or greater than 20% of the bone marrow cel- by flow cytometry (i.e., myeloperoxidase antigen may be seen
lularity.° This criteria of at least 20% monocytes and monocytic in acute monocytic leukemia rather than acute monoblastic
precursors is to separate out cases of AML with or without mat- leukemia), these findings alone are not able to discern specific
uration that have occasional, but not significant, numbers of differences between these two subtypes of leukemia.
monocytes.** Morphologically, monoblasts have round nuclei, Aside from cases of acute monocytic leukemia that fall
prominent nucleoli, and delicate chromatin, surrounded by a under the AML with recurrent genetic abnormalities section,
basophilic cytoplasm that may contain fine azurophilic gran- there are few specific cytogenetic findings that would be
ules. The distinction between monoblasts and promonocytes is suggestive of acute monoblastic or monocytic leukemia. One
often difficult, but promonocytes generally have a more convo- of these is associated with erythrophagocytosis and coagu-
luted nucleus and a lower nuclear:cytoplasmic ratio than do lopathy and is characterized by a t(8;16)(p11;p13) abnormal-
monoblasts; a more granular cytoplasm that is less basophilic ity. In general, acute monoblastic and monocytic leukemia
and more vacuolated than monoblasts. At least 3% of the follows an aggressive course.
myeloid blasts must positively stain for myeloperoxidase. Gen-
erally, monoblasts, promonocytes, and monocytes will stain ACUTE ERYTHROID LEUKEMIA Acute erythroid leukemia,
positively with NSE. which is equivalent to FAB M6, is characterized by an abnor-
AMM expresses myeloid antigens, as would be expected. mal proliferation of erythroid precursors and, generally, an
In addition to these, antigens of monocytic differentiation may aggressive course. Two types are generally recognized. One
also be expressed, including CD11b, CD11c, CD14, CD64 and type of erythroid leukemia is characterized by the neoplastic
CD4.* Excluding the cases of inv(16) AMML that are classified population purely composed of erythroid precursors (pure ery-
as AML with recurrent genetic abnormalities, the cytogenetic throid leukemia, FAB M6b) and immature erythroid precursors
features of AMML are diverse and not specific for this diagno- should be greater than 80% of the cellularity with no significant
sis.°° AMML may respond to aggressive therapy, but this myeloblastic component.® Pure erythroid leukemia has previ-
is varied. Again, AMML has a prognosis similar to that of ously been referred to as DiGuglielmo disease or true
other AMLs. erythroleukemia. The other type of erythroid leukemia has ery-
throid precursors and myeloblasts (erythroleukemia M6a),
ACUTE MONOBLASTIC LEUKEMIA AND ACUTE although with a predominance (greater than or equal to 50%) of
MONOCYTIC LEUKEMIA Synonymous with FAB M5a erythroid precursors. ° In the erythroleukemia type, myeloblasts
and M5b, acute monoblastic and acute monocytic leukemias must be greater than 20% of the nonerythroid population.
are leukemias characterized by a preponderance of cells of Patients with either type of erythroid leukemia have hypercel-
monocytic lineage. In these cases, cells of monocytic lin- lular bone marrows with marked erythroid hyperplasia associ-
eage (monoblasts, promonocytes, and monocytes) must ated with abnormal erythroid forms.° Megaloblastoid changes
comprise greater than 80% of the leukemic cells (not overall in the erythroblasts along with other dysplastic features, includ-
marrow cells). 7* Like the FAB criteria, the WHO further ing bizarre multinucleation, markedly vacuolated cytoplasm of
subdivides leukemias of monocytic lineage based on the rel- erythroblasts, and cytoplasmic budding, are common."* Cyto-
ative proportion of monoblasts and promonocytes. In cases plasmic PAS-positive staining in the neoplastic erythroblasts is
of acute monoblastic leukemia (FAB M35a), monoblasts consistent with the diagnosis of erythroleukemia but is not spe-
comprise the majority of monocytic cells (typically cific; and negative staining does not rule it out.° Myeloblasts in
monoblasts are greater than 80% of the monocytic cells in erythroleukemia show myeloid differentiation and they may
monoblastic leukemia).*? In contrast, in acute monocytic have Auer rods. Abnormal megakaryocytes may also be present.
leukemia (FAB MSb) the majority of monocytic cells are In the differential diagnosis of erythroleukemia is a
promonocytes. myelodysplastic syndrome with excess blasts, as both dis-
Acute monoblastic and monocytic leukemia both have dis- eases have dysplastic erythroid precursors and increased
tinctive clinical manifestations associated with the monocyte’s numbers of blasts (see Chap. 19). One may appreciate the
propensity to migrate to extramedullary sites. Skin and gum overlap between these two diagnoses when considering that
involvement are particularly characteristic. Lymphadenopathy the diagnosis of erythroleukemia can be made according to
frequently occurs, and sometimes the spleen and liver are the criteria of erythroid precursors which are greater than
markedly enlarged. CNS involvement also has an increased 50% of all nucleated cells in the BM and myeloid blasts
incidence in these patients.’ which are more than 20% of the nonerythroid marrow cells.°
The morphologic features of the constituent cells have A diagnosis of MDS may be considered with a blast percent-
been described earlier in the section on AMML. Typically, age significantly less than 20% of NEC.°
monoblasts and promocytes are strongly positive for NSE." Also in the differential with erythroleukemia are
Monoblasts are usually not myeloperoxidase positive, whereas vitamin B,, and folate deficiency. Pure erythroid leukemia
354 Chapter 16 Introduction to Leukemia and the Acute Leukemias

should not correct following vitamin B,,/folate therapy. Also, spontaneously regress without chemotherapy, only to recur in
pure erythroid leukemia will exhibit dysplastic changes in the some cases. Care should, therefore, be taken when assessing
erythroid line that far exceeds those typically seen in vitamin increased blasts in Down neonates. Importantly, not every infant
B,,/folate deficiency, although the latter may at times be strik- with increased megakaryoblasts will undergo spontaneous
ing as well. In children, caution must be taken when the diag- remission, and some neonates, as well as infants and children, are
nosis of pure erythroid leukemia is considered owing to the diagnosed with megakaryoblastic leukemia. In this younger pop-
possibility of congenital dyserythropoietic anemia. The diag- ulation, the translocation, t(1;22)(p13;q13) found in acute
nosis of erythroleukemia should be made only with consulta- megakaryoblastic leukemia is associated with a poor prognosis.’
tion of the clinical history and setting, including a history of
MDS and folate and B,, levels.° ACUTE BASOPHILIC LEUKEMIA In this very rare
The erythroblasts of pure erythroleukemia generally lack leukemia, the differentiation is primarily toward the
myeloperoxidase and do not express myeloid antigens. basophilic lineage. Care should be taken to ensure the
Depending on how immature the erythroid blasts are, they absence of the Philadelphia chromosome, which is the hall-
may or may not express glycophorin A.** Other antigens such mark of chronic myelogenous leukemia (CML), because
as CD41 and CD61, which are typically megakaryocytic anti- CML in blast phase may be indistinguishable. The blasts
gens, may be variably expressed.'' Indeed, distinguishing ery- will generally have a moderately basophilic cytoplasm and
throblasts from megakaryoblasts can be difficult. coarse basophilic cytoplasmic granules similar to those
seen in basophils. Numerous immature basophils may also
ACUTE MEGAKARYOBLASTIC LEUKEMIA (M7) Acute be present, as in CML. Mature basophils are often few in
megakaryoblastic leukemia, which is equivalent to FAB M7, number. Cytochemically, acute basophilic leukemia shows
is a relatively uncommon form of leukemia characterized by metachromatic staining with toluidine blue, but are
neoplastic proliferation of megakaryoblasts and atypical myeloperoxidase negative. The blasts express myeloid
megakaryocytes. Recognition of this entity was aided by the markers and also express CD9.** No specific cytogenetic
use of platelet peroxidase (PPO) ultrastructural studies. PPO, abnormality has been found. This is a very rare leukemia;
which is distinct from myeloperoxidase, is specific for the what information exists about the outcome of patients sug-
megakaryocytic cell line.?! Acute megakaryoblastic leukemia gests a poor prognosis.
is defined as an acute leukemia in which greater than or equal
to 50% of the blasts are of megakaryocytic lineage.° ACUTE PANMYELOSIS WITH MYELOFIBROSIS This, too,
The blasts observed in acute megakaryoblastic leukemia is a rare form of acute leukemia representing 1% to 2% of all
display a wide range of morphology, from small cells with cases.° It is characterized by an acute panmyeloid proliferation
scant cytoplasm and dense chromatin, to large cells with a in the setting of bone marrow fibrosis. This may be seen de novo
moderate amount of cytoplasm and a fine reticulated chromatin or following treatment with alkylating agents.** Clinically,
pattern.“ Cytoplasmic projections or blebs are sometimes pre- severe pancytopenia is present without splenomegaly, corre-
sent, and in some cases azurophilic granules resembling early sponding to the relatively acute onset. The evolution of this dis-
granular megakaryocytes can be seen. The presence of ease process is typically rapid. The peripheral blood shows an
megakaryocytic fragments in the peripheral blood is also sug- erythroleukoblastic picture with dysplastic features and a promi-
gestive of acute megakaryoblastic leukemia. nent megakaryoblastic/megakaryocytic population.° The bone
Conventional cytochemistry can suggest the diagnosis of marrow biopsy is hypercellular, with all lineages showing
acute megakaryoblastic leukemia, but is not definitive.'* The varying amounts of hypercellularity. Bone marrow fibrosis is
myeloperoxidase and Sudan black reactions are negative, prominent.°
whereas acid phosphatase and PAS are usually positive.'* Non- AML with multilineage dysplasia with fibrosis and acute
specific esterase shows dot-like positivity with a-naphthy] megakaryoblastic leukemia are in the differential diagnosis, and
acetate.” distinguishing between these may be difficult. Blast percentages
Electron microscopy for platelet peroxidase may be per- and immunophenotype may help, and in acute panmyelosis with
formed, but the diagnosis of acute megakaryoblastic leukemia myelofibrosis, all three cell lines are hyperplastic. The WHO
is usually made by using an immunophenotypic demonstra- committee recommends that if the acute leukemia is predomi-
tion of platelet-specific antigens. Megakaryoblasts will gener- nantly megakaryoblastic with myelofibrosis, it be classified as
ally express at least one of the platelet associated antigens “acute megakaryoblastic leukemia with myelofibrosis”; and if
CD41, CD42b, and CD61. CD36 and factor VIII antigen the blasts represent a panmyelopathy with myelofibrosis present
expression are typical. In addition to this, the megakary- in the BM, the leukemia should be classified as ‘acute pan-
oblasts may or may not express the myeloid markers CD13 myelosis with myelofibrosis.’° The prognosis for all three enti-
and CD33, or CD34 and CD45.” It is important to rule out ties in the differential is poor.**
lymphoid differentiation.
Acute megakaryoblastic leukemia may arise in the context MYELOID SARCOMA Myeloid sarcoma is an extramedullary
of a myelodysplastic syndrome (MDS), myeloproliferative dis- (outside the bone marrow) tumor mass of myeloid blasts or
ease (MPD), or de novo. Increased megakaryoblasts are also immature myeloid precursors. Myeloid sarcoma may occur in
associated with a transient disorder in neonates with Down many sites in the body, including lymph nodes, skin, sinuses,
syndrome. The increased blasts in Down neonates will often brain, and below the periosteum of bones. Myeloid sarcoma is
 Chapter 16 Introduction to Leukemia and the Acute Leukemias = 355

also known as extramedullary myeloid tumor, granulocytic sar- unclassified.''! Typical undifferentiated leukemias show an
coma, chloroma, or if in the skin, leukemia cutis.° This myeloid immunophenotype positive for HLA-DR and CD34, but with
tumor may precede or occur concomitantly with presentation or no lineage-specific markers.** Truly biphenotypic leukemias
signal relapse of AML, MDS, chronic myelogenous leukemia, may also show changes in antigen expression over time and
or other chronic myeloproliferative disease (MPD). If it appear to switch lineages. In adults, most of these leukemias
occurs in the setting of MDS or MPD, it may signal transfor- are treated as AML if the morphology and cytochemistry sug-
mation to blast phase. gest undifferentiated leukemia.”
Three types of myeloid sarcoma are generally recognized
and are based on the degree of maturation: blastic, immature,
and differentiated.’ As their names suggest, blastic myeloid Acute Lymphoblastic Leukemia
sarcoma consists of myeloblasts, while immature myeloid Classification of ALL and Introduction
sarcoma is composed of myeloblasts and promyelocytes, and
differentiated myeloid sarcoma shows maturation to neu- ALL is mostly a disease of children, and treatment has
trophils. Also common is a monocytic form, often seen in skin improved remarkably. About 75% of ALL occur in children
and mucosal surfaces. The genetic abnormalities seen in and most ALL are precursor B cell ALL.*’ Historically, ALL
myeloid sarcoma will typically mirror those of the underlying was universally fatal, but today about 80% of newly diag-
AML or MDS. nosed children with ALL may be cured.** Fewer cases of ALL
Importantly, there is a difference between extramedullary are seen in adults and treatment is significantly less effective.
hematopoiesis, which is typically seen in the liver, spleen, and Today, the classification of ALL relies on immunopheno-
other areas of fetal hematopoiesis, and myeloid sarcoma. The for- typic, molecular, and cytogenetic characteristics of the clonal
mer 1s largely a compensatory mechanism and does not evidence blast population in addition to the morphologic features of
transformation to the blast phase. In contrast, a myeloid sarcoma the blasts. The WHO classification scheme emphasizes
represents uncontrolled spread of a neoplastic proliferation.® immunophenotype and genetics, and does not directly rely on
the FAB morphologic classifications. The focus of the currently
ACUTE LEUKEMIAS OF AMBIGUOUS LINEAGE accepted WHO is on whether the lymphoblastic leukemia is a
The advent of immunophenotyping, molecular, and genetic precursor B or precursor T cell lineage, and whether cytoge-
techniques has resulted in the understanding that some acute netic findings indicate a favorable or unfavorable prognosis.
leukemias lack clear lineage, but show characteristics of both These characteristics, not the morphologic classification of the
myeloid and lymphoid, or both B and T lymphoid lineages.° For lymphoblasts, guide therapeutic decisions. Nonetheless, an
example, lymphoid and myeloid antigens may be coexpressed appreciation of the features of FAB morphologic classification
by a single population of leukemic cells (biphenotypic); there is worthwhile, in part to allow one to help differentiate between
may be biphasic populations with distinct lineage-specific phe- myeloblasts, lymphoblasts and reactive lymphocytes.
notypes (bilineal); or there may be the lack of such markers spe- To fully appreciate the classification of ALL and other
cific for a given lineage (undifferentiated). More importantly, lymphoproliferative disorders (e.g., CLL, lymphoma, and
ambiguous lineage leukemias should be distinguished from multiple myeloma), it is important to understand lymphocyte
AML with aberrant lymphoid antigen expression, or ALL with ontogeny. Lymphoid leukemias and lymphomas are a clonal
one or two myeloid antigens. The hallmark of these rare proliferation of lymphoid cells that have been “frozen” at a
leukemias of ambiguous lineage is that the overall findings are given stage of maturation, typically retaining some features of
insufficient to classify as myeloid or lymphoid (undifferenti- their normally differentiated counterparts. In ALL, the malig-
ated) or the antigen expression is truly ambiguous and shows nant clone has an immunophenotype with features of an early
significant coexpression across lineages.° lymphocyte or “lymphoblast,” typically expressing TdT and
Acute leukemias of ambiguous lineage generally either B or T lymphocyte antigens.
have a poor prognosis, especially in adults. The most
important lineage-specific antigens are cytoplasmic CD79a Review of Lymphocyte Ontogeny
and cytoplasmic CD22 for B-lymphoid lineages, membrane
or cytoplasmic CD3 for T-lymphoid lineages, and myeloper- Lymphocytes originate from pluripotent stem cells that are
oxidase (MPO) for myeloid lineages.** Also important present in the yolk sac, fetal liver, spleen, and bone marrow.
but less specific are CD19 and CD20 for B lymphoid, CD2 At birth and into adulthood, the stem cells are normally found
and CD5 for T lymphoid, CD10 for lymphoid generally, only in the bone marrow, where they respond to specific
and CD13 and CD33 for myeloid lineage.*’ A scoring growth factors (hormone-like substances) that trigger their
system for acute leukemias of ambiguous lineage has been commitment toward B- or T-lymphocyte differentiation. The
proposed by the European Group for the Immunologic microenvironment of these developing cells plays a critical
Classification of Leukemia (EGIL) and is reproduced in role in their maturation: B cells develop in the bone marrow
the WHO classification.2? This scheme assigns different (bursa-equivalent tissue), whereas T cells develop in the thy-
degrees of specificity for various antigens expressed by a mus (from committed stem cells that have migrated there
given leukemia. from the marrow). Lymphocyte maturation in these organs is
With the routine use of flow cytometric immunopheno- antigen independent. After the lymphocytes have matured
typing of acute leukemia, fewer than 4% of cases remain they migrate to the peripheral lymphoid organs, including the
356 Chapter 16 Introduction to Leukemia and the Acute Leukemias

lymph nodes, spleen, and other lymphoid tissues. In these composed of four minigene families including the variable
organs the lymphocytes remain in a resting state until they are (VH), diversity (DH), joining (JH), and constant (CH)
stimulated to undergo antigen-dependent development. regions. The CH region has separate DNA sequences that
encode for the different Ig isotypes including mu (yw), delta
B-LYMPHOCYTE DEVELOPMENT (8), gamma (y), alpha (a), and epsilon (€). The V, D, and
Early B-cell maturation (antigen independent) is divided into J regions are the first to undergo rearrangement, forming a
three stages: early pre-B cell, pre-B cell, and mature B cell VDJ complex. In this process, intervening sequences
(Fig. 16-28). These stages are identified by their expression of (introns) are excised and the V, D, and J regions are spliced
TdT, surface markers (HLA-DR, CD10 [CALLA], CD19, together. Messenger RNA is transcribed from this VDJ
CD20), and immunoglobulin (cytoplasmic or surface Ig).*” The complex along with DNA sequences downstream from it,
early pre-B cell is TdT positive and expresses HLA-DR, CD19, including an intron and the constant u (Cu) region. The
and usually CALLA (CD10). HLA-DR, a histocompatibility- mRNA itself is then spliced to bring the VDJ complex
related antigen, is expressed first, followed by CD19 and then adjacent to the Cu region, creating a template for cytoplasmic
CD10. CD19 is the most sensitive and specific surface marker u-heavy chain synthesis. This process is closely followed by
for early B cells.'' During this stage the immunoglobulin genes a similar rearrangement of the k gene (on chromosome 2),
begin to undergo structural re-arrangement followed by the pro- which, if unsuccessful, is in turn followed by rearrangement
duction of cytoplasmic mu (Cu)-heavy chain.” The presence of of the \ gene (on chromosome 22). As B-cell development
cytoplasmic ul distinguishes the pre-B cell from its predecessor, continues, the heavy chain may undergo additional rearrange-
which otherwise has a similar phenotype. As the cell continues ments in the CH region, initiating an isotype switch from i to
to mature, immunoglobulin light chains are produced, and [gM d. Both IgM and IgD are expressed on the surface of the
is assembled and inserted into the plasma membrane. This sur- majority of mature B cells (Fig. 16-29).
face Ig (slg) is the hallmark of the mature B cell, which no After maturation in the bone marrow, B cells circulate
longer expresses TdT. Each B cell expresses only one type of Ig through the blood to the peripheral lymphoid organs, where
light chain (kappa [k] or lambda [A]). This feature is extremely they remain in a resting state until stimulated by specific anti-
helpful in identifying monoclonal proliferations of mature gens to undergo further development. Activated B cells
B cells, because a normal population of B cells will consist of a undergo clonal expansion, producing daughter cells that
mix of k and A light chain expressing B cells.'* If all the B cells retain the same antibody idiotype (antigen-binding region).
in a population express, for example, k, the population is likely Some daughter cells become memory cells and regain the
clonal, because polyclonal B cells would be a mix of some small mature B-cell morphology and phenotype, whereas oth-
B cells expressing \ along with others expressing k. ers continue development toward a short-lived antibody-
Immunoglobulin genes are rearranged in a unique secreting cell—the plasma cell. During this final development,
process that is normally limited to cells committed to B-cell the Ig heavy chain may undergo another isotype switch, to
differentiation. The Ig genes are composed of discontinu- IgG, IgA, or IgE. The plasma cell produces large quantities of
ous segments of minigene families that, when productively Ig and is characterized by a high concentration of cytoplasmic
rearranged, encode for the heavy chain and the k or A light Ig. It does not express SIg, CD19, nor CD20, although other
chains. The heavy chain gene (on chromosome 14) is antigens are sometimes expressed (e.g., PCA-1, PC-1).!!

~ A

THeL° y
Ny f ay
Antigen & : gy x i
Stem cell DR helper fact
perfactors \ (aa yo
DR pig DR cpig ty
nla
CD19
\CD20
Plasma cell

rN
—_—

Early pre-B Pre-B Mature B

VDJ rearrangements of Cytoplasmic mu Surface
mu-heavy chain gene immunoglobulin
‘es
pet cycells
<————_ Antigen-independent development —————————__> <——— -Antigen- dependent —————>

> Pre-B = cytoplasmic mu

Y Mature B = surface immunoglobulin

Figure 16-28 m B-cell development. Heavy chain (H) and light chain (L) are designated as H®, L° if in embryonic form, and H*, L° if
rearranged. > = cytoplasmic mu; Y = immunoglobulin.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias ee)Nn~—

V D J Cu
Germline DNA
(non-rearranged)
| DNA rearrangement

i a] i

| DNA rearrangement Rearranged DNA

sequences
VY DD J Cu f 4 y
9 eS
ey, i
i idea
7 bine

|Transcription
RNA splicing
Y DPD st Cy
Messenger RNA

|Translation

L\J\J\J,\/ mheavy chain Figure 16-30 @ E-rosette formation in a T-cell.

Figure 16-29 @ Schematic of immunoglobulin y-heavy chain

gene rearrangement. The variable (V),.diversity (D), and joining
(J) regions of germline DNA are linked through rearrangement and T-LYMPHOCYTE DEVELOPMENT
loss of intervening sequences. The VD] complex, intervening In the past, T cells were identified by incubating lymphocytes
sequences, and a constant (Cu) region are then transcribed. The with sheep red blood cells and observing for E-rosette formation
resulting RNA is spliced, linking the VDJ and Cu regions and creat- (Fig. 16-30). Now, with the availability of monoclonal antibod-
ing a template for the Ig u-heavy chain. (Lines between blocks ies, T cells are identified and subclassified via immunologic
represent intervening sequences.)
reagents. T-cell development (antigen-independent) in the thy-
mus is divided into three main stages: stage I, early thymocyte;
stage II, common thymocyte; and stage II, mature thymocyte.
Stages I and II occur in the thymic cortex, and the last stage
occurs in the thymic medulla. Similar to early B cells, thymo-
cytes express TdT and unique surface markers (Fig. 16-31).

Peripheral
Bone marrow Thymus lymphoid
Cortex Medulla organs

Early Common Mature

thymocyte thymocyte thymocyte

Suppressor cell

Thymocytes)
Helper cells)
Suppressor cells)
Mature T cells)

Figure 16-51 i Normal T-cell maturation. T-cell receptor (TCR) 8 and « genes are designated as B° and a° if in the embryonic form, and B*
and a’ if rearranged. The colored blue bars indicate where the antigens are present in the stages of maturation. No color indicates the antigens
are not present. Please note that the CD1 antigen disappears on mature thymocytes. The orange color indicates that as the cells mature they
lose the expression of either CD4 or CD8, with only one of these antigens remaining on each mature thymocyte or T-cell, and not both.
358 Chapter 16 Introduction to Leukemia and the Acute Leukemias

CD7, which is present on early thymocytes, is one of the earli-

est T-cell markers to be expressed; it is also the most sensitive
marker for T-cell ALL. Its expression is followed by that of CD2
and CDS." As the thymocytes move into stage II of thymic
development, they express CD1, a marker of common thymo-
cytes, along with both CD4 and CD8. CD3 is the next marker to
be expressed; it is usually absent or only weakly expressed at
stage II, but it is fully expressed in the mature thymocyte (stage
III). At this stage CD1 and CD4 or CD8 are lost, giving the
mature thymocyte a CD4 or CD8 phenotype.
During thymic maturation, the T cell synthesizes an
antigen-receptor molecule called the T-cell receptor (TCR),
which is closely associated with the CD3 molecule on the
plasma membrane. Two TCR isotypes have been discovered,
TCR-aB and TCR-y6. The genes that encode for the a, B, y,
and 8 polypeptides undergo rearrangement in a manner that Figure 16-32 @ ALL, L1, bone marrow.
parallels Ig gene rearrangements in the B cell. The TCR-B
gene (on chromosome 7) rearrangements precede TCR-a (on
chromosome 14) rearrangements. Less is known about the y
exhibit L1 features, whereas others are larger and have more
and 6 genes or about the function of the TCR-y6, but it is
abundant cytoplasm, a variable chromatin pattern, and promi-
clear that rearrangement of the y gene precedes that of the «
nent nucleoli. Nuclear clefting and indentation are characteris-
and B genes. The majority of mature T cells express the TCR-
tic. This type may be difficult or impossible to distinguish
a isotype.
morphologically from AML-M1 or MO. L3 ALL (Fig. 16-35)
is composed of a uniform population of relatively large
FAB Classification of ALL blasts that are characterized by moderate to abundant, deeply
basophilic, vacuolated cytoplasm. The nucleus has a round to
The FAB classification system separates ALL into three mor-
oval contour without indentations. L3 ALL is referred to as
phological groups (Table 16—13):
“Burkitt’s type” because its morphology is the same as that
L1: Small, uniform lymphoblasts seen in Burkitt’s leukemia/lymphoma.
L2: Large, pleomorphic lymphoblasts The distinction between L] and L2 ALL is not always
clear. Individual L1 and L2 lymphoblasts can be differenti-
L3: Burkitt’s type (vacuolated and deeply basophilic
ated most reliably by evaluating the N:C ratio (high in L1,
cytoplasm in the lymphoblasts)
lower in L2) and the absence (L1) or presence (L2) of nucle-
The morphology of these groups is evaluated on a bone mar- oli. Cases of “pure L1” ALL (more than 90% LI blasts)
row aspirate smear rather than peripheral blood. L1 ALL were associated with the best prognosis, cases of “pure L2”
(Fig. 16-32) exhibits a uniform population of small blasts with ALL (more than 50% L2 blasts) were associated with a
scant cytoplasm, a homogeneous chromatin pattern, and incon- worse prognosis, and cases with mixed cell types had
spicuous nucleoli. The nuclear shape is regular, but occasional an intermediate prognosis.'* L3 morphology was historically
clefting may be present. L2 ALL (Figs. 16-33 and 16-34) is associated with the worst prognosis.'* As mentioned
characterized by cellular heterogeneity. Some blasts may previously, however, cytogenetic findings better predict

Cytologic Features

Cell size Predominantly small Large, heterogeneous Large, homogeneous

Nuclear chromatin Homogeneous in any one case Heterogeneous Finely stippled, and homogeneous
Nuclear shape Regular, occasional clefting Irregular, clefting and Regular (round to oval)
indentation common
Nucleoli* Inconspicuous One or more, often large One or more, prominent
Cytoplasm* Scanty Variable; often moderately Moderately abundant; strongly
abundant basophilic
Variable Prominent
*The most useful cytologic features in separating L1 from L2 lymphoblasts are quantity of cytoplasm and presence and
prominence of nucleoli.
Source: Adapted from Bennett, JM, et al: Minimally differentiated AML (FAB MO). Br J Haematol 78:325, 1991.
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 359

rete
aaa

Immunologie

Precursor B- Si ALL : tbr ehh 1 Sita

Early-Pre-B-cell ALL L1, L2 morphology
- Pre-B-cell ALL seh L1, L2 morphology
B-cell ALL | eer aeules? morphology S
_Teell 7ANGIS San tb edt es pecteteptolG 12 morphology
FAB = - French-American-British.

Figure 16-33 m ALL, L2, bone marrow.

prognosis. Today, most treatment centers have abandoned

the FAB classification system of ALL and rely strictly on
immunophenotypic, cytogenetic, and molecular findings for
therapeutic decisions.*° The classifications of ALL are listed
in Table 16-14, comparing the immunologic classification
to the FAB classification.

WHO Classification of ALL

As mentioned, the WHO classification of ALL is based
on whether the clonal lymphoblast population is of B-
or T-lymphocyte lineage and whether the leukemic popula-
tion has the attributes of an “early lymphocyte” or
lymphoblast.~ As a _ result, the WHO classification
includes: Precursor B-lymphoblastic leukemia/lymphoma
and precursor T-lymphoblastic leukemia/lymphoma.** This
determination relies heavily on the immunophenotype of
the blast population, which allows one to identify whether
Figure 16-34 m ALL, L2, peripheral blood. the blasts are “frozen” at an earlier or later stage of lympho-
cyte development. B-lymphoblast phenotypes are summa-
rized in Table 16-15. Lymphoblasts are phenotyped using
monoclonal antibodies (see Table 16-9). The nuclear
enzyme (TdT), which is found in immature lymphoid cells

Phenotype
|e oo I OT NIE

Precursor - HLA-DR, CD34, Bel CD19, CD20 ors

B-cell CD10
Pre-B ALL HLA-DR, TdT (+), CD19, CD20 (+),
@DiONcls Vine
B-ALL HLA-DR, CD19, CD20, CD22,
CD10 (4), slg
Precursor HLA-DR (+), CD1, CD2, cCD3, dual
T-ALL PE GCDAECDSeCDS EDT Cs):
CD10 (2), CD34 (&), idl
‘Figure 16-35 W ALL, L3, bone marrow.
360 Chapter 16 Introduction to Leukemia and the Acute Leukemias

independently associated with an unfavorable prognosis.

26
(both B and T lineage), is critical for the diagnosis of lym-
phoblastic leukemia. This rearrangement is not generally found in cases of early
pre-B-cell ALL. Basing the distinction on cytogenetic, rather
PRECURSOR B-CELL ALL (EARLY PRE-B AND PRE-B) than blast immunophenotype, results in a more precise
Precursor B cell ALL is seen predominantly in the pediatric distinction.
age group, although it may occur at any age. Its peak incidence The WHO’s discussion of precursor B-cell ALL includes
is between the ages of 3 and 5 years, with 75% of cases pre- several genetic alterations that have important prognostic
senting in children younger than 6 years old.** Most childhood implications. About one third of cases show the leukemic
cases have L1 morphology. L2 morphology is more common blasts to have an increase in chromosome numbers.” If the
in adults. Patients with precursor B-cell ALL usually present hyperdiploidy is greater than 50, the ALL is associated with
with disease predominantly localized to the blood and bone sensitivity to chemotherapy and is considered to have a favor-
marrow. Prominent splenomegaly, hepatomegaly, and lym- able prognosis.** In contrast, hypodiploidy has an unfavorable
phadenopathy are infrequently seen at presentation. It is also prognosis. In addition, the following specific genetic abnor-
uncommon for these patients to exhibit a markedly elevated malities are mentioned in the WHO classification as having
white blood cell count (greater than 100 X 10°/L).'* The two specific prognostic value.**
most important clinical predictors of how a patient will
respond to treatment are age and white blood cell count at ALL WITH t(12;21): TEL-AMLI Generally considered as the
diagnosis, with age 10 or greater, age younger than | year, and most common translocation in precursor B-cell ALL in chil-
WBC count greater than 50,000 predictive of worse out- dren, the t(12;21) occurs in about 16% to 28% of unselected
come." Other risk factors, such as the genetic characteristics cases.*! It is rarely found in adults and the blasts typically
of the lymphoblasts, are discussed below. show strong expression of CD10, but not CD20. The translo-
Classification prior to the WHO often subdivided precur- cation results in fusion of the TEL gene on chromosome 12
sor B-cell ALL into various stages based on the degree of and the AML/ gene on chromosome 21q.** AML/ is part of a
maturation, reflecting B cell ontogeny. Prior classification dis- transcription factor complex. This translocation is more com-
tinguished between early pre-B and pre-B ALL using blast monly detectable by RI-PCR than by routine cytogenetic
immunophenotype. For example, early pre-B-cell ALL analysis and identifies a subgroup of pediatric ALL patients
expresses HLA-DR and CD19. Pre-B-cell ALL has the same with a good prognosis.*°*°?! The peak incidence of this
surface phenotype but also expresses Cu.** Most precursor translocation is between 2 and 5 years of age.*4
B-cell leukemias have CALLA (CD10) on their surface and
are referred to as “common ALL.” Some of these, particularly ALL WITH t(1;19): PBX/J-E2A This translocation results in
pre-B-cell ALL, also express CD20, a pan-B-cell marker that the fusion of the PBX/ gene on chromosome | with the E2A
first appears during the midstage of B-cell development. gene on chromosome 19, and the formation of an abnormal
Although immunophenotype is no longer routinely used to fusion protein.*! It is closely associated with strong CD10 and
distinguish between pre-B-cell and early pre-B-cell ALL, it negative CD34 expression on the leukemic blasts. Detectable
remains useful for identifying ALL and distinguishing it from by RT-PCR methods, and in many cases FISH, this transloca-
AML. Surface « and X light chains are typically absent on tion is associated with a poor prognosis in pediatric ALL.
lymphoblasts, and TdT is positive.'’ Rarely to occasionally, Recent intensification of therapy has shown an improved
myeloid associated antigens like CD13 or CD33 are survival.*!
expressed on precursor B lymphoblasts, and CD45, an anti-
gen routinely used to identify hematopoietic cells, is typically ACUTE LYMPHOID LEUKEMIA (ALL) WITH t(9;22):
dimly expressed on lymphoblasts or may be negative.!! BCR-ABL The t(9;22) is associated with high-risk B-lineage
The WHO’s classification relies on immunophenotype ALL (as well as CML) and is seen in about one quarter of adult
for the classification of ALL and helps distinguish between ALL cases.” This form of ALL is almost uniformly fatal in all
myeloid versus lymphoid lineage, as well as between B- age groups. Blasts usually express CD10 and CD34, and may
versus T-lymphoblastic leukemias. Whereas flow cytometry express some myeloid antigens like CD13 and CD33. The
is the clinical mainstay for diagnosing precursor B-cell ALL, translocation results in fusion of the ABL oncogene on
molecular assays to identify particular cytogenetic abnormal- chromosome 9 with the BCR gene on chromosome 22, and pro-
ities have become important for determining prognosis and duction of a fusion protein with strong transforming activity.
guiding treatment.*'** For example, it was previously noted Treatment for BCR-ABL, or Philadelphia chromosome positive
that the difference between children with pre-B-cell ALL (Cu ALL, is evolving with the advent of imatinib and other therapy
present) and early pre-B ALL (Cu absent) was important targeted to the product of this chromosomal abnormality which
because the former was associated with a poorer outcome. is used in combination with other chemotherapy. This abnor-
The distinction was based on blast immunophenotype. Both mality may be detected by RT-PCR and FISH.!
groups showed a high rate of achieving complete remission;
however, patients with pre-B-cell ALL appeared to have a ALL WITH t(4;11): AF4-MLL This translocation is seen in
shorter duration of remission.*? Subsequent analysis estab- the most common form of ALL in infancy and involves fusion
lished that 20% to 30% of pre-B ALL pediatric patients have of the AF4 gene into the MLL gene (also called ALL/ and
a translocation of chromosomes | and 19 (t{1;19]), which is HRX) on chromosome 11q23.*° MLL gene is a transcription
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 361

factor and is involved in other translocations. Many acute Burkitt’s Leukemia/Lymphoma (Mature
leukemias with an MLL gene abnormality are CD10 negative
B-Cell ALL)
by flow cytometry.*° The t(4;11) may be detected by RT-PCR
method. There is a FISH probe for 11q23, which allows iden- Recall that the FAB classification scheme had three categories
tification of a translocation involving the MLL gene and some of ALL based on blast morphology. L3 lymphoblasts were
other chromosome. The 11q23 abnormality is also seen in Burkitt-like and morphologically had the features of lym-
secondary AML in adults, which occurs when adults get AML phoblasts, so were logically classified as ALL. The WHO
after they have been treated with certain chemotherapy. In Classification, with its emphasis on immunophenotype and
both AML and pediatric ALL, 11q23 abnormalities are asso- cytogenetics, does not include B cell ALL, or the FAB L3
ciated with a poor prognosis.*> subtype of ALL, in its precursor B cell ALL classification,
because Burkitt-like lymphoblasts lack the immunopheno-
PRECURSOR T-CELL ALL typic characteristics of an early B cell, namely the TdT is
About 15% to 25% of all patients with ALL have precursor negative. The leukemic B cells in Burkitt’s leukemia, previ-
T-cell ALL.*°*’ These T-ALL cases can be further categorized ously called B-cell ALL, are derived from a transformed or
into one of the three stages of intrathymocyte development stimulated B cell; they are more mature B cells than the
based on immunophenotype, but the prognostic significance lymphoblasts in precursor B ALL.
of where the lymphoblasts are in these stages of development Burkitt leukemia/lymphoma is rare and most typically
remains uncertain.*°*° Cytoplasmic CD3, CD2, and CD7 are represents the leukemic phase of Burkitt lymphoma in
expressed early in development. Surface CD3 is expressed patients with bulky disease (see Chap. 21). Only rarely will
after cytoplasmic CD3. CD7 is a very reliable marker of patients present with blood and marrow disease clinically
T-cell ALL, but CD2 and CD5 are also expressed in many resembling pre-B ALL and not have bulky lymphoma. Burkitt
cases.'' Approximately 10% of T-cell ALL cases express or L3 morphology can reliably predict the immunophenotype,
CD10 or CALLA, which is also seen on many pre-B ALLs."! which is that of a mature B-cell. There is typically a high con-
Interestingly, CD4 and CD8 are occasionally coexpressed in centration of surface immunoglobulin (usually slgM) and
T lymphoblasts, corresponding to a later stage of thymocyte monoclonal light-chain restriction. CD19, CD20, and HLA-
development, but before selection of either CD4 or CD8 on DR are positive; TdT is negative; and most cases do not
mature T cells. Like the lymphoblasts of pre-B ALL, the lym- express CD10. Virtually all cases have a characteristic
phoblasts of T-cell ALL are TdT positive and are associated translocation involving a rearrangement of the c-myc onco-
with both L1 and L2 morphology (see Table 16—14).°’ gene on chromosome 8 with the immunoglobulin heavy-chain
Patients with T-cell ALL often present with a mediastinal gene on chromosome 14, or the light-chain genes on chromo-
mass, a high white blood cell count (greater than 100 x 10°/L somes 2 or 22 (t[8;14], t[2;8], or t[8;22]).*!
in 50% of cases), hepatosplenomegaly, and early meningeal Patients with Burkitt’s leukemia are treated differently
involvement. Males are affected more often than females, and from patients with pre-B ALL, which has a relatively good
the disease occurs more often in older children. These patients prognosis. The prognosis for Burkitt’s leukemia (B-cell ALL)
generally have a poorer prognosis than those with common has traditionally been worse than for most precursor B-cell
pre-B ALL. Features that most consistently correlate with a ALL, but has improved with newer targeted therapies.
better response to therapy include a low white blood cell level,
younger age (less than 15 years), and L1 morphology.** There
is also some evidence that certain surface markers may be Childhood versus Adult ALL
associated with a better (CD5) or worse (CD3) prognosis.*”**
When patients present with a mediastinal mass, it may be The incidence and prognosis of ALL differs markedly with
difficult to distinguish T-cell ALL from lymphoblastic lym- age. Recall that one of the most important clinical predictors
phoma. The cytologic features of this type of lymphoma are of outcome in ALL 1s age, with patients 10 years old or older,
similar to those of ALL. Although the lymphoma is generally and infants younger than | year having a worse prognosis.
associated with a more mature immunophenotype, both the Overall, about 80% of pediatric patients are alive and disease
lymphoma and the leukemia are composed of TdT positive free at 5 years, compared with significantly lower rates in
lymphoblasts with evidence of T cell lineage.*°® After a vari- adults with ALL.*’ For this reason ALL may be considered in
able time, patients with lymphoblastic lymphoma almost two subcategories: childhood ALL and adult ALL.
always develop bone marrow involvement, which renders There are cytogenetic and morphologic differences
their condition indistinguishable from T-cell ALL. The dis- between adult and pediatric ALL. Ll morphology occurs
tinction between T cell or B cell lymphoblastic leukemia and most frequently in the pediatric age group, whereas the L2
lymphoma is generally based on clinical criteria. Patients who type tends to be seen in adults. The t(9;22), a poor prognosis
first present with prominent marrow and peripheral blood translocation also known as the Philadelphia chromosome,
involvement are usually diagnosed with T-cell ALL, even if has a significantly greater frequency in adult precursor B-cell
they have concomitant mediastinal involvement. In contrast, ALL.*° MLL or mixed lineage leukemia gene abnormalities
a patient who presents with mediastinal involvement but no are typically seen in infant precursor B ALL patients and
marrow or peripheral blood involvement is diagnosed with largely account for the disappointing prognosis for ALL in
lymphoma. patients less than | year old.*? The most common cytogenetic
362 — Chapter 16 Introduction to Leukemia and the Acute Leukemias

abnormality in pediatric ALL is t(12;21), seen in about 25% cytopenias, including severe infections and hemorrhages.
of pediatric cases and associated with a favorable prognosis. Improved supportive care has reduced mortality and morbidity
Many patients diagnosed with ALL will achieve a com- from AML therapy; hematopoietic growth factors given during
plete remission, and many are potentially cured.*? Unfortu- induction accelerate recovery from myelosuppression and are
nately, some patients relapse, and some eventually die from pivotal in timely drug administration during dose-intensive
their disease. The therapy used to achieve a cure has toxic treatment regimens. Results of AL therapy have improved over
effects that are a special concern in growing and developing the past 20 years, but therapies producing cures consistently
children. To select the patients who will have an optimal have yet to be identified for most subtypes of AL. Intensive
response to less aggressive therapy, investigators have identi- investigation in this area is still ongoing, with the goal of devel-
fied prognostic factors and defined clinical risk categories. oping therapies that are more patient- and disease-specific, and
These tools allow clinicians to better select the type and inten- also less toxic. Many new “molecular” markers with potential
sity of therapy to most optimally match each patient’s risk of value in prediction of prognosis and therapeutic decision-
relapse against long-term sequelae of treatment. To date, indi- making have been identified by recent microarray gene expres-
cators of a poor prognosis include older age, a high white blood sion profiling and comparative genomic hybridization. The
cell count (more than 20 < 10°/L), and particular chromosomal integration of these new markers into treatment schema is dif-
abnormalities like t(9;22) BCR-ABL, t(4;11) and other MLL ficult and must be done very carefully; however, although their
abnormalities, t(1;19) PBX—E2A and hypodiploidy.* T-cell or clinical utility is currently limited, these new observations are
mature B-cell phenotypes and L2- and L3-type morphology providing pivotal information about disease mechanisms and
have also been associated with less favorable prognosis.** Chil- potential new drug targets.
dren, with hyperdiploid lymphoblasts (more than 50 chromo-
somes), a low white blood cell count, and t(12;21) TEL-AMLI
have a more favorable prognosis.**
Treatment of ALL
The effective treatment of pediatric ALL is one of the great
successes of clinical oncology, with long-term survival in
Treatment of Acute Leukemia
over 80% of children.'?**°? However, over the same time
Therapy for acute leukemia (AL) is among the most complex of period, adults with ALL have had a much lower cure rate of
any anticancer programs. The goals of therapy for AL are to erad- 20% to 40%.?7837° Although well-recognized age-related dif-
icate the leukemic clone, reconstitute normal hematopoiesis, and ferences in the biology of ALL exist, recent studies suggest
prevent any emergence of resistant leukemic subclones. Anti- that the survival difference between adults and children with
leukemic therapy generally consists of three distinct phases: ALL is due more to differences in treatment than disease
induction, consolidation, and maintenance.*'*? Induction biology or patient characteristics. Most children with ALL are
therapy is designed to attain complete remission as quickly as treated on clinical trials, in academic centers with experi-
possible; its success is the best predictor of long-term disease- enced support teams; children also receive higher doses of
free survival. Remission occurs when the patient’s blood counts some important drugs, and adherence to prescribed drug
return to normal, and bone marrow samples show no sign of dis- doses and schedules is very high in pediatric clinical trials. In
ease. Induction therapy achieves a remission in more than contrast, most adults with ALL are not treated on clinical tri-
95% of children and in about 75% to 89% of adults.*° Induction als or in academic medical centers, and dose reductions are
therapy is usually very intense and lasts about | month.** After much more common. Newer studies find that cure rates of
induction chemotherapy, the next step is usually consolidation adult ALL patients treated on pediatric treatment regimens is
chemotherapy, depending on the patient and type of leukemia. similar to that seen in children, suggesting that ALL therapy
The goal of consolidation therapy is to reduce the number of based on disease biology, not age, should be the standard for
disease cells left in the body. The drugs and doses used during all ALL patients in the future.*”~?
consolidation therapy depend on the patient’s risk factors. Con- Central nervous system (CNS) prophylactic treatment is
solidation therapy is also intense, lasting approximately 4 to standard in ALL therapy during induction and consolida-
8 months.” Maintenance therapy usually starts after a patient tion.’° Although unusual at diagnosis (<10%), CNS disease
stays in remission after induction and consolidation therapy. The can be as high as 50% to 75% at | year in the absence of in-
goal is to destroy any leukemic cells that may remain in order to trathecal (IT) chemotherapy. Craniospinal irradiation is asso-
maintain a disease-free state. Maintenance therapy is less ciated with many neurologic adverse events, including
intense than the other two phases and may last 2 to 3 years.” seizures, dementia, and intellectual dysfunction, so IT and
However, the specific drugs used and their timing and high-dose systemic chemotherapy are typically used today for
doses differs remarkably among different AL subtypes.*°34 CNS prophylaxis.****
Their modes of action differ but, in general, they poison and Intensification of induction therapy by using additional
kill dividing cells, usually by blocking DNA or RNA synthesis. drugs has improved survival in some ALL subtypes. Intensive
The toxicity of chemotherapy for AL must always be balanced consolidation is standard in post-remission ALL therapy, but
with the efficacy of treatment. The consequences of AL therapy specific drug cycles are variable. Regardless of the regimen
can be very serious; treatment-related early death is not uncom- used, strict adherence to protocols, without significant dose
mon and there is often significant morbidity from prolonged delay, appears to be very important for long-term survival of
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 363

ALL patients. Post-remission maintenance therapy in ALL is of >10,000, and others have an intermediate risk. APL therapy
also standard, and is sometimes given over 2 to 3 years.*° The is curative in most patients; anticipated cure rates are close to
only exception is mature B-ALL, in which relapses beyond 100% in low-risk, 90% in intermediate-risk, and 70% in high-
the first year are rare.*° There are still many questions about risk patients. The addition of all-trans-retinoic acid (ATRA) to
the optimal drugs, doses, and schedules used for maintenance AML chemotherapy increases the CR rate and dramatically
therapy in the different subtypes of ALL. reduces the relapse rate in APL patients.'*!7°°
The utility of allogeneic stem cell transplant (SCT) as
front-line therapy in ALL is not yet well validated. In some AML WITH INV(16) OR t(8;21)
clinical trials, allogeneic SCT has been done in first complete Appropriate treatment is curative in about 50% of cases in
remission (CR1) and it appears to be of some benefit in high- this group, using standard AML regimens for induction, con-
risk ALL.**°° However, the indications, scheduling, and pro- solidation, and maintenance therapy that include high dose
cedures for SCT in CRI are not yet standardized and it is not cytarabine.©°*? Transplant should not be considered in this
clear that SCT-based therapy in CR1 is associated with an im- AML subtype unless relapse occurs.
proved outcome over chemotherapy alone in most subtypes of
ALL. Larger cooperative group studies are required to evalu- AML IN THE ELDERLY
ate the optimal integration of SCT in front-line ALL therapy. Standard AML chemotherapy in the elderly (>60 years old) is
Targeted therapies have been developed for some ALL associated with a high rate of treatment-related mortality and
subtypes. One example of a targeted therapy is the use of ima- a poor outcome in patients who do survive initial therapy.”” CR
tinib, a tyrosine kinase inhibitor, in addition to chemotherapy rates in these patients are <50%, and the probability of sur-
for Philadelphia chromosome-positive ALL. This has resulted vival at 2 years is about 10%. Patients <80 years of age have
in a considerable improvement in outcome in this formerly a mortality rate of <50%, so transfusion support or palliative
very unfavorable subgroup of ALL patients, without signifi- care may be reasonable options for this age group. Investiga-
cantly increasing toxicity.°’ Another example of a targeted tional drugs and/or targeted therapies at initial diagnosis may
therapy is the addition of an anti-CD20 monoclonal antibody be appropriate for these poor prognosis patients. Less inten-
to therapy for mature B-ALL; this has significantly improved sive induction approaches may also be used in the elderly,
outcome for these patients.°? Recent trends in treatment of such as reduced intensity allogeneic stem cell transplant.’”"™
ALL are focusing on’ immunostimulatory oligonucleotides
and other interventions at the genetic level.**?
Measurement of Response to Therapy
for Acute Leukemia
Treatment of AML
Response to therapy for AL is measured by a number of para-
AML is a very heterogenous disease biologically, and the cyto- meters. Traditionally, a complete remission (CR) has been
genetic and molecular abnormalities detected at the time of defined as normalization of peripheral blood counts and a
diagnosis provide the best information for predicting clinical return to normal bone marrow hematopoiesis. The blast percent-
outcome and stratifying therapy.*?AML is typically divided age should be normal, and there should be no persistence of
into the following subgroups for therapy decisions: (1) acute disease morphologically or with flow cytometric analysis.
promyelocytic leukemia (APL), (2) AML with inv(16) or Cytogenetic analysis is typically performed before and after
(8:21), (3) AML in younger patients with no adverse risk fac- therapy, and the absence of a previously identified cytogenetic
tors, (4) AML in younger patients with adverse risk factors, and abnormality is another important component of CR. However,
(5) AML in the elderly. Within each of these groups, there these traditional measures of treatment response are relatively
are further risk factors that can affect initial and subsequent insensitive. New techniques for detection of a low level of resid-
therapy. These include the presence of high white blood cell ual disease, below the level detected by morphologic review,
counts of >50,000 and leukemic lung infiltration, ®’ which are immunophenotyping, or standard karyotyping, suggest that the
indications for immediate therapy without waiting for results of achievement of a molecular complete remission may be the best
karyotyping. In all AML patients, regardless of therapy, achieve- measure of treatment efficacy in AL. Persistence of detectable
ment and maintenance of a CR is the goal of therapy. Once CR disease results in significantly worse survival.’*”°
is maintained for 3 years, the risk of relapse declines markedly Molecular remission is defined as a level of minimal resid-
to only 5% to 10%.™ Some specific treatment considerations for ual disease (MRD) that is below the detection limit of
different AML treatment groups are discussed below. PCR analysis, which is generally | blast in 10,000 (10+) or
100,000 (10°) normal bone marrow cells. The presence of
APL MRD is felt to be an independent prognostic factor that can
Early recognition of APL is crucial, for a number of reasons. identify primary drug resistance and other factors playing a role
APL patients are at risk for fatal hemorrhage due to dissemi- in therapy response. MRD testing is being used in some AL clin-
nated intravascular coagulation, so the appropriate treatment for ical trials to aid in decision-making about the need for mainte-
APL is different from that employed for other types of AML." nance or other more aggressive therapy following induction.
Low-risk APL patients present with WBC counts <10,000 and However, the predictive value of MRD analysis in ALL depends
platelet counts >40,000; high risk patients have a WBC count on the technical quality of the testing, which is time-consuming,
364 Chapter 16 Introduction to Leukemia and the Acute Leukemias

Table 16-16 Some Targeted Drugs count, 79.2 X 10°/L. A peripheral blood smear was manu-
| sGurrently. ines se oe ally reviewed in the hematology laboratory and the differen-
tial diagnosis included 80% blasts.
_ Development | forAMILaie toe A bone marrow aspirate and biopsy were obtained, which
both showed virtually total replacement of normal elements
Type
with sheets of poorly differentiated cells (Fig. 16-36). The
Epigenetic Therapy aspirate smears showed sheets of blasts. The blasts were rel-
DNA methyltransferase Reverse gene silencing by atively large (15 to 20 um) with a moderate amount of cyto-
inhibitors inhibiting DNA methylation plasm. The nuclei varied in shape from round to oval, and
Histone deacetylase Reverse gene silencing by some were indented or folded; most had several distinct
(HDAC) inhibitors inhibiting histone small nucleoli. An occasional blast had azurophilic granules,
deacetylation but this was the exception. No Auer rods were seen. The
FLT3 Inhibitors AML patients (50%) biopsy specimen was hypercellular, approaching 100% cel-
overexpress FLT3
lularity in some areas. The morphology of the aspirated cells
was similar to those seen in the peripheral blood except that
fewer of the cells had folded nuclei, and, in general, the
expensive, and requires highly-trained staff. In most clinical tri- nuclear-to-cytoplasmic (N:C) ratio was higher. Cytochemi-
cal studies of the aspirate smears were positive: the
als, MRD testing must be done by designated central reference
myeloperoxidase stain was positive in approximately 30%
laboratories.” Detection of MRD at any time point after the start
of the blasts and the NSE (a-naphthy] butyrate) was positive
of consolidation therapy is associated with a high relapse risk in in occasional cells (less than 5%).
most AL subtypes, particularly ALL.” Patients with MRD after
consolidation therapy are candidates for stem cell transplanta-
tion (SCT) or other targeted therapies.
The rapid discovery of novel molecular aberrations and
global gene expression profiles have led to the development
of targeted drugs for AML.” These genomic markers confirm
the heterogeneity of this disease and support the need for
individualized patient treatment. Several of these genomic
markers serve not only as prognostic factors but also as ther-
apeutic targets for smali molecules and other novel com-
pounds. Many new targeted agents are currently in clinical
testing. Some of the classes of targeted drugs currently in
development are listed in Table 16-16.” These targeted
approaches are being investigated for relapsed and refractory
AML of initial complete remission duration of less than
1 year. However, the role of these new targets in leukemoge-
nesis remains to be elucidated.
Figure 16-36 @ Case study—bone marrow (AML).

A portion of the aspirate was analyzed using flow cytom-

Case Study I etry. The blast immunophenotype was as follows: CD7,
CD13, CD33, CD34, dim CD45, and HLA-DR. Importantly,
A 42-year-old woman presented with a 2-month history of TdT, CD19, CD3, and immunoglobins were all negative.
fatigue and weakness and a 2-week history of a sore throat. Another portion of the aspirate was sent for conventional
She reported a 15-pound weight loss over the past month or karyotyping and showed normal karyotype. FISH analysis
two. One week prior to admission she started antibiotics for for a genetic abnormality involving 11q23 MLL showed
her sore throat, but she reported little improvement; she sub- a majority of cells with a translocation involving the
sequently developed a peritonsillar abscess. On admission MLL gene.
to the hospital she was found to have an elevated white Diagnosis: The WHO diagnosis would be AML with
blood cell count with a large number of circulating blasts. 11q23 MLL abnormality. The diagnosis without the FISH
The physical examination of the patient showed an anx- analysis would be acute myeloid leukemia without matura-
ious, middle-aged woman whose vital signs were normal, tion (FAB M1).
aside from a slightly elevated temperature (37.6°C). Her Follow-up: HLA matching was performed on the patient’s
right tonsil was enlarged and inflamed. She had no adenopa- brother and sister, but neither had a compatible tissue type.
thy, and her liver and spleen were not palpable. The possibility of bone marrow transplantation was subse-
Laboratory studies were ordered, and the following quently ruled out. The patient was placed on a standard pro-
results were reported: hematocrit, 19.5%; hemoglobin, tocol for acute myeloid leukemia. During her induction
6.3 g/dL; platelets, 64 < 10°/L; and white blood cell (WBC) chemotherapy she developed anemia, thrombocytopenia,
continued
continued
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 365

and leukopenia. She required platelets and packed red blood A specimen was also sent for cytogenetic studies. Inter-
cell transfusions. She also required broad-spectrum antibi- phase FISH studies showed 95% of cells positive for the
otics for fever resulting from neutropenia, although no PML-RARa fusion, indicative of the t(15;17) translocation.
specific pathogen could be identified. Three weeks after her Diagnosis: The WHO diagnosis is acute promyelocytic
induction chemotherapy, a repeat bone marrow biopsy was leukemia with t(15;17). The FAB classification would cate-
performed. There was no evidence of residual leukemia by gorize this as AML M3. Importantly, there is a microgranu-
morphologic examination of the aspirate smears and biopsy; lar variant of APL, in which the abnormal promyelocytes
or flow cytometric analysis of the aspirate; or by the FISH have a paucity of visible granules and the nuclei are typi-
probe looking for MLL abnormalities. She remained in com- cally bilobed. These atypical cells may be confused with
plete remission for 11 months, but then relapsed with monocytic blasts, but an MPO is strongly positive.
20% morphologically atypical blasts in her bone marrow Follow-up: Standard treatment with all-trans retinoic acid
confirmed by abnormal immunophenotype identified by (ATRA) was initiated, followed by cytotoxic therapy.
flow cytometry. Attempts to induce a second remission were Through the course of hospitalization, the patient showed
unsuccessful. She developed progressive hepatomegaly, laboratory evidence of DIC, and required blood support
jaundice, and persistent neutropenia. She also developed from the beginning of therapy to maintain appropriate
multiple infections and died from her disease several hemoglobin, platelet, and fibrinogen levels. However, the
months later. patient’s CBC and clinical status began to improve. A month
This case illustrates a typical course of acute myeloid later, no PML—RARa fusion transcripts were detected by
leukemia. Although the patient was not cured, she did RT-PCR on a bone marrow specimen. He then received
achieve and maintain a complete remission for nearly a full maintenance therapy with ATRA in a standard dose for the
year. The 5-year survival rate for adult AML patients with next 2 years.
MLL abnormalities is about 45%, but it is important to note The case underscores the importance of recognition of
that there are many different kinds of AML with MLL abnor- APL. Proper morphologic diagnosis is imperative, so that
malities. Currently, more than 50 different genes have been the appropriate molecular studies can be requested for con-
identified that translocate with 11q23 and the characteristics firmation and so the patient is treated appropriately as soon
of each of these have yet to be fully described. Therefore, as possible. A delay in treatment can lead to complications
MLL abnormalities have an intermediate to poor prognosis. due to APL-associated coagulopathy, including cerebral
«
hemorrhage. The response to induction therapy with ATRA
and standard chemotherapy has been shown to be in the
range of 90% to 95% in genetically confirmed APL either by
conventional cytogenetics, FISH for t(15;17) or by RT-PCR
for PML-RARa.
Case Study 2
A 32-year-old man presented to the ER complaining of
fever, lethargy, and mucosal bleeding. He said that these
symptoms began 5 to 6 days ago, but that before he had been
in a general state of good health. The patient’s past medical
history was unremarkable. On physical exam, the patient
appeared ill, pale, and in moderate acute distress. Numerous
purpura were found on his upper and lower extremities, Case Study 3
which the patient said erupted over the past several days. No
hepatosplenomegaly or enlarged lymph nodes were noted. T. J. was 4 years old when he first presented to his family
CBC and examination of the peripheral blood smear doctor with a 3-week history of fatigue, weakness, and a
showed mild anemia with numerous schistocytes (red blood persistent sore throat. On physical examination he had a pal-
cell fragments). Severe thrombocytopenia and many circu- pable spleen but no evidence of lymphadenopathy. He
lating atypical mononuclear cells were also seen. Additional appeared pale and had multiple bruises over his lower
laboratory studies showed a markedly prolonged prothrom- extremities. A CBC, platelet count, and differential were
bin time and decreased fibrinogen, indicating a profound carried out, with the following results:
coagulopathy.
A posterior iliac crest bone marrow aspirate and core CBC
biopsy were performed. The bone marrow biopsy and aspi-
WBC DAO 107/15 MCV 87.0 fL
rate smear revealed a markedly hypercellular bone marrow
RBC Alex 102 /E MCH 29.0 pg
with 80% promyelocytes, many with prominent azurophilic Het 21.0% MCHC 33.3 g/dL
granulation and abnormal kidney-bean shaped nuclei. Some Hgb 7.0 g/dL Platelets 6.7 X 10°/L
of the promyelocytes contained multiple Auer rods (“faggot
cells”). Cytochemical staining with myeloperoxidase Differential
(MPO) and Sudan black-B showed strong staining in these
promyelocytes. Flow cytometric analysis of bone marrow PMN 3%
Blasts 97%
showed a myeloid population expressing CD13, CD33,
CD45(dim), CD117, while lacking HLA-DR. continued
continued
366 — Chapter 16 Introduction to Leukemia and the Acute Leukemias

Diagnosis: Precursor B-cell ALL.

Follow-up: This patient was placed on a protocol for ALL
to which he responded very well. His physical examination
A bone marrow examination was performed that revealed 2 months after induction chemotherapy was unremarkable
sheets of small blasts having scant cytoplasm and indistinct except for some hair loss. His CBC was entirely normal,
nucleoli (see Fig. 16-30). Cytochemical and cell marker stud- although his white blood cell count was low-normal. A bone
ies of the bone marrow aspirate gave the following results: marrow examination showed no evidence of residual
leukemia by morphologic and flow cytometric analysis.
Cytochemistry Flow cytometry did show a population of hematogones,
which are immature B cells often seen in regenerating mar-
Peroxidase Negative rows and having a similar immunophenotype to precursor
NSE Negative B-cell ALL blasts. He was continued on therapy, including
one reintensification phase followed by maintenance ther-
Immunohistochemistry
TdT Strongly positive
apy. Eight years later, he is free of any signs of leukemia and
is living a normal life.
Surface Markers .

CD3 Negative CD10 100%

CD5 Negative CD19 98%
CD7 Negative CD20 Negative

continued

Qu estions
Ye e

: AN 4-year-old boy presents with bruising, fever, and

coughing. His white blood cell count is 15 X 10°/L;
3. The white blood cell count is 15 X 10°/L with 90%
blasts, 6% segmented neutrophils, and 4% monocytes.
hematocrit, 23%; and platelets, 53 =< 10°/L. A bone mar- The blasts are relatively large and have abundant cyto-
row aspirate is obtained that reveals sheets of immature plasm. More than 90% of them are positive with the
cells. Cytochemical studies for peroxidase and NSE are
nonspecific esterase stain, and an occasional blast is pos-
negative; the TdT is positive, Surface markers studies
itive with Sudan black. What is the diagnosis?
are done that show the following phenotype; HLA-
a. AML, with maturation, FAB M2 type
DRe CDS eCDi0s Cu—- sle— CD) — A portion
b. AML, acute promyelocytic leukemia, FAB M3 type
of the aspirate is sent for cytogenetics and molecular
c. AML, acute myelomonocytic leukemia, FAB M4 type
studies. What is the most likely diagnosis?
d. AML, acute monoblastic leukemia, FAB M5 type
a. AML
b. Precursor-B-cell ALL with t(9;22) BCR-ABL 4. Cytochemical stains were performed on bone marrow
c. Precursor-B-cell ALL with t(12;21) TEL-AMLI1 smears from an acute leukemia patient. All blasts were
d. T-cell ALL TdT negative. The majority of the blasts showed varying
amounts of Sudan black B positivity. Fifty percent of
2. A 42-year-old woman present with an elevated white
them stained positive for nonspecific esterase. What type
blood cell count, anemia and thrombocytopenia. The
of leukemia is indicated?
white blood cell count is 50 < 10°/L with 80% blasts,
a. Acute myeloblastic leukemia
15% segmented neutrophils, and 5% lymphocytes. The
b. ALL
bone marrow reveals sheets of immature cells. Cyto-
c. Acute myelomonocytic leukemia
chemical studies show MPO positive (20%) and the non-
d. Acute erythroleukemia
specific esterase negative staining on the immature cells.
Flow cytometry shows the blast population positive for 5. Bone marrow examination reveals a hypercellular mar-
CD13, CD33, dim CD45, CD34, and CD117. What is row with 60% blasts. Flow cytometry shows the follow-
the diagnosis? ing blast immunophenotype: CD3, CD7, CD10, CD13,
a. AML, without maturation, FAB M1 type CD34, CD45, and TdT positive. Surface immunoglobins
b. AML, with maturation, FAB M2 type are negative. The diagnosis is:
c. AML, acute myelomonocytic leukemia, FAB a. Burkitt’s leukemia
M4 type b. AML
d. AML, acute monoblastic leukemia, FAB M5 c. ALL, precursor B-cell type
type d. ALL, precursor T-cell type
 Chapter 16 Introduction to Leukemia and the Acute Leukemias 367

6. A 49-year-old woman was admitted to the hospital for 10. What cytochemical stain is best for differentiating AML
easy bruising and menorrhagia. She had evidence of dis- from ALL?
seminated intravascular coagulation. Her white blood cell a. Alpha-naphthyl acetate
count is 3 X 10°/L with 95% large, atypical mononuclear b. Nonspecific esterase
cells and some atypical bilobed cells. Many of these cells c. Myeloperoxidase
are packed with larger, purple-staining granules; some d. Periodic acid—Schiff
have multiple Auer rods; and all are strongly peroxidase-
. How many blast cells in a bone marrow aspirate smear
positive. What is the diagnosis?
are necessary for a diagnosis of acute leukemia using
a. AML, FAB M2
FAB criteria?
b. AML, FAB M3
a. 20%
c. AML, FAB M4
b. 15%
d. AML, FAB M5
C2570
. What is the likely genetic abnormality referring to ques- d. 30%
tion 6?
. How many blast cells in a bone marrow aspirate smear
a. ((8;21) AML/I-ETO
are necessary for a diagnosis of acute leukemia using
b. (4511) AF4-MLL
WHO criteria?
c. t(9;22) BCR-ABL
a. 20%
d. t(15;17) PML—-RARa b. 15%
. A 21-year-old patient’s bone marrow and peripheral Cnzove
blood has medium sized blasts with basophilic cyto- d. 30%
plasm and numerous lipid vacuoles. Cytogenetic studies . Which chromosome abnormality occurs in the FAB M2
show t(8;14) involving MYC on chromosome 8 and the
type of AML?
Ig heavy chain region on chromosome 14. Flow cytome- a. t(8;21)
try is most likely to show which of the following? b. t(9;22)
a. CD37 CD77, CD10, CD45, and TdT Cant Clsyailii/))
b. CD10, CD19, CID20, CD45, and surface « light chain d. t(1;19)
c. CD10, CD19, CD45, TdT, and surface « light chain
d. CD10, CD19, CD34, and no surface light chain or TdT 14. Which cytochemical stain is useful in separating
myeloblasts from monoblasts?
. Which of the following is true about the prognostic a. Myeloperoxidase
implications of cytogenetic findings seen in precursor b. Sudan black B
B-cell ALL? c. Specific esterase
a. Hypodiploidy as determined by flow cytometry DI is d. Nonspecific esterase
associated with a favorable prognosis
b. t(1:19) PBX—E2A is associated with an unfavorable . Which chromosome abnormality occurs in the FAB M3
prognosis type of AML?
. (9:22) BCR-ABL is associated with a favorable
a. t(8;21)
(o)

prognosis bet:22)
Cat@lo: iy)
d. Hyperdiploidy of greater than 50 chromosomes as
determined by flow cytometry DI is associated with a ericlelien)
poor prognosis.
See answers at the back of this book.
36 8 Chapter 16 Introduction to Leukemia and the Acute Leukemias

mwAuer rods are pathognomonic for a myeloproliferative

process, which is usually acute promyelocytic leukemia
(AML).
m Acute leukemia tends to present with a sudden clinical
m Using cytochemical stains, myeloblasts are positive for
onset and a rapidly fatal course if untreated. myeloperoxidase and Sudan black B.
g Chronic leukemia has a more insidious onset and an indo- a Lymphoblasts are positive for terminal deoxynucleotidyl]
lent clinical course. transferase (TdT) by immunologic methods and usually
m The neoplastic cells in acute leukemia are immature negative for myeloperoxidase and Sudan black B.
(blasts, early myeloid cells), whereas the neoplastic cells mwNonspecific esterase (NSE) is positive in blasts with
in chronic leukemias are more mature.
monocytic differentiation.
g A diagnosis of acute leukemia is made using current WHO
w Flow cytometry is an extremely useful tool for analyzing
criteria by the presence of more than 20% blast cells in
cell surface and cytoplasmic antigen expression in
bone marrow aspirate smears or in peripheral blood.
leukemia blasts and can be effective in monitoring resid-
w Acute leukemia is divided into two basic categories based ual disease.
on cell lineage: myeloid and lymphoid.
mw Cytogenetic analyses of leukemic cells can often identify
m The distinction is critically important because the treat- chromosomal abnormalities, which can be critical in
ment and prognosis are different. directing therapeutic decisions.
a Myeloblasts are usually large in size and have fine chro- mw Molecular diagnostic studies are becoming increasingly
matin and prominent nucleoli. Lineage is confirmed by important and are used to confirm a suspected chromoso-
cytochemical staining and immunophenotyping. mal abnormality or to monitor minimal residual disease
Ud Lymphoblasts tend to be small- to medium-sized blasts following treatment or bone marrow transplantation.
with dense chromatin and less distinctive nucleoli.
Lineage is confirmed by cytochemical staining and
immunophenotyping.

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SB) Yoshiyuki, K, et al: Infant acute lym- cyclophosphamide, vincristine, doxoru- trated adults with acute myeloid
phoblastic leukemia with MLL gene bicin, and dexamethasone (Hyper- leukemia younger than 60 years: Final
rearrangements: outcome following in- DVAD), a dose-intensive regimen, in induction results of Cancer and
tensive chemotherapy and hematopoietic adult acute lymphocytic leukemia. Leukemia Group B study 9621. J Clin
stem cell transplantation. Blood Cancer 101:2788, 2004. Oncol 22:4290, 2004.
104(12):3527, 2004. . deBont, JM, et al: Significant difference G3: Giles, FJ, et al: Leukapheresis reduces
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phoma Epidemiology Consortium . Schiffer, CA: Differences in outcome in 64. DeLima, M, et al: Implications of
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phoblastic leukemia. Rev Clin Exp mens? Better doctors? Both? J Clin cancer and return to work. Blood
Hematol 6:161, 2002. Oncol 21:7600, 2003. 90:4719, 1997.
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phoblastic leukemia in older patients: adult gap in cancer care and outcome. acid APL: long-term outcome and prog-
Semin Hematol 43:126, 2006. Curr Probl Pediatr Adolsec Health Care nostic factor analysis from the North
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MLL/AF4 fusion protein. Blood phoblastic leukemia at diagnosis: Results longed remission after high-dose cytara-
98(12):3398, 2001. from the international ALL trial MRC bine intensification in AML varies by
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t(8;21) acute myeloid leukemia (AML): Blood 108:63, 2006. Clin Oncol 21:4642, 2003.
A survey of 161 cases from the French . Goldstone, AH, et al: Attempts to . Marcucci, G, et al: Abnormal cytogenet-
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oO . Delaunay, J, et al: Prognosis of myeloid leukemia (AML) in older remission predicts short overall and
inv(16)/t(16;16) acute myeloid leukemia patients: The results of the United disease-free survival, and higher relapse
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French AML Intergroup. Blood 102:462, AML 11 trial. Blood 98:1302, 2001. Results from Cancer and Leukemia
2003. . Buchner, T, et al: Treatment of older Group B study 8461. J Clin Oncol
. Mrozek, K, et al: Comparison of cytoge- patients with AML. Crit Rev Oncol 22:2410, 2004.
netic and molecular genetic detection of Hematol 56:247, 2005. . Bruggemann, M, et al: Clinical signifi-
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A Cancer and Leukemia Group B study. total-body, irradiation-based condition- dard-risk acute lymphoblastic leukemia.
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. Estey, E: How I treat older patients with tion from related and unrelated donors. . Kolitz, JE: Current therapeutic strategies
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 Chapter

Chronic
Myeloproliferative
Disorders I
Chronic Myelogenous Leukemia
Carl R. Schaub, MD

Introduction to Chronic OBJECTIVES

Myeloproliferative
Disorders At the end of this chapter, the learner should be able to:
Historical Perspective | . Name the diseases included in the chronic myeloproliferative disorders.
Definition and Classification
2 Describe the relationship of chronic myelogenous leukemia to the other chronic
Chronic Myelogenous myeloproliferative disorders.
Leukemia i
Historical Perspective . List risk factors for the development of chronic myelogenous leukemia.
Definition . Describe the relationship between the Philadelphia chromosome, the translocation
Incidence and Etiology between chromosomes 9 and 22, and the BCR-ABL fusion gene.
Pathogenesis
Clinical Features . Describe the role of the BCR-ABL fusion gene and its protein product in the
Laboratory Findings pathogenesis of chronic myelogenous leukemia.
Differential Diagnosis . Describe the clinical presentation of chronic myelogenous leukemia, including the
Prognosis common presenting signs and symptoms.
Treatment
. List the three phases of chronic myelogenous leukemia, along with the diagnostic
Case Study
criteria for each.

. List the common laboratory findings in chronic myelogenous leukemia. Which

findings are most specific for the diagnosis?
. Describe the cytogenetic analysis of chronic myelogenous leukemia, including the
analytic techniques used, and the advantages of each.

. List the important differential diagnoses of chronic myelogenous leukemia.

. Describe the factors most important in determining the prognosis of patients with
chronic myelogenous leukemia.

. Briefly describe the current treatment of chronic myelogenous leukemia.

37]
Shy) Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia

Introduction to Chronic dominant expansion of one or more “myeloid” (non-lymphoid)

cell lines, with overproduction of one or more of the formed
Myeloproliferative Disorders
elements of the peripheral blood (i.e., granulocytes, erythro-
Historical Perspective cytes, and platelets). They are most commonly diseases of
adults, affecting middle-aged and older individuals. Classi-
The term myeloproliferative disorders was first proposed by cally, CMPD have included four clinical entities: chronic
William Damashek in 1951 to emphasize similarities between myelogenous leukemia (CML), polycythemia vera (PV),
chronic myelogenous leukemia, polycythemia vera, essential essential thrombocythemia (ET), and chronic idiopathic
thrombocythemia, and chronic idiopathic myelofibrosis.' This myelofibrosis (IMF). A specific CMPD can be classified based
term was proposed to describe a heterogeneous group of on the predominant increased cell type in bone marrow and
acquired clonal disorders of the bone marrow, characterized by peripheral blood.* In CML, the bone marrow shows a granulo-
bone marrow hyperplasia related to excessive proliferation of cytic hyperplasia with a dominant increase in granulocytes
one or more of the hematopoietic cell lines, with overproduction (neutrophils, eosinophils, and basophils) in the peripheral
of the corresponding cell(s) in the peripheral blood. These dis- blood. In PV, an erythroid hyperplasia in the bone marrow is
orders, now referred to as the chronic myeloproliferative disor- accompanied by a striking increase in erythrocytes in the
ders, have in common the clinical finding of splenomegaly, as peripheral blood, while in ET, megakaryocytes are dominantly
well as (frequently) extramedullary hematopoiesis. A number of increased in the marrow and platelets in the peripheral blood.
subsequent discoveries, including the fusion gene (BCR-ABL) IMF would seem to be the exception because it is character-
in chronic myelogenous leukemia and a point mutation in the ized by a decrease in most cells (pancytopenia) in the periph-
Janus kinase 2 gene (JAK2) in other chronic myleoproliferative eral blood, especially late in its course. However, early IMF
disorders,> have expanded our knowledge of the molecular may show an increase in all cell types, at least before bone
pathogenesis of various myeloproliferative disorders, resulting marrow fibrosis supervenes to cause pancytopenia. The simi-
in significant improvements in therapy. The World Health Orga- larities and differences between the CMPD are summarized in
nization (WHO) has recently proposed a new classification sys- Table 17-1. It should be noted that, although each CMPD is
tem for the chronic myeloproliferative disorders that, while characterized by a dominant increased cell type, any cell type
remaining true to Damashek’s original concept of a family of can be increased to a greater or lesser extent in any CMPD. In
related diseases characterized by bone marrow proliferation, has addition, frequent transitions between these diseases as well as
added a few new entities to the family. * overlapping characteristics sometimes make precise classifica-
tion of an individual case difficult (Fig. 17-1). Splenomegaly,
Definition and Classification the characteristic physical finding in all CMPD, occurs in 40%
to 99% of patients depending on their specific disorder.
The chronic myeloproliferative disorders (CMPD) are a hetero- Extramedullary hematopoiesis is also commonly found, par-
geneous group of diseases that originate from the clonal ticularly in autopsy studies.*
expansion of a hematopoietic pluripotent stem cell. This clonal In 2001, the WHO classification of hematologic neo-
expansion results in bone marrow hyperplasia related to a plasms expanded the list of CMPD from the four entities named

Essential Chronic Idiopathic

Disease Polycythemia Vera Thrombocythemia Myelofibrosis

Dominant increased Granulocytes: neutrophils Erythrocytes Platelets Neutrophils and

cell type and basophils megakaryocytes
early, fibrosis late
WBC count (X 10°/L) TTT, usually > 50 T, usually 12-25 sf ey early, J J late
Hemoglobin Normal to J J ate th Normal to J J Normal to T early,
late
Platelet count seat TtoTT T TT, 600-2500 Dt early, | J late
(X 10°/L)
LAP ev Usually T T Usually T LtoT
Bone marrow Marked granulocytic Erythroid Erythroid Hypercellular
hyperplasia hyperplasia, hyperplasia early, fibrosis
J iron stores J iron stores late
Fibrosis None-T None-? T None-T Tava

WBC = white blood cell; LAP = leukocyte alkaline phosphatase.

Reference range: WBC = 5-11 X 10°/L; platelets = 150-450 X 10°/L; ee slight decrease; LL = moderate
decrease; | = slight increase; T T = moderate increase; T T = marked increase.
 Chapter 17. Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia 373

Chronic Myelogenous Leukemia

Historical Perspective
Chronic myelogenous leukemia (CML) was_ originally
15-20%
10-15% described in 1845, almost simultaneously by Rudolf Virchow
in Berlin and John Hughes Bennett, an Englishman practicing
in Edinburgh.*® Bennett’s publication in October 1845 pre-
a CS) ceded Virchow’s by nearly 6 weeks, but Virchow described an
increase in blood leukocytes (Weisses Blut, white blood) with-
out attributing a cause, while Bennett believed that this repre-
sented pus in the blood (“pyemia”), perhaps secondary to
some unknown infection. Virchow coined the word Leukamie
(“leukemia”) 2 years later in 1847.”*
In 1960, CML was found to be associated with a con-
60-70%
sistent chromosomal abnormality: the Philadelphia (Ph)
chromosome, so called because it was first identified at the
University of Pennsylvania School of Medicine, in
Philadelphia.’ This was the first identification of a specific
chromosomal abnormality associated with a malignancy.
The abnormality was described as a shortened G group
Figure 17-1 @ Relationship between the various myeloproliferative chromosome, subsequently identified as an abnormal chro-
disorders. Frequencies of transitions between each of these bone
marrow stem cell disorders are shown. AML = acute myeloblastic
mosome 22 (22q-) seen in metaphases recovered from
leukemia; PV = polycythemia vera; IMF = idiopathic myelofibrosis; the bone marrow of patients with CML. In 1973, Rowley
CML = chronic lymphoblastic leukemia; CNL = chronic neutrophilic observed that the Ph chromosome resulted from a recipro-
leukemia. (A solid line indicates a strong relationship between disorders; cal translocation between the long arms of chromosomes
a dashed line indicates a weak relationship.)
« 9 and 22 (written as t(9;22)).'° In the 1980s, the Ph chromo-
some was shown to carry a unique fusion gene termed
in the preceding text, adding chronic neutrophilic leukemia, BCR-ABL, the protein product of which is now thought to
chronic eosinophilic leukemia/hypereosinophilic syndrome, and be the cause of CML.!'!:”
unclassifiable myeloproliferative disease (Table 17-2). In addi-
tion, a separate crossover category of myelodysplastic/ Definition
myeloproliferative diseases was created to include related dis-
eases such as atypical chronic myeloid leukemia, chronic Chronic myelogenous leukemia, also referred to as chronic
myelomonocytic leukemia, juvenile myelomonocytic leukemia, myeloid leukemia or chronic granulocytic leukemia, is a
and myelodysplastic/myeloproliferative diseases, unclassifiable. chronic myeloproliferative disorder characterized by a marked
The reader is referred to the WHO monograph Pathology and increase of granulocytes in peripheral blood, including neu-
Genetics of Tumours of Haematopoietic and Lymphoid Tissues trophils with immature forms, eosinophils, and basophils, as
for a discussion of these diseases.* well as a marked granulocytic hyperplasia of the bone marrow.
CML was the first human disease in which the pathogenesis
could be directly traced to a specific chromosomal abnormal-
ity, the Ph chromosome, which can be identified in approxi-
mately 90% to 95% of patients with the typical characteristics
of CML." Its presence is characteristic of the disease, but not
diagnostic, since a similar abnormal chromosome is also found
in 5% of children and 20% of adults with acute lymphoblastic
leukemia and in 2% of patients with acute myeloblastic
leukemia.'*

Chronic myelogenous leukemia [Ph chromosome,

t(9;22)(q34;q11), BCR-ABL-positive] Incidence and Etiology
Chronic neutrophilic leukemia
Chronic eosinophilic leukemia (and the hypereosinophilic CML accounts for 20% of cases of leukemia in adults, with
syndrome) approximately 4200 new cases in the United States in 2004
Polycythemia vera and an annual incidence of 1.6 cases per 100,000 popula-
Chronic idiopathic myelofibrosis (with extramedullary
tion.'° There is a slight male predominance (male to female
hematopoiesis)
ratio 1.4:1). The median age at diagnosis is between 45 and
Essential thrombocythemia
Chronic myeloproliferative disease, unclassifiable 55 years, with 12% to 30% of patients of age 60 years or
older at diagnosis.'° CML is unusual in children. Identified
374 Chapter 17 Chronic Myeloproliferative Disorders I: Chronic Myelogenous Leukemia

leukemogens known to increase the risk of development of cluster region) gene on chromosome 22 (Fig. 17-3). The
CML include exposure to ionizing radiation’? (such as was resulting chimeric gene, located on the Ph chromosome and
seen in radiologists prior to the use of safety shielding tech- referred to as the BCR-ABL gene, is transcribed, producing a
niques, patients treated with radiation therapy, and survivors 210-kDa protein with tyrosine kinase activity (called
of nuclear explosions), to cytotoxic drugs, especially alkylat- p2108°*48/). This new BCR—ABL fusion protein causes aber-
ing agents, and to biologically active chemicals such as rant activation of several cellular signaling pathways and is
benzene.'* Detailed studies of atomic bomb survivors in Japan considered essential in the pathogenesis of CML.” The
after World War II found that in Hiroshima, 34 of the BCR-ABL protein causes leukemogenic cell growth in
78 leukemia cases were CML, while only three of the hematopoietic cell lines, and can generate a syndrome in mice
20 leukemias in the survivors in Nagasaki were CML." that is similar to CML.'*** The 5% to 10% of CML patients
Development of CML follows the initial leakemogenic event without an identifiable Ph chromosome will nevertheless have
by several years. However, the vast majority of CML patients a BCR-ABL gene when studied via molecular techniques'®;
report none of the above risk factors or exposures; the cause these patients have the same clinical course and outcome as
in more than 95% of CML cases is unknown. CML is not an Ph chromosome positive patients. Those who do not have an
inherited disease; rather it appears to be acquired, as sug- identifiable BCR-ABL gene are considered atypical CML, a
gested by the rarity of familial aggregations of CML"? and separate disease with a different prognosis and treatment (see
the failure of the second member of pairs of identical twins to Differential Diagnosis below).
develop the chronic leukemia.*? The Ph chromosome is not Depending on the precise break point in the BCR gene,
present in patients’ non-hematopoietic tissues, nor is it found the BCR—ABL fusion protein can vary in size from 185 kDa to
in the parents or offspring of patients. 230 kDa. As stated in the preceding text, nearly all patients
with typical CML in chronic phase have the 210-kDa protein.
However, a subset of CML patients with a 230-kDa protein
Pathogenesis present with a lower WBC count, slower progression to blast
phase, and a better prognosis.”° Patients with Ph chromosome-
CML is a clonal stem cell disorder?! in which the abnormal
positive acute lymphoblastic leukemia may present with either
clone is characterized by the presence of the Ph chromosome.
the 210-kDa protein or a 190-kDa protein. This suggests that
The chromosomal translocation that creates the Ph chromo-
not all Ph chromosomes or BCR-ABL genes are identical, and
some is t(9;22), a reciprocal translocation between the long
also that fusion proteins of different sizes can be correlated
(q) arms of chromosome 9 and chromosome 22. A smaller
with different clinical outcomes.*’
piece of the long arm of chromosome 9 is broken off at band
The Ph chromosome has been found in neutrophil,
q34.1 and translocated to the long arm of chromosome 22; at
monocyte, B lymphocyte, erythrocyte, megakaryocyte, and
the same time, a larger piece of the long arm of chromosome
basophil precursors from CML patients’ blood and bone mar-
22 is broken at band g11.21 and moved to chromosome 9.
row. This suggests that the original cell in which the muta-
This results in a small, abnormal chromosome 22 (the
tion arose was a pleuripotential stem cell, before separation of
Ph chromosome; Fig. 17—2). The notation describing this
the lymphoid and the myeloid lineages. It is the presence
translocation, t(9;22)(q34.1;q11.1), defines both the translo-
of the Ph chromosome carrying the BCR-ABL gene that gives
cation and the specific break points in chromosomes 9 and 22.
the abnormal CML clone its growth advantage over normal
In the translocation, the ABL-/ gene, the cellular homologue
cells and allows them to replace the normal bone marrow
of the Abelson murine leukemia virus oncogene from chro-
elements.”
mosome 9, is brought into contact with the BCR (break
All leukemias appear to be maintained by a pool of self-
renewing malignant cells. In CML, immature granulocytes fill
the bone marrow and are released prematurely into the
peripheral blood in various stages of maturation.*? At the mol-
ecular level, it has been established that it is the chimeric
BCR-ABL fusion protein that plays the central role in
myeloid proliferation and transformation in CML. There is
direct evidence from in vitro studies that the BCR-ABL pro-
tein (p210°***") causes uncontrolled cell growth in
hematopoietic cell lines.”’ Introduction of the BCR-ABL gene
into the bone marrow of mice leads to the development of a
myeloproliferative syndrome that closely resembles human
CML, proving that the BCR-ABL gene alone is sufficient to
establish the leukemic phenotype in vivo. Effects of the
BCR-ABL fusion protein in the CML cells include increased
Figure 17-2 @ A metaphase spread from a patient with CML proliferative capacity, a slight delay in maturation,?! and a lack
showing t(9;22). The abnormal chromosomes 9 and 22 are marked of responsiveness to the normal regulators of growth (e.g.,
with arrows. The Philadelphia chromosome is the abnormal chro- cytokines or the bone marrow microenvironment).*?*° The
mosome 22 on the right; the normal chromosome 22 is on the left. abnormal protein also seems to prevent apoptosis (normal
 Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia aD

Philadelphia MOLECULAR PRODUCT

(Ph)
chromosome

9 Hybrid
gene
ber-abl
123 4
Hybrid
mRNA
(spliced)
ber-abl
Normal
c-abl
mRNA

Inactive
Selective Tyr-phosphorylation protein kinase
advantage of protein(s) substrates AcIve
CML @ of Ph- regulating growth Sort
positive and/or differentiation kinase ber-abl c-abl
clone of hematopoietic 210 kD 145 kD
progenitor cells
A
Figure 17-5 ® Molecular basis of the Philadelphia (Ph) chromosome. (A) Sequence of molecular and biochemical events involved in gener-
ating the Ph chromosome and its phenotypic consequences. (B) Southern blot analysis of DNA from CML cells analyzed with a bcr probe to
show clonal rearrangements in the ber region. Lane 7: Ph-positive CML DNA showing one rearranged band; lane 2: Ph-positive CML as in
lane 1 but with a different break point in the bcr region; lane 3: Ph-negative leukemic cell DNA showing no rearranged bcr; and lane 4:
molecular weight markers. (From Greaves, MF: Cellular identification and markers. In Zucker-Franklin, D, et al [eds]: Atlas of Blood Cells.
Lea & Febiger, Philadelphia/EE edi-ermes, Milano, Italy, 1988, p 43, with permission.)

programmed cell death) in the CML clone.'*** The BCR-ABL sweats and weight loss; bone tenderness and aching related to
fusion gene also leads to defective adhesion of the CML clone marrow expansion; and complaints related to an accompanying
to bone marrow stromal components, resulting in the early anemia." On physical examination, patients usually have mini-
release of immature forms into the bloodstream.***° mal to moderate splenomegaly, and occasionally hepatomegaly
The genetics of CML can occasionally be even more as well. Lymphadenopathy, which is rarely seen except late in
complex. Approximately 5% of CML patients have the deleted the course of the disease, is associated with a poor prognosis.
portion of chromosome 22 translocated to other chromosomes, Some patients will have bleeding complications related to qual-
such as t(4;22), t(12;22), and t(19;22).*’ These are simple vari- itative or quantitative platelet disorders. Rarely, patients may
ant Ph chromosome translocations, whereas other patients show manifestations of leukostasis (increased blood viscosity
have complex variant translocations involving two or more secondary to a very high WBC count) with vaso-occlusion, such
chromosomes in addition to chromosome 22 (e.g., t(9;11;22)). as cerebrovascular accident, myocardial infarct, venous throm-
On occasion, the Ph chromosome may be “masked” by the bosis, priapism, visual disturbances, and pulmonary insuffi-
presence of a larger piece of chromatin material translocated to ciency. In the later stages, patients may occasionally present
the abnormal chromosome 22 from one of the other chromo- showing effects of basophilia with increased serum histamine,
somes involved in the rearrangement, giving a normal sized, including pruritus, diarrhea, and refractory peptic ulcer disease.
but abnormal, chromosome 22.*° The CML clone is geneti- On rare occasions, the presenting sign of a patient with CML is
cally unstable, and may accumulate additional mutations as it a granulocytic sarcoma (chloroma), a soft tissue tumor com-
progresses to accelerated and blast phase. posed of immature myeloid cells, so named because of the char-
acteristic green color of the tumor mass when exposed to air.
CML is characterized by three phases:
Clinical Features
1. Chronic phase
Patients with CML may be asymptomatic or symptomatic
2. Accelerated phase
at presentation. It is common for the disease to be discovered
3. Blast phase
incidentally during a routine physical examination, or routine
hematologic evaluation of an asymptomatic patient.” When Most patients (85%) are diagnosed in the chronic phase. Dur-
symptoms do occur, the most common complaints are general ing this phase, the disease may remain stable for several years
malaise; fullness in the upper abdomen with early satiety, and and is usually responsive to conventional chemotherapy. Pro-
loss of appetite related to splenomegaly and hepatomegaly; gression of CML to accelerated phase or blast phase, in the
complaints resulting from a hypermetabolic state such as night absence of therapy, usually occurs 3 to 5 years after onset.’
376 Chapter 17 Chronic Myeloproliferative Disorders I: Chronic Myelogenous Leukemia

The accelerated phase is heralded by a variety of signs

and symptoms of disease progression that do not meet the cri-
teria for blast phase. Associated worsening symptoms may
include unexplained fevers; significant weight loss; progres-
sive leukocytosis; worsening splenomegaly; requirement for
higher doses of myelosuppressive agents; bone and joint pain;
bleeding; thrombosis; and infections.*! Blast phase (previ-
ously called blast crisis or blast transformation) represents the
conversion from CML to an aggressive form of acute
leukemia that is difficult to treat.*! This will be acute myel-
ogenous leukemia (AML) in approximately 50% of cases and
acute lymphoblastic leukemia (ALL) in 20% to 30%, with the
remainder classified as acute undifferentiated leukemia or
biphenotypic leukemia.* The criteria for diagnosing progres-
sion to accelerated phase and blast phase are discussed in the
paragraphs that follow. CML may be a triphasic disease with Figure 17-5 m Pergeroid (pseudo-Pelger—Huét) neutrophil in CML.
progression from chronic phase through accelerated phase to Note that the cytoplasm is mature and the nucleus is small and
round with condensed chromatin. This cell can be mistaken for a
blast phase, or may be biphasic with progression directly from
myelocyte. A basophil is in the upper left corner.
chronic phase to blast phase.*”
critical significance to the diagnosis that basophilia has.
Laboratory Findings Thrombocytosis is present in about one half of cases, and is
usually of moderate degree (less than 1,000,000/uL).*?
The most important laboratory finding in the peripheral
Decreased platelet counts are uncommon, at least in the
blood is an increased white blood cell count (frequently
chronic phase.*? Micromegakaryocytes can be seen in circula-
above 100,000/uL), reflecting neutrophilic leukocytosis with
tion in as many as 25% of CML patients (Fig. 17-6). Normo-
basophilia (Fig. 17-4). Neutrophils will include virtually all
cytic anemia is commonly present, although occasional
cells in the neutrophilic series, from segmented neutrophils to
patients may have normal or even increased hemoglobin
occasional blasts (fewer than 10%).° The presence of a greater
levels.?’ The severity of anemia present is commonly propor-
percentage of myelocytes than the more mature metamyelo-
tional to the degree of leukocytosis.** A summary of the labo-
cyes is a typical finding (the “myelocyte bulge’”’).** Most neu-
ratory features of chronic myelogenous leukemia at diagnosis
trophilic precursors appear morphologically normal, although
is provided in Table 17—3.% The degree of leukocytosis is
occasional cells with pseudo-Pelger—Huét anomaly may be
correlated with the appearance of symptoms and laboratory
seen late in the disease (Fig. 17—5). Increased basophils are
findings in Table 17-4. With progression to the accelerated
invariably seen in CML, such that if the absolute basophil
phase, patients may develop worsening anemia; worsening
count is not increased, a diagnosis of CML should be seri-
thrombocytosis or thrombocytopenia; and increasing periph-
ously questioned. Basophilia may precede the other findings
eral blood basophils (220%). There may be a shift to the
of CML by several years. Increased absolute eosinophil and
more immature myeloid forms with an increasing number of
monocyte counts are also usually present, but do not have the
blasts (10% to 20%). Blast crisis is heralded by an increase in
blood or bone marrow blasts to more than 20%.

Soe
We.

Figure 17-4 @ Peripheral blood from a patient with CML shows

numerous mature neutrophils along with bands, metamyelocytes,
and myelocytes. Note the two basophils at the upper right and Figure 17-6 A micromegakaryocyte with giant platelets, from
bottom center. the peripheral blood of a patient with CML.
 Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia 377

Sas of Chronic.
. Myelogenous Leukemia
Peaokeal Blood Bone Marrow
Neutrophilic leukocytosis Myeloid hyperplasia
with immature forms Blasts < 10% (in chronic
Basophilia/eosinophilia phase)
Thrombocytosis Minimal/no dysplasia
Anemia Increased megakaryocytes
Blasts < 10% (in Myelofibrosis (mild/
chronic phase) moderate)
Decreased LAP score Monocytes usually <3%
Increased lactate
dehydrogenase Genetic Studies
Increased uric acid Philadelphia chromosome
Increased vitamin B,,/ positive (90-95%) Figure 17-7 mA bone marrow aspirate from a patient with CML
transcobalamin BCR-ABL-positive in chronic phase shows granulocytic hyperplasia with orderly matu-
a 99%) ration through segmented neutrophils. Blasts are not significantly
LAP = fenocyte alkalineees
increased. A basophil is at upper left.

The bone marrow (BM) is hypercellular with a marked

granulocytic hyperplasia (Fig. 17—7). Maturation from blasts
to segmented neutrophils is fairly orderly, although the rela-
tive increase in myelocytes as seen in the peripheral blood is
also present. Blasts constitute less than 10% of the marrow
elements in the chronic phase. Basophils and eosinophils are
also increased, as in the peripheral blood. Megakaryocytes are
also typically increased and clustered in groups of three or
more. Gaucher-like histiocytes (histiocytes with blue pigment
in the cytoplasm, “sea-blue” histiocytes) can be seen in one
third of patients (Fig. 17—8).*° As the disease progresses, vary-
ing degrees of marrow fibrosis may develop (Fig. 17—9).*°
Leukocyte alkaline phosphatase (LAP), also called neu- A
trophilic alkaline phosphatase, is usually decreased in CML.
Compare Figure 17-10 showing decreased LAP in a patient Figure 17-8 @ Pseudo-Gaucher cells (sea-blue histiocytes) in the
marrow of a patient with CML.
with CML with Figure 17-11 showing increased LAP activ-
ity in a normal patient with leukemoid reaction. However,

Table 17-44 Chronologic Sea E TEE y

of Appearance Oe ye
Findings in CML }
WBC Counts Chimica or Laborator yF inding

Normal AGeaeae of Ph chromosome

12,000/L Basophilia
Thrombocytosis
Decreased LAP score
22,000/.L Immature granulocytes > 5%
35,000/L Increased serum vitamin B,, levels
50,000/j2L Splenomegaly
70,000/pL Constitutional symptoms
RAN
LAP = nee dialing Oe daa! ROE.
Source: Adapted from Kamada, N, and Uchino, H: Chronologic
sequence in appearance of clinical and laboratory findings charac Figure 17-9 ti This bone marrow biopsy from a patient with CML
teristic of chronic myelocytic leukemia. Blood 51:843, 1978. is stained with a reticulin (silver) stain to show reticulin fibrosis,
which in this case is quite extensive.
5
378 Chapter 17. Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia

cytogenetics, as in a patient with an interstitial translocation.

The RT-PCR assay is extremely sensitive, and is used to detect
residual disease or recurrence after treatment.’”*°
Accelerated phase is suspected when patients become more

3 symptomatic as described in the preceding text. The laboratory

and clinical findings used to diagnose accelerated phase are sum-
marized in Table 17-5. Blast phase represents conversion of
CML to acute leukemia. The laboratory findings in blast phase
are summarized in Table 17—6.° Clonal evolution will occur in at
least 50% of patients in accelerated phase and in up to 80% of
those in blast phase.! This refers to the acquisition of additional
cytogenetic abnormalities, such as multiple Philadelphia chro-
mosomes, isochromosome 17, trisomy 8, and other complex
karyotypes.'> Mutations or deletions of tumor suppressor genes
such as p/6 and p53 are also found in blast phase, and presum-
Figure 17-10 @ Leukocyte alkaline phosphatase (LAP) is decreased ably play a role in its development.*”~”
in peripheral blood neutrophils in this patient with CML. Compare
with Figure 17-11.
Differential Diagnosis
LAP may return to normal or may even be increased when a
patient with CML becomes infected, during remission of dis- CML must first be distinguished from reactive granulocytic
ease with therapy, or when a patient enters blast phase.* leukocytosis, or leukemoid reaction, which can occur with a
Although neutrophils have abnormal biochemical reactivity, variety of stresses including infections and occult malignan-
as reflected by low LAP, bactericidal and phagocytic func- cies. Both are characterized by neutrophilic leukocytosis with
tions of the leukemic granulocytes remain largely intact.*°*? immature forms, but CML will also show basophilia and
Patients may also have elevated levels of uric acid and eosinophilia, as well as the “myelocyte bulge.” CML will also
lactate dehydrogenase (LDH). A marked increase in serum show the Ph chromosome and/or BCR-ABL gene, as well as
vitamin B,, levels as well as B,, binding capacity reflects the a decreased LAP score. The differential laboratory diagnosis
marked leukocytosis.'° between leukemoid reactions and CML is summarized in
A definitive diagnosis of CML requires the detection of Table 17-7.
the t(9;22)(q34.1;q11.2) and/or the BCR-ABL fusion gene. The CML may be confused with any of the other chronic
t(9;22) (Ph chromosome) can be detected by routine cytogenet- myeloproliferative disorders (CMPD), especially in its early
ics. This technique has the advantage of detecting not only the stages, because all the CMPDs are characterized by some
Ph chromosome, but also any other genetic abnormalities that
may indicate disease progression. The BCR-ABL gene can be
detected by fluorescence in situ hybridization (FISH) or reverse
transcriptase polymerase chain reaction (RT-PCR). These have
the advantage of specifically detecting the BCR-ABL gene
when no structural chromosomal abnormality is seen on routine

Blasts 10%-—19% in peripheral blood and/or bone marrow

Peripheral blood basophils = 20%
Persistent thrombocytopenia (< 100 x 10°/L) unrelated to
therapy, or persistent thrombocytosis (> 1000 x 10°/L)
unresponsive to therapy
Increasing spleen size and increasing WBC count unresponsive
to therapy
Cytogenetic evidence of clonal evolution

Blasts = 20% ofeee blood WBC or nucleated bone

marrow cells
Figure 17-11 @ Leukocyte alkaline phosphatase (LAP) is increased Extramedullary blast proliferation (granulocytic sarcoma)
in peripheral blood neutrophils in this patient with a leukemoid Large foci or clusters of blasts in the bone marrow biopsy
reaction related to infection.
 Chapter 17. Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia 379

ATYPICAL CHRONIC MYELOID LEUKEMIA

Atypical chronic myeloid leukemia (aCML) is a leukemic
disorder that has features of both a chronic myeloproliferative
disease and a myelodysplastic syndrome. aCML resembles
CML because it is characterized by peripheral blood neu-
trophilic leukocytosis with immature forms and granulocytic
Leukemoid hyperplasia in the bone marrow. However, aCML does not
Reaction show basophilia, does show significant dysplastic features in
Toxic vacuoles the granulocytic line, and always lacks the Ph chromosome
Toxic granules and the BCR-ABL gene. Cases of Ph-negative CML that also
Dohle bodies Frequent lack the BCR-ABL gene via molecular techniques are more
Eosinophilia 0 appropriately classified as aCML. Patients with aCML seem
Basophilia 0
to have a poorer prognosis with median survival times of less
Pseudo-Pelger—Huét O-1+ Occasional
Karyorrhexis O-1+ Lee than 20 months. From 25% to 40% of aCML patients will
Giant bizarre nuclei O-1+ 1-3+ develop acute leukemia.**>**
Myelocyte bulge 0 Present Chronic myelomonocytic leukemia and juvenile chronic
LAP score High Low myelomonocytic leukemia are two other myeloprolifera-
Ph chromosome Negative Positive
tive/myelodysplastic syndromes that may resemble CML but
LAP = leukocyte alkaline phosphatase. lack both Ph chromosome and BCR-ABL gene.*

Prognosis
degree of neutrophilic leukocytosis and basophilia, while There are significant differences in clinical aggressiveness and
CML can present with increased platelet count, and occasion- prognosis between patients presenting with CML in chronic,
ally increased RBC count. The LAP score will be decreased accelerated, and blast phases. In addition, there can be signifi-
in CML and may sometimes be decreased in IMF as well, cant survival differences between patients presenting in the
while it is usually increased in PV. When the Ph chromosome same phase. Several systems have been proposed to predict
or the BCR-ABL gene is present, CML can be diagnosed with prognosis in CML patients. The system in widest use currently
confidence; otherwise, it may be better to diagnose chronic
is the Sokol score. The Sokol score takes into account age,
myeloproliferative disease, unclassifiable, and re-evaluate the splenomegaly, platelet count (platelets >700,000/uL), and blast
patient frequently. count to stratify patients into three prognostic groups with
median survivals of approximately 2.5, 3.5, and 4.5 years.
CHRONIC NEUTROPHILIC LEUKEMIA Although the Sokol score was derived by studying patients
Chronic neutrophilic leukemia (CNL) is a rare but related treated with older therapies in the 1970s and 1980s, recent stud-
chronic myeloproliferative syndrome that is characterized by ies suggest that it remains largely accurate in predicting out-
splenomegaly, peripheral blood neutrophilia, and neutrophilic come in patients treated with modern therapeutic regimens.
hyperplasia of the bone marrow.*° The absolute neutrophil Several factors may predict early blast transformation in
count in the peripheral blood is usually greater than CML. These poor prognostic indicators include the presence
25,000/uL, and bands are present, but the percentage of of karyotypic abnormalities in addition to a single Ph chromo-
immature myeloid cells (metamyelocytes, myelocytes, some or BCR-ABL gene; hepatosplenomegaly; thrombocy-
promyelocytes, and blasts) is usually low. Basophilia and topenia (less than 150,000/uL) or thrombocytosis (more than
eosinophilia are not seen. Unlike CML, CNL never shows a 500,000/uL); extreme leukocytosis (more than 100,000/uL);
Ph chromosome, and LAP score is usually increased.’ CNL peripheral blood blasts greater than 1% or bone marrow blasts
must be distinguished from CML and from reactive neu- greater than 5%; and peripheral blood basophils above
trophilia, which is far more common. 15%.*°* The tumor suppressor gene p53 is altered in 20% to
30% of CML patients in blast crisis. Mutations in the p53
CHRONIC EOSINOPHILIC LEUKEMIA gene may have prognostic significance because these muta-
Chronic eosinophilic leukemia (CEL) is another related syn- tions may induce drug resistance by interfering with normal
drome that is characterized by a clonal expansion of programmed cell death (apoptotic) pathways in leukemic
eosinophil precursors with increased peripheral blood CML cells.*”
eosinophils of greater than 1500/uL.*! There is no Ph chromo-
some, and blasts are less than 20% in blood and bone marrow.
If there is no evidence of clonality in the eosinophil line and Treatment
blasts are not increased, the term “idiopathic hypere-
osinophilic syndrome” is preferred.°’ Tissue damage occurs The treatment for CML has evolved hand in hand with increased
as a result of infiltration by eosinophils with release of understanding of the underlying molecular pathogenesis. Before
cytokines. CEL must be distinguished from CML and from the mid-1980s, patients with CML in chronic phase were treated
reactive eosinophilia. with oral chemotherapy in the form of hydroxyurea or
380. Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia

busulfan, intended to reduce leukocytosis, thrombocytosis, and However, in a multicenter randomized trial in unrelated mar-
splenomegaly. Although such therapy frequently resulted in row donor transplantation, T-cell depletion did not reduce the
hematologic remission, Ph chromosome remained detectable in incidence of chronic GVHD or improve the survival in
blood and marrow cells, and the onset of blast phase was not patients who developed chronic GVHD.” In addition, long-
delayed.* term outcome of patients given transplants of mobilized blood
Interferon-alpha was the first agent to produce cytoge- or bone marrow reported that patients with advanced chronic
netic as well as hematologic remission in CML. When used in myeloid leukemia had higher leukemia-free survival rates
combination with low-dose cytarabine, it was able to induce with peripheral blood stem cell (PBSC) transplants than bone
complete hematologic response in 69% of patients and major marrow (BM) transplants.”? Leukemia-free survival rates
cytogenetic remission (defined as =66% normal metaphases) were lower for patients in the first chronic phase after PBSC
in 35% of patients.°° Although complete cytogenetic remis- transplants in comparison to BM transplants due to higher
sions (absence of Ph chromosome) were rare (13%), those rates of late transplant-related mortality.” Allogeneic stem
who obtained major cytogenetic remission had significant cell transplantation still remains the only cure for CML; how-
prolongation of survival as well as a lengthened time to blast ever, impressive results of imatinib in newly diagnosed
phase. One study showed that patients who achieved a com- patients has made imatinib the first line of treatment.’' The
plete or major cytogenetic remission exhibited a 72% 10-year effects of imatinib mesylate treatment before allogeneic trans-
survival rate.°’ However, the majority of patients, including plantation for CML is now being investigated.”
those who developed complete cytogenetic remissions, still
had molecular evidence of the BCR-ABL gene by polymerase
chain reaction (PCR) testing. Fade 3 alas: lod
Imatinib mesylate (Gleevec®), the first drug to be devel-
oped against a specific molecular target, has significantly
changed the treatment and prognosis for CML. Imatinib directly A 58-year-old white man presented to his physician for eval-
inhibits the mutant tyrosine kinase activity of the BCR-ABL uation of leukocytosis found incidentally on a complete
fusion gene, and is utilized to treat all phases of CML. In blood count performed at a health fair. He had no current
chronic phase CML patients, imatinib produces up to a 95% complaints, but when questioned, reported feeling of full-
complete hematologic response rate, complete cytogenetic ness in the left upper quadrant of his abdomen. He reported
responses in up to 81% of patients, and improved survival no recent fevers, night sweats, or weight loss. He worked as
rates.°? Patients who achieve complete cytogenetic response a supervisor in an automobile assembly plant. On physical
show 10-year survival rates of 78%.°' In the accelerated phase, examination, he was found to have splenomegaly, with the
one study reports a hematologic response rate of 82% and a spleen tip barely palpable below the costal margin. No
hepatomegaly or lymphadenopathy was found.
major cytogenetic response of 24%.°° However in blast phase,
Complete blood count (CBC) showed WBC 51,200/uL,
the overall hematologic response rate decreases to 55% in the
hemoglobin 13.1 g/dL, hematocrit 38.4%, mean corpuscular
myeloid variant and 70% in the lymphoid variant, with duration
volume (MCV) 90.7 fL, and platelet count 610,000/uL.
of response that is shorter than in the chronic phase.°? Whether WBC differential showed 64% segmented neutrophils, 8%
imatinib can cure patients with CML remains to be determined, bands, 5% lymphocytes, 4% monocytes, 4% eosinophils,
but it is likely that the attainment of cytogenetic remission in the 5% basophils, 2% metamyelocytes, 7% myelocytes, 1%
majority of patients receiving imatinib will translate into signifi- promyelocytes, and 1 nucleated RBC. LAP score was
cantly prolonged overall survival. Some patients’ disease has, over decreased.
time, become refractory to treatment with imatinib; however, A bone marrow aspirate and biopsy were performed, and
newer kinase receptor inhibitors have already been developed that showed a markedly hypercellular bone marrow with marked
can overcome imatinib resistance and restore cytogenetic remis- granulocytic hyperplasia and moderate megakaryocytic hyper-
sion, for example Dasatinib (BMS-354825).%° Imatinib will be plasia. Blasts constituted 6% of the cells present. Bone marrow
cytogenetics performed showed 46,XY,t(9;22) (q34.1;q11.2);
the mainstay of therapy for patients who are not candidates for
that is, a Philadelphia chromosome was present.
bone marrow transplant, or for whom no suitable donor can be
Treatment was begun with interferon-alpha and low-dose
found.
cytarabine with good hematologic response. However, the
At present, allogeneic bone marrow transplantation with patient subsequently had difficulty tolerating the treatment
an HLA-matched related or unrelated donor offers the only because of fever and malaise, and was temporarily lost to
proven cure for CML. Survival is improved if the transplant follow-up.
is performed earlier in the course of the disease, and in the He returned 18 months later complaining of fatigue and
chronic phase. Transplantation-related mortality is high fevers. Physical examination showed increasing splenomegaly
(14%), mostly due to graft-versus-host disease (GVHD) and as well as hepatomegaly. At this time, his CBC showed WBC
infection, and outcome is worse in patients older than the age 98,800/uL, hemoglobin 11.6 g/dL, hematocrit 34.9%, MCV
of 40.*’ Long-term survival rates as high as 78% have been 89.0 fL and platelet count 1,070,000/uL. WBC differential
showed 48% segmented neutrophils, 8% bands, 3% lympho-
reported.*’ Moreover, survival rates using unrelated matched
cytes, 4% monocytes, 3% eosinophils, 21% basophils, 3%
donors are approaching the rates seen with related donors.®
metamyelocytes, 8% myelocytes, 2% blasts, and 2 nucleated
The incidence of acute GVHD can be reduced by depleting
continued
the donor marrow of T-lymphocytes using various techniques.
 Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia 38|

RBCs. A repeat bone marrow aspirate again showed marked 6. When the above patient returned after being lost to
granulocytic and moderate megakaryocytic hyperplasia, now follow-up for 18 months, he was in:
with 14% blasts. a. Chronic phase
Treatment was begun with imatinib mesylate (Gleevec®), b. Accelerated phase
which had recently become available. The patient’s WBC c. Blast phase
count decreased and symptoms disappeared within a few d. Jersey City
weeks. He subsequently achieved complete hematologic 7. The worst prognosis in patients with CML is associated
and complete cytogenetic remission. RT-PCR analysis of the with:
patient’s bone marrow specimens continued to show a. Thrombocytosis
the presence of BCR-ABL fusion gene product, but he b. Blast phase
remained asymptomatic and in complete hematologic remis- c. The presence of the Ph chromosome
sion 3 years later. d. Splenomegaly

QUESTIONS ANSWERS
1. The translocation that results in the Philadelphia chro-
mosome is between chromosomes:
a. 21 and 22
b. 9 and 22
c. 8 and 14
d. 9 and 21
2. The blast phase of CML is: Sl
OS
see
ANAS
@)
>@n
i
a. Followed by the chronic phase and the accelerated
phase DISCUSSION
b. Seen only in atypical CML
c. The terminal phase of CML, characterized by The Philadelphia chromosome, the hallmark of CML, is
increased blasts (>20%) in the blood and bone marrow formed by the translocation of chromosomes 22 and
d. The earliest phase seen in typical CML 9, which results in the formation of a fusion gene
3. All of the following favors a diagnosis of CML over (BCR-ABL) and subsequent activation of the ABL tyrosine
leukemoid reaction except: kinase which results in uncontrolled myeloproliferation.
a. Absence of basophils and eosinophils in the peripheral Aside from the Philadelphia chromosome, splenomegaly
blood and a low LAP score within myeloblasts through neutrophils
b. A low LAP score in the peripheral blood favors a diagnosis of CML rather
c. Ph chromosome than a leukemoid reaction. Blast crisis in CML is the termi-
d. An enlarged spleen nal phase of CML, characterized by increased numbers of
4. The only proven curative treatment for CML at this blasts in the bone marrow and peripheral blood.
time is: Although it is clear that Gleevec® has prolonged the
a. Combination interferon-alpha with low-dose chronic phase of CML, blast crisis still occurs. Blast crisis
cytarabinel remains refractory to standard chemotherapy approaches.
b. Imatinib mesylate (Gleevec®) Allogeneic bone marrow transplant offers the best chance
c. Bone marrow transplantation for cure when performed during the chronic phase.
d. CML is not curable
5. At the molecular level, the aberrant conjoining of
genetic material from chromosome 9 and chromosome
22 results in a fusion gene called:
a. BCR-ABL gene
b. JAK2 gene
C.j03
d. The blast transformation gene
continued
382 Chapter 17. Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia

Orestiand
i. What is the M:E ratio in patients with CML (chronic ~c. Viruses
myelogenous leukemia)? d. All of the above
a. 1:10 7. Which of the following myeloproliferative disorders is
bales characterized by a decreased LAP score?
esOzt a. CML
aoe b. IMF
i) . What is the chromosomal abnormality in CML? CET
a. t(8;14) d. PV
Dat.22) 8. Most patients (85%) are diagnosed in which phase of
ceitiel2) CML?
d. Trisomy 12 a. Accelerated phase
. Which of the following is not consistent with leukemoid b. Chronic phase
reaction? c. Blast phase
a. High WBC count d. All of the above
b. Associated with a bacterial infection 9. The blast phase of CML is defined by what percent of
c. Presence of Philadelphia chromosome blasts found in the peripheral blood or bone marrow?
d. High LAP a. 50%
. All of the following are characteristics of chronic b. 10%
leukemias except: c. 80%
a. Insidious onset d. 20%
b. Mature leukemia cells 10. Which of the following is characteristic of the acceler-
c. Found in adults ated phase of CML?
d. Found in all ages a. Persistent thrombocytosis or thrombocytopenia
. Which phase of CML carries the worst prognosis and is b. Blasts greater than 5% in the peripheral blood or
generally unresponsive to treatment? bone marrow
a. Chronic c. Basophilia greater than 20%
b. Accelerated d. All of the above
c. Blast
d. Refractory See answers at the back of this book.
. Environmental factors that are associated with an
increased risk of developing leukemia include:
a. Ionizing radiation
_b. Chemicals and drugs

-') SUMMARY CHA w Chronic myelogenous leukemia (CML) may be asympto-

matic or symptomatic at presentation. Common symptoms
include general malaise; fullness in the upper abdomen
wg The chronic myeloproliferative syndromes are a family of
related to splenomegaly; symptoms relating to a hyperme-
acquired clonal disorders of the bone marrow that are
tabolic state such as night sweats and weight loss; bone
characterized by excessive proliferation of one or more
tenderness and aching related to bone marrow expansion;
non-lymphoid cell lines, with increase in the correspond-
and complaints related to anemia. Splenomegaly is a com-
ing cell type in the peripheral blood. All are characterized
mon physical finding.
by splenomegaly.
m CML is characterized by an increased WBC count in
w The chronic myeloproliferative syndromes include chronic
peripheral blood, which may be severe. This is related to
myelogenous leukemia, polycythemia vera, essential throm-
neutrophilic leukocytosis with immature forms, and
bocythemia, chronic idiopathic myelofibrosis, chronic
basophilia. Eosinophilia and monocytosis may also be
neutrophilic leukemia, chronic eosinophilic leukemia/
present. Platelets are commonly increased. Anemia is
hypereosinophilic syndrome, and unclassifiable myelopro-
sometimes also present, and is usually normocytic.
liferative disease.
Peripheral blood neutrophils have a low LAP score.
continued
 Chapter 17 Chronic Myeloproliferative Disorders |: Chronic Myelogenous Leukemia 383

m The bone marrow in CML shows hypercellularity related a CML must be distinguished from the other chronic
to a marked granulocytic hyperplasia and a moderate myeloproliferative disorders. It must also be distinguished
megakaryocytic hyperplasia. Pseudo-Gaucher cells and from atypical chronic myelogenous leukemia, which
marrow fibrosis may also be present. lacks the BCR-ABL fusion gene.
m The Philadelphia chromosome (an abnormal chromosome = Imatinib mesylate (Gleevec) currently provides an effec-
22) results from a reciprocal translocation between chro- tive treatment for CML, which can produce long-term
mosomes 9 and 22, and contains the BCR-ABL fusion remissions. However, the only proven cure for CML is
gene. The product of this gene is a protein with tyrosine allogeneic bone marrow transplantation.
kinase activity that is the direct cause of the granulocytic
proliferation that characterizes CML. The BCR-ABL
fusion gene is almost invariably present in CML.

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nNHES, shows promise. JAMA 295:369, 2006.
leukemia: recommendations from an phase of chronic myeloid leukemia with 65. Hochhas, A, et al: Dasatinib induces
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nous leukemia in blast crisis: Analysis of ah HehlImann, R, et al: Randomized com- tinib therapy. Blood 109(6):2303, 2007.
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cal Diagnosis of Hematologic Disorders: . O’Brien, SG, et al: Imatinib compared GyE Radich, JP, et al: HLA-matched related
2 Malignant Disorders. ASCP Press, with interferon with low-dose cytarabine hematopoietic cell transplantation for
American Society for Clinical Pathology, for newly diagnosed chronic-phase chronic phase CML using a targeted
Chicago, 2006, pp 539-560. myeloid leukemia. N Engl J Med busulfan and cyclophosphamide prepara-
44. Armitage, J: Atlas of Clinical Hematol- 348:994, 2003. tive regimen. Blood 102:31, 2003.
ogy. Lippincott Williams & Wilkins, 57. Bonifaz, F, et al: Chronic myeloid 68. Davies, S, et al: Equivalent outcomes in
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5. Dosik, H, et al: Acquired lipidosis: of complete cytogenetic responders. leukemia after early transplantation of
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46. Castro-Malispina, H, and Moore, MA: reaction in Philadelphia chromosome- 69. Pavletic, SZ, et al: Influence of T-cell
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7
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49. Randolph, TR: Chronic myelocytic 6 i . Kantarjian, H, et al: Complete cytoge- after allogeneic stem cell transplantation.
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80:75, 2005. leukemia and acute lymphoblastic
 Chronic
Myeloproliferative
Disorders II
Polycythemia Vera, Essential
Thrombocythemia, and Idiopathic
Myelofibrosis
Kathrina Chua, MD
Meyer R. Heyman, MD

Introduction to OBJECTIVES
Myeloproliferative
Disorders . At the end of this chapter, the learner should be able to:
Historical Perspective . Describe the origin of myeloproliferative disorders.
—"

Definition and Classification

. Identify the most common myeloproliferative disorders.
Polycythemia Vera
Historical Perspective . List the characteristics of polycythemia vera.
Definition . Identify the major and minor Polycythemia Vera Study Group (PVSG) criteria for
WV
WH
&
Incidence polycythemia vera.
Pathogenesis
Clinical Features . List laboratory findings for polycythemia vera.
Laboratory Findings . Identify the major and minor PVSG criteria for essential thrombocythemia.
Differential Diagnosis
Treatment . List the characteristics of essential thrombocythemia.

Essential Thrombocythemia . List laboratory findings for essential thrombocythemia.

Historical Perspective
. List the features of idiopathic myelofibrosis.
Definition
Incidence . List laboratory findings for idiopathic myelofibrosis.
Pathogenesis
Clinical Features
Laboratory Findings
Differential Diagnosis
Treatment
idiopathic Myelofibrosis
Historical Perspective
Definition
Incidence and Etiology
Pathogenesis
Clinical Features
Laboratory Findings
Differential Diagnosis
Treatment
Case Study 1
386 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

Case Study 2
Case Study 3

Introduction to Myeloproliferative clonal expansion of the hematopoietic pluripotent stem cell

resulting in the overproduction of one or more of the formed
Disorders elements of the blood (i.e., erythrocytes, leukocytes, and
Historical Perspective platelets in the peripheral blood). Classically, CMPDs have
encompassed four clinical entities: chronic myelogenous
The term myeloproliferative disorders (MPDs) was first pro- leukemia (CML), polycythemia vera (PV), essential thrombo-
posed by Damashek in 1951 to describe a group of acquired, cythemia (ET) and idiopathic myelofibrosis (IMF). These
malignant disorders characterized by excessive proliferation specific myeloproliferative disorders are distinguished by the
of hematopoietic cells and fibroblasts in the bone marrow. predominant cell type involved. The most prominent feature
Damashek further theorized that these disorders were likely of CML is excessive production of granulocytes; in PV, over-
influenced by a common myelostimulatory factor, prompting production of erythrocytes; and in ET, overproduction of
the finding of similar clinical and hematologic features platelets. IMF is characterized by a prominence of marrow
including extramedullary hematopoiesis.' In recent years, a fibrosis and extramedullary hematopoiesis in the liver
number of discoveries have provided further enlightenment and spleen. The hallmark physical finding of CMPDs is
on the molecular pathogenesis of myeloproliferative disor- splenomegaly, which occurs in 40% to 99% of patients
ders. However, Damashek’s concept of the myeloproliferative depending on their specific disorder. These four disease enti-
disorder continues to be universally accepted. ties each have distinctive features, but also overlapping
characteristics (Table 18-1). CML has been discussed in
Chapter 17. This chapter focuses on PV, ET, and IMF.
Definition and Classification
In 1999, the new World Health Organization (WHO) clas-
By definition, chronic myeloproliferative disorders (CMPDs) sification of myeloid neoplasms expanded the list of CMPDs to
are a group of heterogenous diseases that originate from the seven entities, now including chronic neutrophilic leukemia,

Table 18-1
Idiopathic Polycythemia Essential
Myelofibrosis Vera Thrombocythemia

Hemoglobin slL Marked T N-sl J

WBC count (X 10°/L) Variable, usually 12-25 Variable, often
s] T early on mildly T
Platelet count (X 10°/L) Variable— sl T 450-1000, 450-800 600-2500
J advanced disease
NRBCs Common Common Rare Rare
LAP Mod J N- mod 7 Usually sl T N
Bone marrow Marked myeloid Fibrosis, dry tap Hypercellular, Hypercellular, marked T
hyperplasia mod — marked T sl J iron stores megakaryocytes
Fibrosis None — sl 7 mod T — marked T None - sl T None — mod T
Splenomegaly 60%-80% 80%-99% 80% 40%-50%
(% patients)
Ph chromosome Positive Negative Negative Negative
(BCR/ABL gene fusion)
JAK2 (V617F) mutation Negative Positive (35%-57%) Positive (65%-97%) Positive (23%-57%)
Special studies Ph chromosome Magnetic resonance, T RBC mass Abnormal platelet
cytogenetics, bone marrow determination, function tests
FISH, BCR/ABL imaging J erythropoietin
by PCR level
WBC = white blood cell; NRBCs = nucleated red blood cells; LAP = leukocyte alkaline phosphatase;
FISH = fluorescent in situ hybridization; PCR = polymerase chain reaction; Ph = Philadelphia; JAK = Janus kinase gene
Reference range: WBC = 5-11 X10°/L; platelets = 150-450 X 10°/L; N = normal; sl J = slight decrease; mod | = moderate
decrease; sl T = slight increase; mod T = moderate increase; marked T = marked increase.
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 387

chronic eosinophilic leukemia (CEL)/hypereosinophilic syn- Study Group (PVSG) in 1967.’ Unfortunately, the group was
drome (HES) and unclassifiable myeloproliferative diseases. In disbanded when they were unable to obtain funding. However,
addition, a category of myelodysplastic/myeloproliferative dis- the data they generated are still used to make decisions regard-
eases was created to encompass juvenile myelomonocytic ing the management of PV.
leukemia, atypical chronic myeloid leukemia (lacking the 9;22
translocation) and chronic myelomonocytic leukemia.’ A dis- Definition
cussion of these rare additional entities is beyond the scope of
this chapter. The reader is referred to the WHO Classification PV, polycythemia, or polycythemia rubra vera is a chronic
of Tumours of Haematopoietic and Lymphoid Tissues.” The abnormality of the hematopoietic stem cell characterized by
2008 WHO classification has replaced the term CMPD with uncontrolled proliferation of erythroid, granulocytic, and
“myeloproliferative neoplasms (MPN)”. In addition, the 2008 megakaryocytic cells. PV should be differentiated from sec-
WHO diagnostic criteria for the traditional BCR-ABL negative ondary polycythemia, in which only the erythrocytes are
CMPDs (PV, ET, IMF) and the CEL/HES CMPDs have been increased in number and from relative erythrocytosis, where the
revised. Table 18—2 compares the 2001 and 2008 WHO classi- increase in hematocrit is secondary to a decrease in plasma vol-
fications of MPN. ume. PV is compared with secondary (hypoxic erythrocytosis)
and relative erythrocytosis in Table 18-3. The updated PVSG
criteria for the diagnosis of PV include the following: (1) an
Polycythemia Vera increase in red cell mass (>36 cc/kg in men and >32 cc/kg
Historical Perspective in women), (2) absence of causes of secondary polycythemia,
(3) splenomegaly, (4) thrombocytosis, (5) leukocytosis, and
Vaquez in 1892 and Osler in 1903 were the first to describe (6) normal or decreased erythropoietin levels (Table 18—-4).*
polycythemia vera (PV) as a chronic disease characterized by The WHO revised the criteria for the diagnosis of PV in
cyanosis, polycythemia, and splenomegaly.** The accompany- 1999, with the addition of other criteria including bone mar-
ing increase in granulocytic and megakaryocytic precursors was row panmyelosis with erythroid and megakaryocytic prolifer-
described by Turk a year later in 1904.° It was not until 1935 that ation and erythropoietin independent endogenous erythroid
marrow fibrosis was added to the hematologic findings by colony growth both in vivo and in vitro.* The discovery of the
Hirsch.° Wasserman formally founded the Polycythemia Vera genetic abnormality (JAK2 exon 12 mutations) in almost all

Table
18-2 World Health Organization Classification a
2001 28

WHO Classification of Chronic WHO Classification for Myeloid

Myeloproliferative Diseases ‘Neoplasms

¢ Chronic myelogenous leukemia (CML) Acute ae arent

[Ph chromosome, t(9;22)(q34;q11), BCR-ABL-positive] Myelodysplastic syndrome (MDS)
¢ Chronic neutrophilic leukemia (CNL) Myeloproliferative neoplasms (MPN)
* Chronic eosinophilic leukemia (CEL) * CML °ET * ‘CNL
(and the hypereosinophilic syndrome) e PV ¢ Primary myelofibrosis * CEL (not otherwise categorized)
¢ Polycythemia vera (PV) ¢ Hypereosinophilic syndrome ¢ Mast cell disease
¢ Chronic idiopathic myelofibrosis ¢ MPNs, unclassified
(with extramedullary hematopoiesis)
¢ Essential thrombocythemia MDS/MPN
* Chronic myeloproliferative disease, unclassifiable ¢ Chronic myelomonocytic leukemia (CMML)
¢ Juvenile myelomonocytic leukemia
e Atypical chronic myeloid leukemia
¢ MDS/MPN, unclassifiable

Myeloid neoplasms associated with eosinophilia and

abnormalities of PDGFRA, PDGFRB, or FGFRI
¢ Myeloid neoplasms associated with PDGFRA rearrangement
¢ Myeloid neoplasms associated with PDGFRB rearrangement
¢ Myeloid neoplasms associated with FGFRI rearrangement
Bpll tayelopr oliferative syndrome) _

PDGERA =2 latelerabved ussite

factor receptor oie:gene; PDGERB == ‘platelet derived as factor receptor beta gene;
FGFRI= fibroblast growth factor I gene.
Source: Modified from Tefferi, A, and Vardiman, JW: Classification and diagnosis of myeloproliferative neoplasms: The 2008 World
Health Organization criteria and point-of-care diagnostic algorithms. Leukemia advance online publication 20 September 2007;
doi: 10.1038/sj.leu.2404955
388 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

Hype
Stayea , ad
i leiaeih adbof

Manifestations Secondary Erythrocytosis Relative Erythrocytosis

Clinical Features
Cyanosis Present May be present
Heart or lung disease Absent Present Absent
Splenomegaly Present in 75% Absent Absent
Hepatomegaly Present in 35% Absent Absent

Laboratory Features
Red cell mass Increased Increased Normal
Erythropoietin Decreased (rarely normal) Increased (rarely normal) Normal
Arterial O, saturation Normal Decreased Normal
Leukocyte count Increased in 80% Normal Normal
Platelet count Increased in 50% Normal Normal
NRBCs, poikilocytes Often present Absent Absent
LAP Increased in 70% Normal Normal
Bone marrow Hypercellular; increased Increased erythropoiesis Normal
erythropoiesis and myelopoiesis;
increased megakaryocytes; fibrosis
Serum vitamin B,, Increased in 75% Normal Normal
Culture studies Autonomous, erythroid proliferation EPO-dependent colony Not applicable
formation 4
NRBCs 2 peclenrea red blood oe EPO ==eee Tape epee alkalineHela

of the patients with PV, led to the revisions found in the 2008 predominance in men.’ PV is seen in all age groups, includ-
WHO diagnostic criteria for PV. Table 18—5 compares the ing young adults and occasionally children.'’ Although rare,
2001 and 2008 WHO criteria for the diagnosis of PV. there have been reports of familial incidence of PV.'' The
disease also appears to be more common in Jewish individu-
Incidence als of Eastern European descent.'*

PV has an incidence ranging from 0.5 to 2.3 per 100,000 pop-

ulation. The median age at diagnosis is 60 years, with a slight Pathogenesis
The clonal nature of PV was initially discovered in studies
of red blood cells (RBCs) in women who normally were
heterozygous for the two different glucose-6-phosphate
dehydrogenase (G6PD) isoenzyme types. In patients with
clonal hematopoiesis, RBCs containing either only the
wild-type or the mutant enzyme only, but not both, were
Category A (Major Criteria) found. In PV patients, a clonal pattern was observed.'*
Recently, X-linked inactivation-based clonality analysis
1. Elevated red cell mass
2. Normal arterial oxygen saturation, O, > 92%
also confirmed the presence of clonal hematopoiesis in
3. Splenomegaly patients with PV.'*
Current insights into the molecular mechanisms of
Category B (Minor Criteria)
PV have come from studies on a single somatic activating
1. Leukocytosis point mutation found on the short arm of chromosome 9
2. Thrombocytosis
3. Elevated leukocyte alkaline phosphatase (LAP) score
in the majority of patients with PV.'*?° Endogenous
4. Increased serum vitamin B,, or vitamin B,,-binding proteins erythroid colony formation (EEC) refers to the ability of
marrow- or blood-derived erythroid progenitors to form
To establish a diagnosis of polycythemia vera, 3 major
criteria are needed, or an elevated red cell mass and nor- colonies in serum-containing cultures in the absence of ery-
mal arterial oxygen saturation and two minor criteria. thropoietin. Although a hallmark of PV, EEC growth can
be seen in other myeloproliferative disorders such as ET
ee Reprinted from Pearson, TC: Evaluation ofidamone criteria
in polycythemia vera. Semin Hematol 38:21—24, 2001, with
and IMF.
permission from Elsevier. Examinations into the mechanism of EEC by PV prog-
enitor cells revealed that EEC formation was abolished by
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 389

table 18-5 WHO Criteria for Diagnosis of Polycythemia Vera .

2001 WHO Criteria for Diagnosis of Polycythemia Vera 2008 WHO Criteria for Diagnosis of Polycythemia Vera

Al. Elevated RBC mass >25% above mean normal Major Criteria:
predicted value, or Hb >19.5 g/dL in men, 16.5 g/dL 1. Hgb >18.5 g/dL (men)
in women >16.5 g/dL (women)
or
A2. No cause of secondary erythrocytosis, including: Hgb or Het >99th percentile
Absence of familial erythrocytosis of reference range for age,
No elevation of erythropoietin because of: sex or altitude of residence
Hypoxia (arterial po, 92%) or
High oxygen affinity hemoglobin Hgb >17 g/dL (men),
Truncated erythropoietin receptor or >15 g/dL (women)
Inappropriate erythropoietin production by a tumor if associated with a sustained
increase of =2 g/dL from baseline
A3. Splenomegaly that cannot be attributed to
A4. Clonal genetic abnormality other than Ph chromosome correction of iron deficiency
or BCR-ABL fusion gene in marrow cells or
AS. Endogenous erythroid colony formation in vitro Elevated red cell mass
>25% above mean normal
B1. Thrombocytosis >400 * 10°/L predicted value
BEV iB Ce De al O2/le
B3. Bone marrow biopsy showing panmyelosis with promi- 2. Presence of JAK2V617F
nent erythroid and megakaryocytic proliferation or similar mutation
B4. Low serum erythropoietin levels
Minor Criteria:
Diagnose PV when Al + A2 and any other category A are 1. BM trilineage myeloproliferation
present or when Al + A2 and any two of category B are 2. Subnormal serum erythropoietin level
present. 3. EEC growth

Diagnosis of PV requires meeting either both major

criteria and one minor criterion or the first major
criterion and 2 minor criteria.

EEC = endogenous erythroid colony; Hct = hematocrit; Hgb = hemoglobin.

Source: Modified from Tefferi, A, and Vardiman, JW: Classification and diagnosis of myeloproliferative neoplasms:
The 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia advance online
publication 20 September 2007; doi: 10.1038/sj.leu.240495

n
IMF (35% to 57%) depending on the progressio
a pharmacological inhibitor of the Janus kinase, as well as
75%), and
of the disease. '620!
short interfering RNAs (siRNA) against JAK2. JAK2 is a
cytoplasmic tyrosine kinase encoded by a gene on the short
arm of chromosome 9. A unique mutation in the JAK2 gene Clinical Features
(found in greater than 90% of PV patients) leads to a pheny-
lalanine to valine substitution at position 617 (V617F) and PV is a chronic disease that usually has an insidious onset.
to constitutive activation of the JAK2 kinase, which results Many patients are asymptomatic and are diagnosed incidentally
in activation of the downstream pathways STATS, PI3K, on routine blood work drawn for other evaluations. Patients may
AKT (also known as protein kinase B), mitogen activated present with thrombosis and/or bleeding secondary to erythroid
protein kinase (MAPK), and extracellular signal-regulated expansion, hyperviscosity, and elevated platelets. Symptoms
kinase (ERK), all of which are implicated in erythropoietin may include headache, epistaxis, ischemic or hemorrhagic
signaling.”' It is likely that activation of these downstream stroke, angina, myocardial infarction, or claudication (cramping
pathways as a result of the JAK2 mutation allows for or pain in leg muscles). Patients with underlying atherosclerosis
the erythropoietin independent production of red cells as are most likely to experience these symptoms. Thrombosis is the
seen in PV.*° most common complication in PV, and occurs in one-third of
Discovery of this mutation may allow for the design of the patients.*? Unusual sites of venous thrombus formation,
an inhibitor of Janus kinase, which could result in pharmaco- including hepatic vein, mesenteric venous, and portal vein
logic control of the erythroid, myeloid, and platelet prolifera- thrombosis are common complications of untreated PV. Hepatic
tion characteristic of PV. This acquired mutation was found vein thrombosis, leading to Budd—Chiari syndrome, has been
by various investigators, not only in the majority of patients reported in up to 10% of PV patients in one series that evaluated
with PV (65% to 97%), but also in patients with ET (23% to 140 PV patients.
390 Chapter 18 Chronic Myeloproliferative Disorders Il: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

In one review, up to 30% of PV patients had, in decreas- are measures of concentration, it is important to separate
ing order of frequency, headache, weakness, pruritus, dizzi- hemoconcentration from a true increase in red cell mass. The
ness, and sweating. Pruritus, especially noted after a warm correlation between Hct and red cell mass at hematocrits
bath, is thought to be secondary to cutaneous mast cell between 42 and 55 is poor, and in some cases determination
degranulation causing the release of histamine, fibrinolytic of the red cell mass by chromium hemodilution is required.
factors, or prostaglandins, and is almost diagnostic of PV.” This test is not always available in many centers. Particular
Erythromelalgia (erythema and painful burning in the hands attention to patients on diuretics or others at risk for hemo-
and feet) is common in PV and is thought to be secondary to concentration (i.e., diabetics, elderly) is necessary to avoid
platelet thromboxane secretion.*° Characteristically, these inappropriate diagnosis. Attention must also be paid to the
symptoms are ameliorated by the administration of low-dose mean corpuscular volume (MCV), because in PV, the absence
aspirin (81 mg). Other complications of PV include gout and of iron may slow but not stop the continued production of
an increased risk for peptic ulcer disease. Acute gouty arthri- microcytic red cells. A normal hematocrit with profound
tis occurs in 5% to 10% of patients with PV and is secondary microcytosis is virtually diagnostic of PV patients with iron-
to increased nucleoprotein turnover. Peptic ulcer disease was deficient erythropoiesis secondary to gastrointestinal blood
thought to possibly be due to increased histamine release loss, which is frequent in patients with PV (Fig. 18-1).
from basophils. Patients with increasing splenomegaly may Assays for serum erythropoietin levels are now readily
develop early satiety (fullness) and abdominal pain symptoms available and are accurate and reproducible. They can be
provoked by splenic infarction. There is a marked increase in helpful in separating PV from other causes of polycythemia.
perioperative morbidity and mortality due to increased post- In PV erythropoietin levels are characteristically low. In one
operative hemorrhage and thrombosis in patients operated on study, the sensitivity and specificity of serum erythropoietin
without proper control of red cell mass. below the reference range of normal for the diagnosis of PV
Splenomegaly is the most common physical finding in was 64% and 92%, respectively. When patients were tested
PV (50% to 80%). However, in keeping with other myelopro- on two occasions the sensitivity increased to 72%. Some
liferative disorders, the manifestations of splenomegaly, patients with normal levels initially were found to have low
myeloid metaplasia, and myelofibrosis are variably expressed values on repeat testing. Therefore, when erythropoietin lev-
at diagnosis and throughout the course of the disease. Most els are low, one can be relatively certain of the diagnosis of
commonly at the time of initial presentation, the degree of PV. Interestingly, the serum erythropoietin level was found
extramedullary hematopoiesis is usually mild, and marrow to be low even when hemoglobin levels were normal after
fibrosis is most often minimal. In the polycythemic phase of venesection.””
the disease, modest hepatomegaly may be observed as well as Characteristically, at presentation there is increased red cell
facial plethora (ruddy cyanotic complexion) secondary to production in intramedullary sites. The erythrocytes are normo-
increased vascular congestion. Hypertension can occur in cytic, normochromic, and have a normal life span. As the dis-
50% of patients, again secondary to vascular congestion. ease progresses, extramedullary ineffective hematopoiesis leads
Patients may also have skin excoriations (abrasions), optic to an increasing anisocytosis and poikilocytosis, as well as
fundi vessel engorgement, and gouty arthritis and tophi. How- shortened red cell life span secondary to splenic sequestration.
ever, 15% to 20% of patients transform to a spent phase with Many patients demonstrate the microcytosis and hypochromia
progressive anemia and increasing splenomegaly. The spent associated with iron deficiency, with low serum iron, decreased
phase of PV is associated with increased marrow fibrosis and mean corpuscular volume (MCV), and decreased mean corpus-
extramedullary hematopoiesis primarily in the liver and cular hemoglobin concentration (MCHC) occurring in about
spleen. The spent phase of PV is often referred to as post poly-
cythemic myeloid metaplasia and myelofibrosis, and it can be
indistinguishable from idiopathic myeloid metaplasia and
ER
0:5,
myelofibrosis both clinically and by laboratory findings. The
spent phase is characterized by constitutional symptoms (fever,
night sweats, and weight loss), worsening hepatosplenomegaly
a
e
secondary to increased extramedullary hematopoiesis,
and resultant portal hypertension with ascites, variceal hemor- % rs
rhage, and portal systemic encephalopathy. The risk of
leukemic transformation in PV at 20 years was found to be
approximately 15%.*°

Laboratory Findings
7. Se
The most diagnostic laboratory findings in PV is an elevated 2
RBC count, hemoglobin (Hgb), and hematocrit (Hct) with
ee
accompanying moderate thrombocytosis (60% of cases) and
leukocytosis (40% of cases). The Het is often higher than Figure 18-1 @ Peripheral blood smear seen in polycythemia vera.
58% in men and higher than 52% in women. Because these Note hypochromia and increased cellularity. (magnification x 400)
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis Bed |

one-half of patients. This iron deficiency is attributed to a shift platelets can occasionally be found. The bone marrow charac-
of iron into the expanding erythroid mass and also to gastroin- teristically shows panmyelopathy with erythroid, myeloid,
testinal blood loss, possibly aggravated by platelet dysfunction. and megakaryocytic hyperplasia, in contrast to secondary
The reticulocyte count is usually normal, and only rarely are erythrocytosis where only erythroid hyperplasia is evident
immature erythrocytes found in the peripheral blood. (Fig. 18-2). The megakaryocytes are often increased in
Relative and absolute granulocytosis occurs in approxi- number and are atypical, with deeply lobated nuclei. They are
mately two-thirds of the patients. The elevation in the total often arranged around marrow sinusoids or in a paratrabecu-
WBC count is usually moderate, with counts in the range of lar ___location. In addition to the striking increase in the number
12 to 25 X 10°/L, as per the PVSG criteria. However, because of megakaryocytes, these cells are often increased in size.
only the neutrophils are increased in PV, the total WBC may Marrow iron stores, demonstrated by Prussian blue staining,
not accurately reflect disease activity. Therefore, newer crite- are reduced or absent. This decrease results from the shift in
ria have replaced the total WBC count with an absolute neu- iron into the expanded red cell mass. Stainable iron may be
trophil count > 10 x 10°/L.*8 Occasionally, basophilia and entirely absent in patients with chronic blood loss or after
eosinophilia are apparent, and a few metamyelocytes, myelo- multiple phlebotomies. Fibrosis is rare (less than 5%) early in
cytes, and even more immature cells may occasionally be the course of the disease, but may increase to 20% at 10 to
seen on examination of the peripheral smear. 15 years, and can reach up to 50% after 20 years with progres-
Thrombocytosis is present at the time of diagnosis in sion to the spent phase. Serial biopsies performed over a
about one-half of the patients with PV. The platelet count is period of many years showed progressive increase in reticulin
most often moderately elevated, with counts between 450 and deposits even before the spent phase develops. As the disease
800 < 10°/L, but in about 5% of the patient’s platelet count runs its course, cellularity usually decreases, although
exceeds 1000 < 10°/L. Platelet life span may be shortened in megakaryocytosis may persist.
proportion to the extent of pooling in the spleen. Morpholog- The leukocyte alkaline phosphatase (LAP) score is ele-
ical alterations of platelets include the presence of giant vated in PV (97% of cases), in contrast to CML in which the
platelets as well as deficient granulation. Most patients with LAP score is decreased (Fig. 18-3). Uric acid, LDH, and
PV form spontaneous megakaryocytic colonies. Platelet vitamin B,, levels can be elevated as well, secondary to high
aggregation studies may be abnormal but are poorly predic- cellular turnover. Two of the three vitamin B,,—binding pro-
tive of the risk of thrombosis or hemorrhage and are not rou- teins, transcobalamin I and III, are frequently elevated in PV,
tinely done as part of the evaluation of PV. In most instances, as in other MPDs. Transcobalamin HI is the binding protein
the bleeding time is normal despite in-vitro platelet functional most commonly elevated in PV, whereas transcobalamin I is
abnormalities. Moreover, in general, the bleeding time is a predominantly increased in chronic myelogenous leukemia.
poor predictor of spontaneous or surgical bleeding. These increased serum values can be attributed to the exces-
The prothrombin time (PT), activated partial thrombo- sive granulocyte turnover seen in myeloproliferative disor-
plastin time (APTT), thrombin time (TT), and fibrinogen lev- ders. Furthermore, the unsaturated B,,-binding capacity
els are generally normal in patients with PV. When performing (UB,,BC) is increased in approximately 75% of patients.
coagulation tests on PV patients, the anticoagulant-to-blood Unfortunately, neither LAP score nor vitamin B,, studies are
ratio must be maintained at a 1:9 ratio. When the hematocrit is sensitive or specific enough to be used as diagnostic tests in
high as in the case of erythrocytosis, the plasma volume is PV and are not included as major or minor criteria in newer
decreased relative to the increase in packed red cells. This classifications and diagnostic algorithms. Hyperuricemia and
results in an increase in the amount of citrate anticoagulant rel-
ative to the smaller amount of plasma per unit volume of
blood. This may result in prolongation particularly of the
APTT and require a reduction in the amount of citrate in the
specimen tube. The clinician should alert the lab to this possi-
bility. The following is a formula to correct the amount of cit-
rate anticoagulant to use when the Hct is greater than 55”:
V(100—Het)
s 2s
9=
a

(595—Hect)

where V = volume of whole blood, C = anticoagulant in mL,

Hct = hematocrit
For example, if a patient has a hematocrit of 65%, the
amount of anticoagulant would be:
5(100-65) 0335018 Se, wee
os aa. We as

(595-65) ot C8? 9.0m SA epee

The RBCs in the peripheral smear are generally normo- Figure 18-2 lf Bone marrow showing panhyperplasia in poly-
cytic, normochromic. Occasionally, basophilia and immature cythemia vera. Note increased number of megakaryocytes (arrows).
granulocytes may be seen, but not circulating blasts. Giant H&E stain (low power)
 Myelofibrosis
392 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic

platelet formation.*! c-Mpl has been found to be overexpressed

(3.8-fold higher) in patients with PV, contrary to earlier
studies that reported reduced expression of Mpl in PV
and IMF.*!*?
yf
git Other relatively new parameters have been described in
association with PV. The mean platelet distribution width
(PDW) is a reflection of the average platelet size and is sig-
nificantly increased in PV as compared with secondary ery-
throcytosis.
Chromosomal analyses of the marrow for a clonal defect
can aid in confirming the diagnosis of PV (WHO classifica-
tion, Table 18—5).? One study of PV patients, in various stages,
showed that 43% had a chromosome abnormality.*? Another
study identified the most frequent karyotypic abnormalities
encountered to be a deletion of the long arm of chromosome
20 (20q—) and an extra chromosome 9 (trisomy 9).** As men-
tioned in the preceding text, greater than 95% of patients
with PV exhibit the JAK2 mutation (V617F) and in its
absence, the diagnosis of PV is unsupported.'®"”

Differential Diagnosis
The differential diagnosis between primary (PV) and secondary
polycythemia may not always be straightforward. The arterial
oxygen saturation should be >92% as indicated in the PVSG
criteria. Patients with arterial oxygen saturations below 92%
should be suspected of having secondary polycythemia related
to hypoxia. Secondary polycythemia may be appropriate (sec-
ondary to hypoxia from chronic obstructive pulmonary disease,
Figure 18-3 ™ Leukocyte alkaline phosphatase (LAP) stain of right to left cardiac shunts, and hemoglobin with increased
peripheral blood showing increased activity in polycythemia vera oxygen affinity) or inappropriate as with erythropoietin secre-
(red staining). LAP negative stain in CML (bottom).
tion secondary to renal cysts, or hypernephroma. Please refer to
Table 18-3 for features that differentiate PV from secondary
polycythemia and relative erythrocytosis. A discussion of the
uricosuria are found in 40% of patients with PV at the time of
causes and management of secondary polycythemia is beyond
presentation. This reflects the increased synthesis and degra-
the scope of this chapter and the reader is referred to other
dation of cellular nucleotides and can be seen in other hyper-
reviews. Unlike secondary polycythemia, PV is a_pan-
proliferative marrow disorders. Most patients with elevated
myelopathy and is usually associated with thrombocytosis and
uric acid levels remain asymptomatic, but uncommonly,
leukocytosis.
clinical gout may develop. A low to normal erythrocyte
sedimentation rate (ESR) is commonly present in PV patients.
The increased Het, as well as the elevated ratio of red cell Treatment
membrane to plasma fibrinogen and globulins, may account
for this finding. The cornerstone of the treatment of PV is to provide sympto-
Peripheral blood assays for erythropoietin independent matic relief, minimize long-term complications, such as
EEC growth in vitro (as part of the PVSG criteria) can be thrombosis and hemorrhage, and to avoid increasing the risk
helpful in confirming the diagnosis, but are not readily avail- of leukemic transformation.*° The most common treatment
able. Another assay that is promising for the diagnosis of PV modality utilized in PV is phlebotomy. Reduction of blood
is the measurement of PRV-] mRNA in granulocytes. PRV-/ volume (usually one unit of whole blood—450 cc), can be
is a novel member of the urokinase-type plasminogen activa- performed weekly or even twice weekly in younger patients
tor receptor superfamily and is overexpressed in mature gran- to control symptoms. The Het target range is less than 45%
ulocytes in greater than 90% of PV patients and correlates for men, less than 42% for women, and less than 37% for
strongly with EEC growth in vitro.*°In one study using quan- pregnant women in the third trimester.*’” Occasionally more
titative reverse transcriptase-polymerase chain reaction urgent cytoreduction may be required as in patients with fre-
(RT-PCR) for PRV-1 mRNA, the assay had a very high sensi- quent transient cerebral ischemic attacks or unstable angina
tivity and specificity for the diagnosis of PV.*° The throm- pectoris. More frequent phlebotomy with smaller volumes
bopoietin receptor (c-Mp/) is essential for hematopoietic stem may be helpful in this situation. Removal of the red cells by
cell survival and differentiation including megakaryocyte and continuous flow centrifugation using some of the newer
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 393

pheresis machines allows for very rapid reduction of the red aggregation (via its inhibition of platelet cyclic AMP phospho-
cell mass with conservation of plasma volume and may be diesterase activity), has also been used in PV primarily for the
very helpful in such situations. Phlebotomy will inevitably control of thrombocytosis. In one study, 0.5 mg to 1.0 mg four
result in iron deficiency and reactive thrombocytosis. The lat- times a day reduced the platelet count by 50%, or less than
ter should not be mistaken as evidence of progressive disease. 600,000/mm°.7 Imatinib mesylate has been reported anecdo-
After achieving iron deficiency, phlebotomies often may be tally to reduce phlebotomy requirements.*°
required less frequently, at every 3 to 6 months. There Low-dose aspirin reduces the rate of thrombosis and
remains considerable controversy over the need for and effi- cardiovascular deaths in patients with PV receiving standard
cacy of the use of myelosuppressive therapy in PV. Although phlebotomy and supportive care. A European study randomized
myelosuppressive therapy (radiation and chemotherapy) may 519 PV patients (with no indication for anticoagulation and no
decrease the number of phlebotomies required, decrease the pre-existing clear indication or contraindication to aspirin) to
platelet count and perhaps control splenomegaly, it is unclear low-dose aspirin (100 mg daily) versus placebo. The aspirin-
whether such treatment will decrease the morbidity of PV treated group had 60% fewer major thrombosis and cardiovas-
(thrombosis and hemorrhage), delay or prevent the onset of cular deaths (3.2% versus 7.9%) after a 3-year follow-up. The
the spent phase, or prolong survival. Moreover, the early use therapeutic benefit was independent of the platelet count. Low-
of radiation and alkylating agents that cause DNA damage dose aspirin can also effectively control erythromelalgia and
have clearly been associated with an increased risk of other vasomotor symptoms. Higher doses (500 to 900 mg/day)
leukemic transformation. An early PVSG_ study, which increased the bleeding risk without any added benefit.“
randomized 400 patients to either phlebotomy alone, phle-
botomy plus chlorambucil, or phlebotomy plus radioactive
phosphorus (*’P), revealed that phlebotomy alone without Essential Thrombocythemia
cytoreductive therapy was associated with an increased risk
for thrombosis, especially in the elderly or those with a prior Historical Perspective
history of thrombosis.** On the other hand, patients who
The earliest descriptions of essential thrombocythemia was
received myelosuppressive therapy had a higher incidence of
made by di Guglielmo in 1920 and by Epstein and Goedel in
secondary malignancies including acute myeloid leukemia
1934 who referred to it as hemorrhagic thrombocythemia.*?°
(AML), lymphoma, and gastrointestinal and skin cancers.**
In retrospect, they likely identified thrombocytosis that was
As a result of this study, radioactive phosphorus and alkylat-
secondary to other disorders. It was not until 1960 that essen-
ing agents such as chlorambucil are no longer used in the
tial thrombocythemia was established as a separate disease
treatment of PV. This study, however, was biased by the fact
entity on a clinicopathologic basis.*”
that many of the patients were not phlebotomized to levels
(less than 45%) that are now known to be safer in PV. Thus
the increased thrombotic risk found in patients treated with Definition
phlebotomy alone in this trial may have been more likely the
result of inadequate phlebotomy. A relationship between the Essential thrombocythemia (ET) is a chronic myeloprolifer-
risk of thrombosis and the extent of thrombocytosis has not ative disorder characterized by marked thrombocytosis asso-
been determined in PV.*° ciated with abnormal platelet function and an increased risk
Currently, cytoreductive therapy is usually reserved for of thrombosis and hemorrhage. It was the last of the MPDs
high-risk patients who are older (greater than 60 years), have to be identified as a distinct entity (since thrombocytosis can
thrombotic risk factors; have had prior thrombotic events; be seen in CML, PV, and IMF), and is frequently thought
have increased platelet counts; or who are symptomatic with of as a diagnosis of exclusion without any defining cytoge-
thrombosis, hemorrhage, hypercatabolic symptoms, severe netic abnormalities. Diagnostic criteria that define ET was
pruritus, and painful splenomegaly. Hydroxyurea has been the proposed by the PVSG in the mid-1970s. These guidelines
traditional cytoreductive agent used in PV. Hydroxyurea can include:
reduce the thrombosis rate, normalize the platelet count and . Platelet count in excess of 600 x 10°/L (and generally
_—
spleen size, and ameliorate hypercatabolic symptoms. How- greater than 1000 x 10°/L)
ever, treatment with hydroxyurea has been associated with an . Megakaryocytic hyperplasia in the bone marrow
increased incidence of AML.*° However, there still remains . Absence of identifiable causes of reactive thrombocytosis
considerable controversy as to whether hydroxyurea increases . Absence of the Ph chromosome
the rate of leukemic transformation in PV. The available stud- Hemoglobin no higher than 13 g/dL or normal red cell mass
ies are contradictory and may be biased by patient selection. If . Absence of significant marrow fibrosis
there is a risk, it is likely considerably less than that of alkylat- . Presence of stainable iron in the marrow, or failure to
YAAK
WY
ing agents. Subcutaneous injections of interferon alpha can respond to oral iron supplements (Table 18-—6).**
also control blood counts, splenomegaly, and hypercatabolic
symptoms. However, side effects such as fever, fatigue, The WHO published a revised set of diagnostic criteria for
depression, anorexia, nausea and vomiting can limit its ET, incorporating the exclusion of certain chromosomal
usefulness.*! Anagrelide, an oral imidazoquinazoline deriva- abnormalities in 2002.* Table 18-7 compares the 2001 and
tive that decreases platelet production and inhibits platelet 2008 WHO diagnostic criteria for ET.
394 Chapter 18 Chronic Myeloproliferative Disorders Il: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

the myeloproliferative disorders, with approximately 6000

Table 18-6 Updated PVSG Criteria people diagnosed annually in the United States. The median
for the Diagnosis of age at diagnosis is 60 years, although as many as 20%
Essential of patients may be younger than 40 years of age. The dis-
Thrombocythemia ease is more common in women, with a female to male
ratio of 1.6. Interestingly, the increased incidence of ET
e Sustained platelet count > 600,000/pL in women is most apparent in women between 30 and
¢ Absence of conditions associated with secondary thrombo-
50 years of age.*®
cythemia
¢ Normal serum ferritin or stainable iron on marrow biopsy
¢ Normal hematocrit and MCV or normal RBC mass
¢ Cytogenetics without t(9;22) and no evidence of Pathogenesis
BCR-ABL gene rearrangement
¢ Marrow collagen fibrosis less than one-third the cross- The clonal nature of ET was elucidated from sex-linked
sectional area on biopsy, without both splenomegaly and G6PD studies, identifying ET as a clonal stem cell disorder,
leukoerythroblastic changes.
supporting its placement within the MPD classification.”
¢ No morphologic or cytogenetic evidence of myelodysplas-
tic syndrome. Subsequent investigations utilizing RT-PCR and DNA methy-
lation of X-linked genes have shown that the majority of
patients with ET (74%) have monoclonal hematopoiesis
Incidence detectable at least in platelets.°' The frequency of cytogenetic
abnormalities in ET is rare.°* Serum thrombopoietin (TPO),
The advent of whole blood platelet counters resulted in a the major megakaryocytic growth and differentiation factor,
marked apparent increase in the incidence of this disease has been reported to be inappropriately normal or elevated,
cohort that is certainly related to case finding. The inci- although confirmatory studies on TPO and its receptor
dence of ET is 2.5 per 100,000.*° It is the most frequent of (c-Mp]) have not supported their role in the pathogenesis of

‘Table 18-7 WHO Criteria for Diagnosis of Essential Thombo ythemiz Ce ey io

2008

Positive Criteria Major Criteria*

1. Sustained platelet count = 600,000/uL 1. Platelet count = 450 = 10°/L

2. Bone marrow biopsy specimen showing 2. Megakaryocyte proliferation with
proliferation mainly of the megakaryocytic lineage large and mature morphology.
with increased number of enlarged, mature megakaryocytes No or little granulocyte or erythroid
proliferation.
Exclusion Criteria 3. Not meeting WHO criteria for CML, PV,
PMF, MDS or other myeloid neoplasm
3. No evidence of PV
4. Demonstration of JAK2V617F or other
Normal red cell mass or hemoglobin <19.5 g/dL
clonal marker or no evidence of reactive
in men, <16.5 g/dL in women
thrombocytosis
Stainable iron in bone marrow, normal serum
ferritin or normal MCV
If the former condition is not met, failure of
iron trial to increase red cell mass or hemoglobin
levels to the PV range.
4. No evidence of CML
No Philadelphia chromosome and no BCR-ABL
fusion gene
5. No evidence of chronic idiopathic myelofibrosis
Collagen fibrosis minimal or absent
Reticulin fibrosis minimal or absent
6. No evidence of myelodysplastic syndrome
No del(5q), t(3,3)(q21;q26), inv93(q21;q26)
7. No evidence that thrombocytosis is reactive because of:
Underlying inflammation or infection
Underlying neoplasm
Prior splenectomy

“Diagnosis of essential thrombocythemia requires meeting all four major criteria for the 2008 WHO diagnostic criteria.
Source: Modified from Tefferi, A, and Vardiman, JW: Classification and diagnosis of myeloproliferative neoplasms: The 2008
World
Health Organization criteria and point-of-care diagnostic algorithms. Leukemia advance online publication 20 September 2007;
doi: 10.1038/sj.leu.2404955
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 395

ET.’** As mentioned earlier, the JAK2 gene mutation has thrombocytosis and platelet functional defects all contribute
been found in 30% to 50% of patients with ET. Essential to the thrombohemorrhagic complications.
thrombocythemia has been divided into two groups based on Other signs and symptoms that have been observed in
the presence of endogenous erythroid colony growth and the this disease are recurrent abortions, fetal growth retardation,
presence of the JAK2 mutation. Those with EEC and the pruritus, gout, and priapism. Modest splenomegaly is present
JAK2 mutation are believed by some to have a greater risk of in approximately 40% of patients with ET.*’ Splenic atrophy
symptomatic hemorrhage and thrombosis.* resulting from splenic vascular thrombosis and silent infarc-
tions occur in up to 20% of patients.
Clinical Features
Laboratory Findings
With the introduction of automated instruments that routinely
perform whole blood platelet counts, asymptomatic patients The peripheral blood smear in ET will characteristically show
with coincidental high platelet counts are being discovered marked thrombocytosis (>600,000/uL) and platelet anisocyto-
more frequently, especially younger individuals. Approxi- sis with platelets ranging in size from tiny to large giant
mately two-thirds of patients are asymptomatic at diagnosis, platelets. Other abnormal morphological findings in the periph-
with the remaining one-third presenting with hemorrhagic or eral blood may include microthrombocytes, platelet aggregates,
vaso-occlusive symptoms, or both. The most common clinical abnormally granulated platelets, and megakaryocytic cytoplas-
complications in ET include thrombosis (11% to 25%) and mic fragments. A megakaryocyte fragment in the peripheral
hemorrhage (3.6% to 37%).°° blood is shown in Figure 18-4. Platelet anisocytosis, when pre-
In most instances bleeding is mild and manifestations are sent, correlates with an elevation of the platelet distribution
primarily mucocutaneous (epistaxis and ecchymoses); how- width (PDW). Enumeration of platelets in whole blood counters
ever, life-threatening hemorrhage may occur after accidental can be problematic due to variations in platelet size and sponta-
trauma or, rarely, after surgery. Bleeding of the gastrointesti- neous platelet aggregation. Large platelets and particularly
nal tract as well as esophageal varices bleeding has also been platelet aggregates may not be counted as platelets resulting in
reported. significant underestimation of the true platelet counts. Review
Thrombosis is the other major manifestation of ET and of peripheral smears for the presence of platelet aggregates is
is caused by intravascular clumping of sludged, hyperaggre- mandatory to avoid errors in platelet counting.
gable platelets. Thrombosis can occur in arterial, venous, or A mild normocytic, normochromic anemia may be present
microcirculatory locations. The most common types of major in up to 50% of patients although the hemoglobin value is not
thromboses include cerebrovascular accidents (stroke and usually less than 10 g/dL. Recurrent mucosal or gastrointestinal
transient ischemic attacks), myocardial infarction, and periph- bleeding leads to iron-deficiency anemia. The MCV and MCHC
eral arterial occlusion. Lower extremity deep venous throm- are decreased, with a microcytic, hypochromic blood picture
bosis and pulmonary embolism account for the majority of becoming apparent on examination of the peripheral smear.
venous events. Intra-abdominal venous thrombosis, including Erythrocyte morphological findings reflective of hyposplenism
mesenteric and hepatic vein thrombosis as seen in PV, is also (which occurs in the occasional patient with splenic infarction
common. Microcirculatory occlusions, causing headache, and atrophy) include the presence of Howell—Jolly bodies,
paresthesia, erythromelalgia (localized painful redness, burn- Pappenheimer bodies (siderotic granules), target cells, and
ing, and “pins-and-needles” tingling sensation), and acral acanthocytes.
cyanosis are often seen in ET. Erythromelalgia of the toes,
feet, and occasionally fingers is a characteristic vaso-occlusive
symptom and may progress to acral cyanosis and/or necrosis.
The involvement of the hands and feet may simulate a diabetic
neuropathy. Erythromelalgia, just as in PV, is a source of con- ~ Oo ote F~
v.

tinual torment and frustration for ET patients. The toxic effect

of the metabolites of platelet arachidonic acid appears to be
responsible for the erythromelalgia, and this may be relieved
by decreasing the platelet count or by use of anti-inflammatory
agents such as aspirin. Thrombotic and bleeding complications = *
te...
are more common when the platelet count is greater than > P * a ~
© 73
sen & §
2000 X 10°/L. oe
’
: ‘ Fear ty
Bleeding manifestations are usually minor, involve the ss - age = 2 ee
> £.
skin and mucous membranes, and include ecchymosis, epis- a =~ ¥ * etd
taxis, menorrhagia, and gingival hemorrhage.°® Neurologic
manifestations are usually of a transient ischemic nature and
include: visual disturbances, headaches, paresthesia, dizzi- Figure 18-4 @ Essential thrombocythemia, peripheral blood
ness, transient ischemic attacks, and, rarely, seizures. Com- megakaryocyte and numerous platelets. (From Hyun, BH: Morphol-
plete stroke is an uncommon occurrence. In older patients, ogy of Blood and Bone Marrow, American Society of Clinical Pathol-
underlying degenerative vascular disease in combination with ogists, Workshop 5121, Philadelphia, 1983, with permission.)
396 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

Leukocytosis is present in about one-third of patients, As mentioned earlier, 23% to 57% of patients with ET
with WBC counts rarely exceeding 50 X 10°/L. Neutrophilia carry a JAK2 gene mutation.”! The finding of the JAK2 muta-
is observed in the majority of patients with elevated WBC tion supports the existence of more than one type of etiology
counts, but a mild eosinophilia or basophilia, or both, may of this disease.2! Red cell mass determinations and erythro-
occasionally be seen. Rarely, nucleated red cells, and imma- poietin levels may be necessary in patients with borderline
ture granulocytes may also be evident. The LAP score is hematocrits to rule out PV.
variable, but most commonly is normal.
The bone marrow in patients with ET demonstrates a
marked increase in the megakaryocytic component. The
Differential Diagnosis
megakaryocytes are typically larger than normal and may be The differential diagnosis of ET from reactive thrombocytosis
dysplastic in appearance with abundant cytoplasm and hyper- can be difficult, and ET often remains a diagnosis of exclusion.
lobulated nuclei, usually occurring in loose clusters or dispersed Essential thrombocythemia must be differentiated from the
in the marrow (Fig. 18-5). There may be increased erythroid various causes of reactive thrombocytosis (Table 18-8). The
and granulocytic proliferation without dysplasia or increased features differentiating ET from reactive thrombocytosis are
myeloblasts. Significant marrow fibrosis points against a diag- summarized in Table 18-9. Thrombocytosis is often present in
nosis of ET, per PVSG criteria (see Table 18-6). Stainable iron patients with a wide variety of inflammatory, malignant, infec-
is normal to slightly decreased in most cases, and increased reti- tious, and trauma-associated conditions. Reactive thrombocy-
culin content is often seen. Marrow karyotype is generally nor- tosis, even when persistently present for weeks or months, is
mal; however, the deletion of the long arm of chromosome usually well tolerated in these patients and is usually unasso-
21 (21q_) has been reported in some patients. ciated with thrombosis or hemorrhage. Bone marrow exami-
Platelet function studies reveal a variety of abnormalities nation may not allow a distinction between ET and reactive
in some patients. Abnormal platelet aggregation to epineph- thrombocytosis and should be avoided when alternative etiolo-
rine, collagen, adenosine diphosphate (ADP), and ristocetin gies are readily apparent. The platelet count in secondary
are quite frequent. Studies have demonstrated both normal (reactive) thrombocytosis seldom exceeds 1000 X 10°/L, and
bleeding times (even in the case of patients with hemorrhagic commonly falls in the range of 500 to 750 X 10°/L; however,
tendencies) and prolonged bleeding times. Reduced platelet extreme thrombocytosis may also be present in reactive condi-
factor 3 (PF3), reduced platelet adhesion, low protein S levels, tions. Platelet morphology and function are generally normal
and nucleotide storage pool defects have all been reported in in chronic reactive states compared to the variety of abnormal-
association with ET. Although qualitative functional platelet ities seen in MPDs.
abnormalities have been described, they are insufficiently ET must also be differentiated from other chronic MPDs
reproducible to be useful in differential diagnosis. These with associated thrombocytosis and from the myelodysplastic
abnormalities are not useful in predicting thrombosis or hem-
syndromes (see Chap. 19) in which the platelet count can be
orrhage. In addition, the severity of thrombocytosis does not markedly elevated. When the platelet count is persistently
necessarily correlate with the presence, absence, or severity of greater than 600 X 10°/L and the bone marrow demonstrates
symptoms. Falsely elevated serum potassium and phosphorus
predominant megakaryocytic hyperplasia, the diagnosis of
levels will sometimes be seen due to platelet lysis in uncen-
ET should be investigated. Because there are no unique clin-
trifuged serum specimens.
ical, hematologic, or histopathologic findings in this disease,
it is by nature a diagnosis of exclusion. At presentation, the
hemoglobin level is normal or mildly decreased. In some
patients a determination of red cell mass may be necessary to
rule out PV with increased platelets. To ensure that masked
PV has not been overlooked in those patients having a normal
or decreased red cell mass as a result of iron deficiency, bone

Acute hemorrhage
Postsplenectomy and Hemolytic anemia
hyposplenism Myelodysplastic diseases
Postoperative Graft-versus-host disease
Malignancy Vitamin E deficiency
Chronic inflammatory disorders Hyperadrenalism
Figure 18-5 @ Essential thrombocythemia, bone marrow. Note Chronic infection Rebound recovery from
increased megakaryocytes. (From Hyun, BH: Morphology of Blood Iron-deficiency anemia thrombocytopenia
and Bone Marrow, American Society of Clinical Pathologists, Work- Drug-induced Exercise
shop 5121, Philadelphia, 1983, with permission.)
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 397

formation (EEC), which is never found in reactive thrombo-

cytosis, and when present, support the presence of an MPD
such as ET.

Treatment
Essential Reactive The therapeutic approach to ET depends on a number of factors,
Thrombocytosis Thrombocytosis including the patient’s age and childbearing potential, the height
Platelet count Frequent Infrequent of the platelet count, and, most importantly, the presence and
> 1,000,000 duration of symptoms. Treatment for ET is usually reserved for
Leukocytosis Frequent Can be present in high-risk patients who are 60 years of age or older, have a prior
reactive states history of thrombosis, and cardiovascular risk factors such as
Anemia Normocytic, Microcytic,
hypertension, hypercholesterolemia, and smoking. Low-risk
normochromic hypochromic
(secondary to patients are younger than 60 years, with no previous history of
iron deficiency) thrombosis, a platelet count of less than 1,500,000/uL, and no
Splenomegaly < 50% Not present cardiovascular risk factors. Intermediate—risk patients are those
Clustering Common Not present who do not qualify for either the low-risk or high-risk groups.**
megakaryocytes
Careful monitoring, without therapy specifically aimed at
in bone marrow
Bone marrow 20% Not present lowering the platelet count, is generally advocated for asympto-
fibrosis matic low-risk patients with extreme thrombocytosis. Young
patients with few or no symptoms do not require treatment
unless surgery is indicated or childbirth is imminent. In these
cases, plateletpheresis or a brief period of myelosuppressive
marrow aspirate with iron stains may be helpful. In addition, therapy may be useful in controlling the elevated platelet count.
if bone marrow aspirate is refused, a 1-month trial of iron Asymptomatic young patients should receive aspirin, which has
therapy should be administered. The response to iron therapy been shown to be beneficial in a randomized trial.“
should be carefully menitored, and if a rise in the Hct level Treatment of high-risk patients (older than age 60, with
and red cell volume is observed, the patient should be evalu- cardiovascular risk factors, or younger symptomatic patients)
ated for evidence of PV, evidence of blood loss, or both. is indicated to control hemorrhage and manitestations of
Chromosomal analysis of the bone marrow should be thrombosis and to control the progressive megakaryocyte pro-
performed to ensure that the Ph chromosome is not present. In liferation. Thrombotic events are far more common than
those patients with CML and thrombocytosis in whom the hemorrhagic events. It is interesting to note that the degree of
Ph chromosome is not demonstrable by standard metaphase thrombocytosis does not correlate with the risk of thrombosis.
cytogenetics, other findings are important for supporting the Acute hemorrhage and occlusive events, which occur in
diagnosis of CML. These include: myeloid hyperplasia, low approximately 30% of patients, indicate the necessity for
LAP score, the presence of BCR-ABL gene rearrangements by immediate therapy. Plateletpheresis achieves a dramatic
molecular techniques and moderate to marked splenomegaly. reduction in the platelet count in a matter of hours; however,
Early IMF is often associated with extreme thrombocy- this reduction is transient and therefore inadequate for long-
tosis. However, there is usually marked splenomegaly, a term control. In addition, the procedure is not cost-effective
leukoerythroblastic blood picture, teardrop poikilocytosis, and involves a difficult process if done four to five times
and the characteristic myelofibrotic involvement of the bone per week.
marrow (increased reticulin and collagen fibrosis). In ET, less Chemotherapy should be initiated in addition to platelet-
than one-third of the biopsy area demonstrates fibrosis. pheresis whenever thrombohemorrhagic complications
Other markers of the acute phase response such as develop, age is greater than 60, and the platelet count is more
interleukin-6 (IL-6), plasma fibrinogen, and C-reactive pro- than 1.5 x 10°/L. A number of effective myelosuppressive
tein are elevated in the acute phase response to infection, agents have been administered in the past (**P, Busulfan), but
trauma and inflammation, and may help in differentiating their use has been associated with an increased potential for
reactive thrombocytosis from ET. induction of acute leukemia. Common treatment options
Myelodysplastic syndromes associated with thrombocy- include cytoreductive agents such as hydroxyurea and anagre-
tosis usually present with a more severe degree of anemia that lide. The antimetabolite hydroxyurea is generally considered
is often macrocytic in appearance as compared with that seen to be the drug of choice because of its efficacy. Hydroxyurea,
in patients with ET. In addition, the presence of either cytoge- compared to no treatment, was shown to reduce the risk of
netic abnormalities (5q-, deletion 7, trisomy 8), or ringed thrombosis in high-risk patients from 24% to less than 4% at
sideroblasts in the bone marrow denotes a myelodysplastic a median follow-up of 27 months.* Reported incidence rates
syndrome as the cause of the associated thrombocytosis rather of leukemic conversion, when hydroxyurea is used alone in
than essential thrombocythemia (see Chap. 19). ET can reach up to 5%. Hydroxyuea should be administered
As previously noted, some patients with ET demonstrate at a daily dosage of 15 mg/kg of body weight, with adjust-
erythropoietin-independent endogenous erythmoid colony ment according to response. In 90% of patients, the platelet
398 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

count will be decreased to less than 600 X 10°/L in 2 to rebound thrombocytosis is inevitable without concomitant
6 weeks. A lower continuous dosage is required to maintain aggressive myelosuppression. If surgery is indicated, the platelet
disease control, because once this treatment is halted the count should be controlled preoperatively by use of platelet-
platelet count rises again. Common side effects of hydrox- pheresis or myelosuppression, or both. Platelet concentrates may
yurea are dose-related, reversible leukopenia, macrocytic red rarely be needed to provide normal functional platelets.
cell changes and anemia, and rarely, fever, rash, and leg ulcers.
The drug anagrelide is also used to treat thrombocytosis.
It is considered an excellent drug for long-term use, because
Idiopathic Myelofibrosis
it does not have mutagenic or leukemogenic potential. Ana- Historical Perspective
grelide can cause significant reduction in the platelet count, as
well as a reduction in both major and minor thromboembolic Idiopathic myelofibrosis (IMF) or idiopathic myeloid meta-
complications.°! Bleeding complications were common only plasia with myelofibrosis, was initially described by Heuck in
in those patients who were taking anagrelide along with low 1879.° He described the case of a 24-year-old butcher who
dose aspirin. Other side effects include headaches, palpita- had been afflicted with severe fatigue for 1 year. On examina-
tions, fluid retention, and diarrhea (which in 15% of patients tion, severe anemia, leukocytosis with myeloid immaturity,
are intolerable).°* However, a recent randomized trial compar- and marked hepatosplenomegaly were noted. The patient sur-
ing hydroxyurea with anagrelide showed that hydroxyurea vived for only 2 years and, on autopsy, was demonstrated to
with aspirin was superior to anagrelide with aspirin in pre- have severe osteosclerosis and extramedullary hematopoiesis.
venting thrombotic and hemorrhagic events in ET patients at Heuck thus concluded, based on the unique features of the
high risk for vascular events. case, that myelofibrosis with myeloid metaplasia was distinct
Alpha interferon has also been advocated for treatment in from leukemia. IMF was formally included as one of the
ET. This agent exerts an inhibitory effect on the growth of myeloproliferatives disorders in 1951.
megakaryocyte progenitors that correlates clinically with a
marked decrease in platelet count following treatment with Definition
interferon. Alpha interferon has also been shown to have an
overall response rate of 88% in ET patients. Aside from control- Chronic idiopathic myelofibrosis (CIMF) is characterized by
ling thrombocytosis and leukocytosis, reversal of splenomegaly the classic triad of findings of (1) fibrosis of the marrow, at first
was also noted.“ Treatment doses of 1 to 5 million units/day patchy and later widespread, that may or may not be accompa-
exert a dose-dependent inhibitory effect on thrombocytopoiesis. nied by bony sclerosis; (2) extramedullary hematopoiesis or
However, the treatment also causes moderate reduction of gran- myeloid metaplasia of the spleen and liver, giving rise to mod-
ulocyte levels and is associated with significant side effects, erate to marked splenomegaly and hepatomegaly; and
which include fatigue, depression, anorexia, weight loss, fevers (3) leukoerythroblastosis and teardrop poikilocytosis of the
and nausea. As with PV, these side effects have precluded wide- peripheral blood. The features of myelofibrosis as defined by
spread use of this agent in treating ET. However, it has been the American PVSG are described in Table 18-10.
effective and safe in pregnant patients with ET, where other CIMF, like the other myeloproliferative disorders, is a
agents such as hydroxyurea are contraindicated. clonal myeloproliferative disease. This has been demon-
Pipobroman (an oral piperazine derivative that functions as strated in female G6PD heterozygotes and also using other
an alkylating agent), is used widely in Europe for the treatment X chromosome-linked polymorphisms. It is characterized
of PV and ET. Although not available in the United States, pipo- by the proliferation of mainly granulocytes and megakary-
broman was found to be as effective as hydroxyurea. Pipobro- ocytes leading to the characteristic finding of bone marrow
man is an alkylating agent and thus may be associated with an fibrosis.
increased risk of leukemic transformation in ET. CIMF is known by at least 20 synonyms. Some of
Low-dose aspirin (100 mg/day) has been effective in pre- the most frequently employed are agnogenic myeloid
venting thrombosis in ET, with an acceptable risk for bleeding, metaplasia, myelosclerosis, osteosclerosis, chronic erythroblas-
if applied to patients with a platelet count <1000 x 10°/L and/or tosis, aleukemic myelosis, and chronic or primary myelofibro-
absence of bleeding history.°° An important role for aspirin may sis. The term myelofibrosis with myeloid metaplasia (MMM),
be found in patients with recurrent miscarriages, because this or just idiopathic myelofibrosis (IMF) for brevity, highlights the
treatment does not carry the risk of teratogenicity. In patients essential features of fibrosis and extramedullary hematopoiesis.
with markedly elevated platelet counts (1520 10°/L) the bind- CIMF can be divided into two stages, according to the
ing of high molecular weight von Willebrand multimers to the WHO criteria: a prefibrotic stage (p-CIMF) and a fibrotic stage
platelets produces in effect a type II von Willebrand’s disease (f-CIMF).* The classic triad of clinical findings is absent or
and its attendant bleeding risks.°’ Therefore aspirin should be minimal in the prefibrotic stage (hypercellular phase) of early
avoided in ET patients with platelet counts of this magnitude, CIMF. The p-CIMF stage is characterized by a granulocytic
until levels are reduced by myelosuppressive therapy. Occasion- and megakaryocytic myeloproliferation, decreased number of
ally patients with ET with life- or organ-threatening symptoms erythroid precursors, lack of fibrosis, and abnormalities in
such as transient cerebral ischemia, unstable angina, or impend- megakaryopoiesis characterized by atypical megakaryocytes.”
ing gangrene may require urgent lowering of the platelet count. Clinically the prefibrotic (p-CIMF) stage of CIMF mimics
Although platelet pheresis may be helpful, in this instance the early stages of ET and PV. The fibrotic (f-CIMF) stage is
 Chapter 18 Chronic Myeloproliferative Disorders Il: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 399

Incidence and Etiology

In the United States, CIMF has an incidence of 1.5 per
100,000 per year. IMF occurs mainly in middle-aged and
elderly patients with a median age of 67 years at presenta-
aoa Myelofirons tion.*? Although rare in childhood, familial occurrences have

(CIM) : been reported in several

absence of any causative
generations within families in the
environmental agent.’” CIMF is said
to be less common in individuals of African or Spanish
1 Ray
2. Fibrosis involving more than one-third of the sectional descent. CIMF has also been linked to exposure to thorium
area of an adequate bone marrow biopsy specimen dioxide, toluene, benzene, and ionizing radiation.’' A high
. A leukoerythroblastic blood picture incidence of CIMF has also been reported in Hiroshima
. Absence of increased red cell mass survivors.’> Myelofibrosis has also occurred secondary to
. Absence of Philadelphia (Ph) chromosome
chronic infections, especially tuberculosis, histoplasmosis,
. Exclusion of systemic disorders
. Adiagnosis of osteomyelosclerosis requiring the presence and after myocardial infarction; however, the fibrosis seen in
of sclerotic changes detected radiologically in axial skeleton these disorders represents a secondary or reactive process.
long bones?

Pathogenesis
Recent evidence has indicated that IMF originates at the level
characterized by fibrosis of the marrow, extramedullary of the CD34* hematopoietic stem cell.”* As mentioned previ-
hematopoiesis (splenomegaly), and leukoerythroblastosis of ously, hematopoiesis in IMF, as in the other MPDs is clonal
the peripheral blood.* The Updated Cologne criteria has been and clonal chromosomal abnormalities were found in as many
used for the diagnosis of IMF as well as for staging purposes as 57% of patients with IMF, in one series.’ The most com-
(Table 18—11).° mon ones reported include: 13q—, 20q—, +8, +9, 12p—, and

or IMFDiagnosis
andStaging
Clinical Criteria
Al. No preceding or allied subtype of myeloproliferative disorders, CML, or myelodysplastic syndromes
A2. Early clinical stages
Normal hemoglobin or anemia, grade I: hemoglobin =12 g/dL
Slight or moderate splenomegaly on palpation or >11 cm on ultrasound scan or CT
Thrombocythemia, platelet count >400,000/,L
A3. Intermediate clinical stage
Anemia grade II: hemoglobin =10 g/dL
Definitive leukoerythroblastic blood picture and/or teardrop erythrocytes
Splenomegaly
No adverse signs (age >70 years, hemoglobin <10 g/dL, myeloblasts > 2% in peripheral blood, pronormoblasts > 2% in peripheral
blood, leukocytosis > 20,000/L, thrombocytopenia < 300,000/L, severe constitutional symptoms, massive splenomegaly,
cytogenetic abnormalities)
A4. Advanced clinical stage
Anemia grade IL: hemoglobin <10 g/dL
One or more adverse signs (age > 70 years, hemoglobin <10 g/dL, myeloblasts > 2% in peripheral blood, pronormoblasts
> 2% in peripheral blood, leukocytosis >20,000/L, thrombocytopenia <300,000/L, severe constitutional symptoms,
massive splenomegaly, cytogenetic abnormalities)

Pathologic Criteria
B1. Megakaryocytic and granulocytic myeloproliferation and relative reduction of erythroid precursors. Abnormal clustering and
increase in atypical giant-sized megakaryocytes containing cloud-like lobulated nuclei and definitive maturation defects

Staging of Marrow Fibrosis

MFO Prefibrotic stage IMF; no reticulin fibrosis
MFI Early IMF; slight reticulin fibrosis
ME2 Manifest IMF; marked increase in reticulin and/or collagen fibrosis
MB3 Overt IMF; advanced collagen fibrosis—osteosclerosis (endophytic bone formation)
Al + B1 = IMF; Al + A2, B1 + MFO= prefibrotic IMF; Al + A3, B1 + MFI, MF2 = early manifestation of IMF;
Al ngA4, BI + MFS3 == end-stage IMF.

Source: Reprinted froth ene we F, and Barosi, G:Payslonbee withRipelold metaplasia: Diagnosis, prognostic
factors and staging. Semin Oncol 32:395, 2005, with permission from Elsevier.
400. Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

abnormalities of chromosomes | and 7.” In a 2006 study, the hematopoiesis, splenomegaly and sometimes hepatomegaly is
JAK2 (V617F) mutation (described previously) was reported present. Splenomegaly related to IMF is present in 85% to 99%
in 56% of the patients with IMF.” Granulocytes of patients of patients at diagnosis and can be massive in about 10%.”
with CIMF had the JAK2 (V617F) mutation which correlated Symptoms due to splenomegaly include left upper quadrant
with the activation of cell signaling.'° This granulocyte acti- pain, early satiety and even left shoulder pain. Palpable
vation leads to abnormal trafficking of CD34* cells with an hepatomegaly is present in 40% to 70% of the cases
increased CD34 count in the peripheral blood.’ Granulocytes (Fig. 18-6). Although hepatomegaly is found in about 50% of
from CIMF are functionally activated as indicated by the patients, it is not generally excessive, but may be accompanied
increased values for LAP. LAP scores are significantly higher by mild to moderate jaundice, ascites, or both. Portal hyperten-
in myeloproliferative disorders than in reactive conditions. sion may develop as a result of increased splanchnic flow due
There is a direct relationship between JAK2 (V617F) muta- to splenomegaly and/or intrahepatic obstruction associated
tion and circulating CD34* cells and fibrosis. CD34* cell with extramedullary hematopoiesis in the liver*® (Fig. 18-7).
counts increase exponentially as the mutant alleles exceed Symptomatic patients complain of fever, anorexia,
50%. In one study, patients carrying the JAK2 (V617F) muta- weight loss, night sweats, pruritus, and bone pain which
tion in their granulocytes had higher PB CD34* cell counts represent the metabolic consequences of myelofibrosis.*!
than individuals without the mutation.’° This study indicated Extramedullary hematopoiesis can occur in almost any organ,
that the JAK2 (V617F) mutation may constitutively activate and may manifest itself as lymphadenopathy, pleural or peri-
granulocytes which mobilizes CD34 cells.’° Transition from cardial effusion, or ascites.**** The central nervous system
heterozygosity to homozygosity for the JAK2 (V167F) muta- can also be involved with increased intracranial pressure,
tion in granulocytes triggers significant CD34* cell mobiliza- altered sensorium, motor and sensory impairment, and even
tion. This results in higher CD34* cell count, which correlates cord compression due to extramedullary hematopoiesis in the
with progression to marrow fibrosis.’°
Unlike PV and ET which are characterized by hyperpro-
liferation, IMF is characterized by ineffective erythropoiesis
and ineffective megakaryopoiesis, suggesting that additional
genetic abnormalities may play a role in the pathogenesis of
IMF. Fibroblasts, however, are not part of the clonal process
and do not share the same clonal chromosomal abnormalities
supporting the theory that bone marrow fibrosis is a sec-
ondary reaction.’
A number of cytokines are involved in the pathogenesis
of fibrosis in IMF. These are all secreted from platelets and
megakaryocytes, although in some instances may also be
secreted from other cellular or stromal sources. These include
transforming growth factor B (TGF-B), platelet-derived
growth factor (PDGF), and basic fibroblast growth factor
(bFGF). TGF-B may possibly be the most important of the
three.” It is capable of promoting secretions of type I and type Figure 18-6 ™ Hepatosplenomegaly, a characteristic finding in
III collagen. Elevated levels of PDGF are found in all patients patients with idiopathic myelofibrosis with myeloid metaplasia
with IMF and are released from platelets and megakaryocytes (IMF/MM).
undergoing intramedullary death. PDGF stimulates fibroblast
proliferation and collagen secretion. bFGF moderates
megakaryocyte-stromal cell interaction resulting in prolifera-
tion of stromal cells such as fibroblasts.
Thrombopoietin promotes megakaryocyte growth and
development. However, neither mutations in the thrombopoietin
receptor nor autocrine stimulation of thrombopoietin has been
shown to play a role in the pathogenesis of CIMF.

Clinical Features

Myelofibrosis is a chronic progressive disorder with an insidi-

ous onset such that the patient may be symptom-free for many
years. A third of IMF patients older than the age of 60 are
asymptomatic at presentation, and the diagnosis is made after
routine physical examination and the incidental finding of
unexplained splenomegaly or abnormal peripheral blood Figure 18-7 @ Extramedullary hematopoiesis in the liver of a patient
results, or both. With progression of extramedullary with IMF.
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 40]

epidural space.** Extramedullary hematopoiesis can occur some cases of IMF, hemolysis is autoimmune in nature, and
within the pulmonary arterial circulation resulting in severe may be DAT positive with deposition of immunoglobulin
pulmonary hypertension. Rapidly developing extramedullary IgG, IgM, or complement on the erythrocyte surfaces.
hematopoiesis may occur in the liver with abnormal liver Hypochromic, microcytic anemia may occasionally occur
function, jaundice, portal hypertension, and hepatic failure.* secondary to gastrointestinal bleeding from peptic ulcer disease.
Symptoms of anemia such as weakness, pallor, lethargy, Megaloblastic anemia with macrocytes and occasionally hyper-
and dyspnea on exertion are common. Approximately 10% of segmented neutrophils, resulting from relative folate deficiency,
patients present with a serious bleeding diathesis secondary may occur due to increased folate utilization.
to thrombocytopenia or thrombocytosis, qualitative platelet The leukocyte count is variable in IMF. In about 50% of
defects, or coagulation abnormalities. Symptoms of bleeding cases the white blood cell (WBC) count exceeds 10.0 x
may be as minor as petechiae or ecchymoses, or as serious as 10°/L; in approximately 35%, the WBC count is normal; and
esophageal variceal bleeding. Infection may ensue related to in nearly 15%, the WBC count is below normal. Marked
immune deficiency, even when neutropenia is absent. leukocytosis (WBC greater than 30 x 10°/L) is initially pre-
Increased cellular turnover of nucleic acids can lead to hyper- sent in 11% to 13% of patients. Conversely, leukopenia can be
uricemia and secondary gout. Osteosclerosis, characterized by a seen in 8% of patients. As the disease evolves, the leukocyte
diffuse or patchy increase in bone density and increased promi- level declines, with an increase of immature myeloid cells
nence of bony trabeculae on radiologic studies, can be seen in dominating the peripheral blood picture, but not to the degree
IMF as well, and may be associated with severe bone pain.” seen in acute myelocytic leukemia. Rarely leukocytosis may
Various scoring systems involving the hemoglobin level, be extreme and may be mistaken for CML. The LAP score is
white count, constitutional symptoms and number of circulat- typically normal or moderately increased. Serum levels of
ing blasts have been used for prognosis in IMF. An example vitamin B,, are increased, but not to the degree found in
of this is the Lille System (Table 18—12).*° untreated CML. Eosinophilia and basophilia may be found
frequently as in other myeloproliferative diseases.
The platelet count may be normal, elevated, or decreased.
Laboratory Findings In approximately 50% of IMF patients, platelet counts range
Normocytic normochromic anemia is present in 50% to 90% of from 450 to 1000 X 10°/L at the time of diagnosis. Occasionally
patients at the time of diagnosis. Twenty-five percent of counts in excess of | million are present. As the disease pro-
patients have severe anemia with hemoglobin less than 8 g/dL. gresses, thrombocytopenia becomes increasingly prevalent.
The cause of the anemia in IMF is multifactorial and results Giant dysplastic platelets are often conspicuous, and megakary-
from additive effects of bone marrow failure, autoimmune
ocytic fragments or even dwarf megakaryocytes may be present
hemolysis, ineffective or dyserythropoiesis, and hypersplenism in the peripheral blood (Fig. 18-10). The disturbed platelet mor-
phology is reflected in abnormal platelet physiology with abnor-
(dilutional anemia).
With increasing disease, the morphological changes mal platelet functions present in as many as 50% of patients.
Spontaneous platelet aggregation may also occur. The bleeding
become increasingly abnormal, and the classic leukoerythro-
blastic blood picture appears (Fig. 18-8). This includes the time (a measure of platelet number and function) is prolonged in
up to 20% of patients, but correlates poorly with the risk of
appearance of abundant nucleated red cells, immature granulo-
bleeding. Although both thrombosis and bleeding may occur in
cytes, and teardrop shaped red cells in the peripheral blood
IMF, bleeding is particularly common, especially after splenec-
(Fig. 18-9). Improvement or even normalization of red cell
tomy. In patients who have undergone splenectomy, the number
morphology after splenectomy supports a causative relationship
of immature WBCs, poikilocytes, and morphologically abnor-
between splenomegaly and red cell morphology.
mal platelets increase because the filtering function of the spleen
Shortened red cell survival may result from ineffective ery-
is no longer present.
thropoiesis and hemolysis. Severe hemolytic anemia develops
Bone marrow aspirations are usually unsuccessful (“dry
in 15% of cases and is evidenced by marked reticulocytosis. It
tap”) secondary to myelofibrosis. The reticulin and collagen
is generally direct antiglobulin test (DAT) negative; however, in
fibrosis requires a needle biopsy for diagnosis. The bone mar-
row biopsy is hypercellular with an increase in neutrophils,
and atypical megakaryocytes in the prefibrotic phase.
Approximately 25% of patients present in the prefibrotic
stage. A left-shift may be observed without significant
increase in myeloblasts. Erythroid precursors are easily
found, but overall erythropoiesis is decreased. Megakary-
ocytes are morphologically abnormal with variations in size
Hemoglobin < 10 g/dL and “cloud-like” or “balloon-shaped” lobulations of the
Leukocyte count < 4000/wL or > 30,000/L
nuclei. Many naked megakaryocyte nuclei are seen as well.*’
Presence: scores | point
As the name implies, reticulin fibrosis is minimal in the
Median survival: Score 0: 93 months
Score |: 26 months prefibrotic phase. In the fibrotic phase, the bone marrow
Score 2: 13 months cellularity is normal or decreased. Areas of hematopoiesis
are separated by regions of loose connective tissue or fat.
402. Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

Figure 18-8 @ Leukoerythroblastosis, teardrop poikilocytosis, and abnormal platelet morphology associated with idiopathic myelofibrosis.
A. Leukoerythroblastosis. Note the myeloblast at the large arrow and the numerous nucleated red blood cells at the small arrows. B. Teardrop
poikilocytosis. C. Dwarf megakaryocyte (or micromegakaryocyte). This pathologic alteration of a megakaryocyte may be found in any of the
myeloproliferative disorders. Although often difficult to distinguish from cells of other lineages, observation of the marked cytoplasmic granu-
larity and further comparison of this cytoplasm to that of other platelets present on the peripheral smear will aid in identification. D. Dwarf
megakaryocyte. The cell at the pointer displays cytoplasmic blebs or budding, which is another characteristic of a micromegakaryocyte. Also
note the giant platelets present on this peripheral blood smear.

Reticulin fibrosis can be prominent with small islands of suggest occult disseminated intravascular coagulation (DIC).
residual hematopoietic precursors. The marrow sinuses can be Extramedullary hematopoiesis in the liver may result in hepatic
increased in size and number with characteristic intrasinu- dysfunction which certainly contributes to the coagulation
soidal hematopoiesis. Atypical megakaryocytes often occur in abnormalities (occult DIC, enhanced fibrinolysis) seen in IMF.
clusters, sheets or within dilated sinuses.*’ Thickening of the Hyperuricemia and elevated liver enzymes are found in
bony trabeculae (osteosclerosis) may be seen (Fig. 18-11). one-third of IMF patients.
Platelet dysfunction can cause troublesome hemostatic Cytogenetic abnormalities are present in approximately
complications in IMF patients. In addition, patients occasionally 30% of cases at diagnosis. The rate increases to approximately
demonstrate prolonged prothrombin and thrombin times, as 90% after leukemic transformation. Chromosomal abnormali-
well as elevated levels of fibrin degradation products and ties are less frequent in younger patients, which may explain
reduced levels of factors V and VIII. These features their better prognosis. Common findings include 13q—, 20q-,

Figure 18-9 @ Teardrop-shaped cells (arrows): peripheral blood in Figure 18-10 ™ Micromegakaryocyte found on the peripheral
a patient with myelofibrosis. blood smear of a patient with essential thrombocythemia.
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 403

of CML, there is marked leukocytosis, whereas in IMF, the

WBC count is usually less than 30 * 10°/L. Red cell mor-
phology in CML is generally normal or may show a slight
amount of anisocytosis and poikilocytosis, compared with the
significant teardrop poikilocytosis in IMF. The presence of
te
the Ph chromosome and low LAP score are the strongest
See ee
3
ay. eee differentiating features that distinguish CML from IMF. Ordi-
Poe
* narily, differentiation from CML is not difficult. However,
atypical cases such as Ph-negative CML, may be virtually
impossible to differentiate from IMF with leukocytosis and
minimal fibrosis (the cellular phase of IMF).
Approximately 15% to 20% of patients with known PV
undergo a transition to terminal myelofibrosis with marked
anemia, bone marrow fibrosis and hypofunction, and progres-
sive splenomegaly.
IMF must be differentiated from secondary causes of
myelofibrosis. Metastatic carcinoma and lymphoma may be
associated with marrow fibrosis. Granulomatous disorders
such as tuberculosis, histoplasmosis, or sarcoidosis of the
marrow can also cause myelofibrosis. Other hematologic dis-
eases, such as acute megakaryocytic leukemia, hairy-cell
leukemia, and myelodysplastic syndromes, may also be
associated with marrow fibrosis (see Table 18—13). Marrow
fibrosis may be seen in autoimmune disorders such as
systemic lupus erythematosus and scleroderma. Granuloma-
tous disorders associated with secondary marrow fibrosis can
be differentiated from IMF by careful examination of marrow
for the presence of granulomas and special stains for fungi
and acid-fast bacilli. The presence of tartrate resistant acid
Figure 18-11 ® Histopathology of the bone marrow in idiopathic phosphatase positive lymphocytes in the marrow and periph-
myelofibrosis. A. Early hyperplastic state without fibrosis. B. Advanced
eral blood, is typical of hairy cell leukemia and allows differen-
stage with a conspicuous increase in reticulin fibers, a still hypercellular
marrow, and a lymphoid infiltrate (arrows). C. Late osteosclerotic tiation from IMF. Multiple myeloma with diffuse marrow
state with endophytic bone formation, a residual cluster of plasmacytosis and the presence of serum and urine monoclonal
hematopoiesis, and large areas of fatty tissue. D. Moderate degree of
reticulin fibers surrounding atypical megakaryocytes in early IMF.
E. Coarse bundles of obvious collagen fibers encompassing a few
hematopoietic elements in terminal stages of IMF. F. Clusters of pleo-
morphic megakaryocytes displaying an abnormal maturation and
mitosis (arrowhead). A-C, magnification «140; D-F x350. A-C and i
F, periodic acid—Schiff (PAS) stain; D and E, Gomori’s silver impregna-
tion. (From Thiele, J, et al: Primary myelofibrosis-osteosclerosis, agno- ¢ Idiopathic Myelofibrosis
genic myeloid metaplasia. AntiCa Res 9:430, 1989, with permission.) ¢ Other Chronic Myeloproliferative Disorders
Chronic myelogenous leukemia
Polycythemia vera
and trisomy 8 and abnormalities of chromosomes 1, 7, and 9. Essential thrombocythemia
Transitional myeloproliferative disorders
These abnormalities make up more than 80% of the chromoso-
mal changes seen in IMF. Patients with specific chromosomal Secondary to Infiltrative Disorders
abnormalities are less responsive to androgens.** As mentioned Metastatic carcinoma
previously, an activation mutation of JAK2 may be found in Hematologic malignancies involving bone marrow
Acute leukemia
20% to 40% of patients with IMF. Myelodysplastic syndromes (preleukemia)
Hairy-cell leukemia
Non-Hodgkin’s lymphoma
Differential Diagnosis Myeloma

IMF must be distinguished from other diseases within the Secondary to Nonmalignant Conditions
spectrum of the CMPDs, as well as differentiated from fibro- Granulomatous disorders Osteoporosis
sis secondary to infiltrative disorders (see Tables 18-1 and Sarcoidosis Vitamin D deficiency
Tuberculosis Systemic lupus
18-13). Clinically, the prefibrotic (p-CIMF) stage of CIMF Histoplasmosis erythematosis
mimics ET. Toxic exposure to chemicals Systemic sclerosis
However, CML is the diagnosis considered most fre- Hypo- and hyperparathyroidism
quently in the differential diagnosis of IMF. In chronic cases
404 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

protein, is easily differentiated from IMF. Myelodysplasia advanced disease with marrow failure and hypersplenism, as
(MDS) with marrow fibrosis can be difficult to differentiate they can be very sensitive to even smal] doses. However, poten-
from IMF. However the marked splenomegaly, extramedullary tial leukemogenicity is again a concern. Interferon alpha
hematopoiesis, and osteosclerosis are indications of IMF. has also been reported to afford a hematologic response in
The WHO has recently changed the designation of IMF to pri- hyperproliferative patients.” Etanercept, a tumor necrosis
mary myelofibrosis (PMP). The 2008 WHO diagnostic criteria factor-alpha (TNF-a) blocker, was shown in a pilot study to
for PME can be found in Table 18-14. palliate constitutional symptoms in IMF, which can be severe
and lead to cachexia (debilitating wasting away).’' Many
patients with severe anemia require transfusion support.
Treatment Approximately one-third respond to treatment with androgens
(fluoxymesterone and oxymetholone), which may be supple-
As with PV and ET, symptomatic treatment of IMF usually
mented initially with corticosteroids usually not to exceed
involves the use of cytotoxic agents to control thrombocytosis,
30 days. Chronic androgen use may be associated with fluid
leukocytosis, and symptomatic splenomegaly. Hydroxyurea
retention, abnormal liver function tests, and an increase in
on average doses of 500 mg twice a day, can aid in reducing
thromboembolic events. Men should be checked for occult
spleen size and control of thrombocytosis and leukocytosis.*”
prostate cancer before use, and women must be appraised of the
However, the benefit is usually temporary. Treatment with
virilizing effects. The use of erythropoietin has not generally
hydroxyurea may be associated with worsening of anemia,
been successful in patients with IMF, although some regressions
which in some instances, can be ameliorated by the concomitant
have been reported in those with low erythropoietin levels. Rare
use of erythropoietin. Early reports that hydroxyurea could
patients with IMF have developed autoimmune hemolytic
cause reversal of marrow fibrosis by suppressing megakary-
anemia which may respond to steroids and, occasionally,
ocyte production and releasing fibrinogenic cytokines, have not
cyclosporine. Thalidomide has also been reported to have
been confirmed in large clinical trials or clinical practice.*?
beneficial effects in some patients with regard to improvement
Doses of hydroxyurea must be carefully titrated in patients with
in anemia and thrombocytopenia, perhaps through its anti- TNF
activity. However, paradoxically, some patients have developed
increased myeloproliferation. Trials using the thalidomide
Beare The2008World derivative lenalidomide are in progress. This drug has been
shown to have immunomodulatory and antiangiogenic proper-
ties that are more potent than thalidomide and have an improved
side effect profile. The drug inhibits TNF-a, induces T-cell pro-
liferation and upregulates [L-10. Its antitumor effects may be
related to its antiangiogenic activity. Activity of the small mole-
cule receptor tyrosine kinase inhibitor SU5416 (Sugen) of vas-
Major Criteria
cular endothelium growth factor receptor 2 (VEGFR-2), c-KIT,
. Megakaryocyte proliferation and atypia’ accompanied by and FLT3, has been minimal in patients with MPD including
either reticulin and/or collagen fibrosis, or in the IMF.” The receptor tyrosine kinase inhibitor, imatinib mesylate
absence of reticulin fibrosis, the megakaryocyte changes
(Gleevec) has not been efficacious in improving anemia. Mini-
must be accompanied by increased marrow cellularity,
granulocytic proliferation and often decreased erythro- mal to moderate activity has been seen in reducing
poiesis (i.e., pre-fibrotic PMF). splenomegaly. In some trials, however, an increase in platelet
. Not meeting WHO criteria for CML, PV, MDS, or other count has been seen. Overall, the activity of imatinib in IMF has
myeloid neoplasm not been impressive. Trials are in progress using the farnesy]l
. Demonstration of JAK2V617F or other clonal marker or transferase inhibitor, Zarnestra.
no evidence of reactive marrow fibrosis
Splenectomy is an option for patients with symptoms
Minor Criteria related to splenomegaly, such as left upper quadrant discomfort,
transfusion-dependent anemia, refractory thrombocytopenia,
1. Leukoerythroblastosis
2. Increased serum LDH hypercatabolic symptoms, or portal hypertension.?* However,
3. Anemia the operative mortality can be significant, and postoperative
4. Palpable splenomegaly thrombocytosis can occur leading to postoperative thrombosis
* Diagnosis of primary prelonnea. (PMF) requires meeting all
and decreased survival.”> Blast transformation following
three major criteria and two minor criteria. splenectomy has been reported as well in a series of patients
* Small to large megakaryocytes with an aberrant nuclear/cytoplasmic with IMF.” Splenic irradiation can provide transient benefit and
ratio and hyperchromatic and irregularly folded nuclei and dense clus-
tering. LDH = lactate dehydrogenase; MDS = myelodysplastic syn-
symptomatic relief for patients who are poor surgical candi-
drome; PV = polycythemia vera dates.” However, it must be done carefully in low doses because
Source: Modified from Tefferi, A, and Vardiman, JW: Classification it can occasionally be associated with severe and irreversible
and diagnosis of myeloproliferative neoplasms: The 2008 World
pancytopenia. Allogeneic stem cell transplantation remains the
Health Organization criteria and point-of-care diagnostic algorithms.
Leukemia advance online publication 20 September 2007; doi: only curative treatment modality for IMF at present; unfortu-
10.1038/s}.leu.2404955 nately, it is often precluded by the patients advanced age, other
comorbidities, and lack of available donors.”
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 405

show the red cell abnormalities associated with fibrosis in the

Case Study 1 matrow and splenic hematopoiesis, and the bone marrow is
easily aspirated. The platelet count in ET is almost always
A 74-year-old man presented with complaints of increasing greatly elevated, often greater than 1000 X< 10°/L, and imma-
weakness, night sweats, shortness of breath, easy bruising, ture granulocytes are rarely prominent.
and a fever of 10 days’ duration. The patient had lost about Hydroxyurea was administered to this patient and contin-
10 Ib over a 6-month period and noted early satiety. On phys- ued over the course of | year. Blood transfusions were
ical examination he was pale, underweight, and had a fever required every 3 to 4 weeks to counteract the impending
of 103°F. Massive splenomegaly, moderate hepatomegaly, anemia. Androgen therapy (danazol) was also initiated. The
and pulmonary congestion were noted. Purpuric lesions were patient’s condition gradually worsened, and it was evident
present on the upper extremities. that the chemotherapy was only mildly effective in decreas-
Initial laboratory studies disclosed the following values: ing splenic size. Splenectomy was performed in an attempt
WBC count: 30.5 < 10°/L; RBC count: 2.9 x 10°/L; Hgb: 8.3 to ameliorate his anemia and relieve the constitutional
g/dL; Het: 25.8%; MCV: 89 fL; MCH: 28.6 pg; MCHC: symptoms of splenomegaly.
32.2%; platelet count: 650 x 10°/L. The differential count Four years after initial presentation, this patient devel-
revealed 45% segmented neutrophils, 6% band neutrophils, oped acute myelogenous leukemia and underwent a rapidly
20% lymphocytes, 9% monocytes, 3% eosinophils, 3% progressive fatal course. This case illustrates a typical
basophils, 4% metamyelocytes, 3% myelocytes, 2% promye- course of myelofibrosis. The median survival is approxi-
locytes, 5% ““dwarf’ megakaryocytes, and 15 nucleated red mately 5 years, and treatment often has little effect in pro-
blood cells (NRBCs) per 100 WBCs. Erythrocyte morphology longing the survival.
demonstrated anisocytosis and poikilocytosis, with prominent
teardrop red cells, polychromasia, and basophilic stippling. QUESTIONS
Platelet number was increased and platelet morphology was
abnormal, as evidenced by the presence of giant platelets and 1. Referring to the WBC differential, is a “left shift” evi-
hyper- and hypogranulated platelets, and) megakaryocytic dent in this case? Why or why not?
fragments. Other laboratory tests included reticulocyte count, 2. What is the corrected WBC count, given the number of
3.6%; LAP score, 132 (normal is 22 to 124); LDH, 3054 U/L; NRBCs?
uric acid, 13.2 mg/dL; stool occult blood, negative; 3. Give reasons why the bone marrow aspirate resulted in a
Ph chromosome, negative; and direct antiglobulin test “dry tap” in this case.
(DAT), negative. A bone marrow aspirate was attempted 4, What is the significance of the teardrop red cells?
several times but was unsuccessful because of a dry tap. The
bone marrow biopsy revealed trilineage hyperplasia with ANSWERS
many clumps of dysplastic atypical megakaryocytes. Exten-
1. Yes. Evidence of a “left shift” in the WBC differential is
sive fibrosis was also noted. Bacterial and fungal bone marrow
indicated by the presence of 4% metamyelocytes, 3%
cell cultures were performed, and all results were negative.
myelocytes, and 2% promyelocytes.
2. The corrected WBC is 26,522/uL. The following calcu-
COMMENT lation is used to derive the corrected WBC.
The diagnosis of idiopathic myelofibrosis with myeloid WBC/uL X 100 _ 30,500 x 100
metaplasia (IMF/MM) was based on the classic findings of Corrected WBCs/uL =
100 + NRBCs — 115
leukoerythroblastic anemia, marked splenomegaly, throm-
bocytosis with circulating megakaryocyte precursors, = 26,522/yL
teardrop erythrocytes, and increased fibrosis of the bone
3. A “dry tap” occurred during the bone marrow aspiration
marrow. Chronic myelogenous leukemia was excluded,
procedure done on this patient because of the extensive
because the majority of these patients have the Philadelphia
fibrosis that is a hallmark feature of myelofibrosis. The
chromosome, a low LAP, a higher proportion of myelocytes reticulin and collagen fibers that cause fibrosis form a
and myeloblasts, and a bone marrow showing predomi-
tight network, locking in the marrow contents. As a result,
nantly granulocytic hyperplasia. In addition, the red cell the bone marrow sinusoidal blood is not aspiratable.
morphological changes (in particular teardrop red cells) are 4. The teardrop cells are significant because they correlate
more prominent in myelofibrosis. with bone marrow fibrosis. The teardrop shape of the red
Of patients with known PV, 15% to 20% undergo a tran- cell occurs as the cell passes through the narrow, fibrotic
sition to IMF; however, because there is no prior history of sinusoids of the bone marrow and spleen.
PV, this disease can be ruled out. Granulomatous disorders
and acute leukemia can be excluded by careful scrutiny of
the bone marrow and the negative microbiologic cultures.
An increased platelet count and marked proliferation of
bizarre megakaryocytes would be highly unusual in acute
leukemia.
Another disorder considered in differential diagnosis is
essential thrombocythemia (ET). Again, these patients rarely
continued
406 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

and normal plasma volume. Further, evidence of trilineage

Case Study 2 involvement, leukocytosis, and thrombocytosis, in addition
to erythrocytosis and bone marrow panhyperplasia, strongly
A 58-year-old white man was admitted to the hospital with suggests.a diagnosis of PV. Abnormal elevation of the
pain and swelling of the left arm suggestive of throm- vitamin B,, and B,,-binding proteins, uric acid, and LAP are
bophlebitis. He had presented to his physician 2 days earlier consistent with a myeloproliferative process and are helpful
with complaints of pounding headaches, blurred vision, tin- in establishing a diagnosis of PV. The low serum iron and
nitus, and generalized pruritus, especially after bathing. The absence of iron stores indicate concomitant iron deficiency.
patient had been treated for gout for the past 2 months. Fam- In most patients this is attributed to occult gastrointestinal
ily history is unremarkable for any hematologic disorders. blood loss and defective platelet function.
The patient is a nonsmoker. This patient fulfills all the diagnostic criteria for PV set
On physical examination, the patient’s face appeared forth by the PVSG. Because this 58-year-old patient has an
flushed, and the retinal veins were engorged. Several elevated Hct and platelet count and is symptomatic (throm-
ecchymoses were apparent on the legs. The spleen tip was bophlebitis), both phlebotomy and myelosuppressive therapy
palpable three fingerbreadths below the costal margin were initiated. Colchicine and allopurinol were used to con-
(indicating moderate splenomegaly). No hepatomegaly or trol the gout experienced by this patient. Pruritus was a per-
lymphadenopathy was observed. sistent complaint despite the management of erythrocytosis
A complete blood count revealed the following values: by phlebotomy and hydroxyurea. Cyproheptadine was pre-
WBC count-.20/3 x" 107“ RBC count? 7.535% 10/2; scribed and found to be successful in controlling the pruritus.
Hgb: 18.2 g/dL; Het: 58.0%; MCV: 77 fL; MCH: 24.2 pg;
MCHC: 31.4%; platelet count: 710 * 10°/L. The differential QUESTIONS
count demonstrated 80% segmented neutrophils, 8% band
neutrophils, 9% lymphocytes, and 3% monocytes. Red cell 1. What laboratory parameters listed in this case indicate a
morphology was consistent with a microcytic, hypochromic microcytic, hypochromic process?
classification. 2. If an erythropoietin level were ordered on this patient,
Subsequent investigations were undertaken as part of the would the expected result be normal, increased, or
diagnostic workup of the erythrocytosis. Determination of decreased?
the red cell mass (utilizing the °'Cr dilution method) 3. What is the reason for the splenomegaly in this patient?
was performed and found to be 41 mL/kg (normal male is
36 mL/kg or less). The plasma volume was 40 mL/kg. Arte- ANSWERS
rial oxygen saturation was 94%. The serum iron was
1. The following red cell parameters indicate a microcytic,
30 ug/dL (normal is 50 to 170) and total iron binding capac-
hypochromic process in this patient: MCV, 77 fL; MHC,
ity (TIBC), 460 ug/dL (normal is 250 to 450). Serum vita-
24.2 pg; MCHC 31.4%. Iron deficiency causes this
min B,, was 925 pg/mL (normal is 205 to 876), and vitamin
process and results from the tremendous increase of iron
B,,-binding capacity was 2600 pg/mL (normal is 1000 to
required for the excessive erythropoiesis seen in PV, and
1022). The LAP score was 198, and the uric acid determina-
is often complicated by blood loss.
tion was 10.3 mg/dL. A bone marrow examination revealed
2. The expected erythropoietin level in this patient would be
95% cellularity, with panhyperplasia and many large
decreased, indicating autonomous production of red cells
megakaryocytes. Iron stores were absent, and the reticulin
by the bone marrow. This is a particularly cost-effective
content was slightly increased.
diagnostic tool for differentiating PV from other secondary
and relative causes of erythrocytosis. Greater than 95% of
COMMENT patients with PV exhibit the JAK2 mutation (V617F),
Several findings in the history and physical examination sug- making this a very useful diagnostic test as well.
gest a presumptive diagnosis of PV. The nonspecific symp- 3. The characteristic mild to moderate splenomegaly seen
toms of headache and blurred vision are a result of cerebral in individuals with PV is attributed to extramedullary
circulatory disturbances caused by hyperviscosity. Throm- hematopoiesis. Splenic enlargement is seen in about
botic episodes, such as the phlebitis recorded in this patient, 75% of patients and is an important finding.
are vascular manifestations resulting from the thrombocytosis
in conjunction with the hyperviscosity and increased blood
volume. The facial plethora and engorged retinal veins are
findings associated with conjunctival and mucosal blood ves-
sel congestion. Generalized pruritus occurs in 30% of patients
with PV and is related to hyperhistaminemia. The lack of car-
diac or respiratory abnormalities and the presence of normal
arterial saturation are helpful in ruling out secondary erythro-
cytosis. The splenomegaly noted is a frequent finding in
myeloproliferative disorders.
The most important clinical findings supportive of PV are
the elevation of the Hgb and Het, increased red cell mass,
continued
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 407

exclude the other chronic MPDs, the PVSG guidelines should

Case Study 3 be followed. To distinguish a patient with ET from an iron-
deficient PV patient, serum iron, TIBC, ferritin, and bone mar-
This 35-year-old white woman initially presented with row iron stains are usually sufficient. A trial of oral iron is
thrombocytosis (platelet count, 1200 x 10°/L) discovered rarely required. In patients with anemia, splenomegaly, throm-
bocytosis, and leukocytosis, the presence of the Ph chromo-
on routine physical examination. Her WBC and Hgb values
were normal. The history was unremarkable except for some is unequivocal evidence of CML.
occasional epistaxis and minor bruising. She was advised to The outlook for long-term survival in ET is encouraging
have a routine follow-up examination and CBC every as long as appropriate measures are taken to minimize
3 months and, despite a continually elevated platelet count, thrombohemorrhagic complications. Many patients can tol-
remained asymptomatic for 3 years. At that time she was erate markedly increased platelet counts for years without
seen by her physician with complaints of dizziness, visual any complications. The introduction of plateletpheresis has
disturbances, and erythromelalgia. She had also had recent allowed dramatic response in life-threatening or urgent sur-
dental surgery and experienced a major perioperative bleed- gical situations. In addition, hydroxyurea has proved to be
ing episode. Mild splenomegaly was noted. Her platelet an effective chemotherapeutic agent.
count was 2500 xX 10°/L. Other laboratory values were as
follows: WBC count: 18.5 < 10°/L; Het: 28.5%; prolonged QUESTIONS
bleeding time; reduced platelet adhesion; and defective
1. Why did this patient experience a major perioperative
platelet aggregation with epinephrine. Bone marrow biopsy
bleeding episode when her platelet count was 2500
demonstrated megakaryocytic hyperplasia with massive
10°/L?
platelet clumping. Erythroid and myeloid hyperplasia, as
2. Erythromelalgia can progress into what clinical manifes-
well as a mild increase in reticulin content, were also
tation?
observed.
3. What is the reason for the megakaryocytic hyperplasia
Plateletpheresis was performed to rapidly reduce the
seen in the bone marrow biopsy?
marked thrombocytosis. The patient was treated with the
myelosuppressive agent hydroxyurea in dosages varying
from | g/day to 500 mg five times per week, depending on
ANSWERS
the platelet counts. Fhe bleeding and vaso-occlusive symp- 1. Although the platelet number is elevated in ET, the
toms were resolved, and coagulation abnormalities were platelets function abnormally. Severe thrombocythemia
corrected. Close follow-up is necessary for this patient to (greater than 1500 x 10°/L) can be associated with defi-
ensure a continued beneficial clinical and _ laboratory ciency of the large von Willebrand multimers and
response. resulting hemorrhage. Besides abnormal platelets func-
tion, hemorrhage in individuals with ET has been attrib-
COMMENT uted to thrombosis with infarction, ulceration of the
infarction, and subsequent bleeding.
This case highlights the common findings in essential
2. The erythromelalgia of the toes, feet, and fingers seen in
thrombocythemia (ET); namely, marked increased platelet
ET patients can progress to cyanosis and/or necrosis of
counts, thrombohemorrhagic events, splenomegaly, and the extremities. This toxic effect is caused by the
bone marrow megakaryocytic hyperplasia. Although this is metabolites of platelet arachidonic acid.
primarily a disease of upper-middle-age (50 to 70 years) a 3. The megakaryocytic hyperplasia of ET results from a
second population of younger, predominantly female neoplastic clonal disorder of multipotential stem cells,
patients exists. Two-thirds of patients are asymptomatic, as which gives rise to excessive numbers of circulating
was this patient initially. With the advent of automated cell platelets. This disease can be contrasted with the various
counters that routinely generate platelet counts, asympto- disorders that can be associated with a reactive, or non-
matic patients are being discovered more frequently. neoplastic thrombocytosis.
The erythromelalgia noted in this patient represents one
of the most characteristic vaso-occlusive manifestations.
Prolonged bleeding after trauma or surgery is a common
finding related to platelet dysfunction.
In an asymptomatic young patient with platelet counts
below 1000 to 1500 X 10°/L, it is advisable to withhold
myelosuppressive therapy, because these patients do well for
many years untreated. When a patient requiring surgery pre-
sents with markedly increased platelet count and hemorrhagic
complications, plateletpheresis will lower the platelet count
dramatically. In addition, myelosuppression is necessary to
control the hyperproliferative process.
Causes of reactive thrombocytosis, such as iron deficiency
anemia, malignancy, inflammatory disorders, splenectomy,
and so on, are generally easy to exclude based on the clinical
and hematologic features of the individual patient. To reliably
continued
408 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

Oi estiomns i
t. What is the origin of MPDs? 9. When is myelosuppression advocated in patients with PV?
a. Fibroid infiltration of major organs a. If the patient is less than 20 years old —
b. Neoplastic transformation of multipotential stem cells b. When thrombosis-associated risk factors are present
c. Widespread deterioration of cellular function c. If the patient shows signs of glossitis
d. Splenic sequestration of normal blood cells d. If the patient has failed iron therapy
. Which of the following is not a characteristic of a 10. Which features help to distinguish secondary erythrocy-
chronic MPD? tosis and relative erythrocytosis from PV?
a. Extramedullary hematopoiesis a. Absence of splenomegaly; normal leukocyte, platelet,
b. Possible termination into acute leukemia and LAP levels
c. Cytogenetic abnormalities b. Presence of splenomegaly; increased leukocyte,
d. Hypoplasia of bone marrow platelet, and LAP levels
. The Philadelphia chromosome involves: c. Presence of hepatomegaly; decreased leukocyte,
a. (9322) platelet, and LAP levels
b. The BCR and c-ABL genes d. Presence of both splenomegaly and hepatomegaly;
c. A 210-kD protein possessing increased tyrosine kinase increased leukocytes and platelets; decreased LAP score
activity ih What is the safest and least expensive treatment for
d. All of the above patients with polycythemia vera?
. What is the predominant abnormal erythrocyte morphol- a. High altitude
ogy ‘associated with idiopathic myelofibrosis? b. Decrease of iron levels
a. Schistocytes c. Therapeutic phlebotomy
b. Ovalocytes d. Decrease of erythropoietin levels
c. Teardrop cells . What condition is defined by a platelet count greater
d. Target cells than 600 < 10°/L, megakaryocytic hyperplasia, absence
. Which features of chronic myelogenous leukemia are the of Ph chromosome, and hemoglobin of 13 g/dL or more
most important characteristics that distinguish it from (or normal red cell mass)?
myelofibrosis? a. Essential thrombocythemia
a. Presence of increased platelets and fibroblasts b. May—Hegglin anomaly
b. Decreased erythrocytes with abnormal morphology c. Acute myelogenous leukemia
c. Increased leukocytes with hypercellular bone marrow d. Polycythemia vera
d. Low LAP score and presence of Ph chromosome . The thrombosis seen in patients with essential thrombo-
. Which of the following factors does not cause fibroblast cythemia is a result of which of the following?
proliferation? a. Protein C deficiency
a. CSE b. Marked fibroblast proliferation
b. TGF-B c. Intravascular clumping of sludged hyperaggregable
c. bFGF platelets
d. PDGF d. Splenic sequestration of platelets

. What are the laboratory findings in PV? . What condition is not characteristically associated with
a. Decreased hematocrit; increased RBCs and granulo- reactive thrombocytosis?
cytes; decreased platelets a. Acute hemorrhage
b. Increased hematocrit; increased RBCs, granulocytes, b. Aplastic anemia
and platelets c. Chronic inflammatory disorders
c. Normal hematocrit; normal RBCs; increased granulo- d. Iron-deficiency anemia
cytes and platelets 1 Which of the following chronic MPDs is associated with
d. Increased hematocrit; increased RBCs; decreased the best prognosis?
granulocytes and platelets a. CML
. What is the expected erythropoietin value in PV? b. IMF
a. Normal Cyn
b. Increased oR Sih
c. Decreased
See answers at the back of this book.
— Cr :
 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis 409

-~ SUMMARY
@ Familial polycythemia is the result of mutations in the
erythropoietin receptor (EPo-R) or mutations in the oxy-
gen sensing pathway regulating erythropoietin production
MYELOPROLIFERATIVE DISEASE (Chuvash polycythemia).
w Myeloproliferative disorders (MPDs) arise from a malig- ESSENTIAL THROMBOCYTOSIS
nant transformation of a single multipotential stem cell
m The platelet count is markedly elevated in essential
that is committed to differentiation of granulocytes,
thrombocythemia (ET), often to more than 1000 10°/L.
monocytes, erythrocytes, and platelets.
m Hemorrhage and thrombosis caused by dysfunctional
m= MPDs are grouped together because of shared characteris-
platelets, splenomegaly, erythromelalgia, and neurologic
tics, the most important being panhyperplasia of the bone
manifestations are clinical features of ET.
marrow, extramedullary hematopoiesis, bone marrow fibro-
sis, and predilection for leukemic transformation. # ET must be differentiated from the many causes of reac-
tive thrombocytosis.
POLYCYTHEMIA VERA
gw Symptomatic patients or high-risk patients should receive
mw Elevation of the hematocrit (above 58% in males and
cytoreductive therapy with hydroxyurea.
above 52% in females) is the most important hallmark of
polycythemia vera (PV). g All patients with platelets < 1,000,000/uL should receive
aspirin if they have no antecedent history of bleeding.
w Elevated red cell mass, splenomegaly, decreased erythro-
poietin, normal arterial oxygen saturation, and increased IDIOPATHIC MYELOFIBROSIS
leukocyte alkaline phosphatase (LAP) are other important w Important features of idiopathic myelofibrosis (IMF) are
features of PV. anemia with teardrop poikilocytosis, leukoerythroblastic
w 90% of patients with PV have a mutation (V617F) in the blood picture, marked bone marrow fibrosis, splenomegaly,
JAK2 gene resulting in activation of downstream path- and variable but often elevated platelet counts.
ways (STATS). a Cytokines secreted by megakaryocytes and platelets stim-
w Approximately 15-20% of patients with PV will enter the ulate bone marrow fibroblastic proliferation in IMF.
spent phase with marrow fibrosis, worsening splenomegaly w Giant, bizarre platelets and micromegakaryocytes (dwarf
and pancytopenia. megakaryocytes) may be seen in the peripheral blood in
w Treatment of PV involves phlebotomy or use of cytotoxic IMF.
myelosuppressive agents, or a combination of both. m Progressive disease is manifested by extramedullary
SECONDARY POLYCYTHEMIA hematopoiesis with increasing splenomegaly, hepatomegaly,
weight loss, cachexia, portal hypertension, and pan-
m Increased secretion of erythropoietin has been implicated
cytopenia.
as the stimulus responsible for all cases of secondary ery-
throcytosis. g@ Hydroxyurea may be helpful in controlling splenomegaly,
leukocytosis, and thrombocytosis but does not alter the
m The arterial oxygen saturation is often decreased in
natural history of the disease.
patients with secondary erythrocytosis.
w Splenic radiotherapy and splenectomy may be helpful in
# Relative erythrocytosis occurs when there is depletion in
carefully selected patients.
circulating plasma volume (causing increased hematocrit
but normal red cell mass), and it is often seen in patients w Allogeneic bone marrow transplant remains the only cura-
with dehydration. tive treatment.
410 Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis

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 Chapter

| q Myelodysplastic
Syndromes
Giovanni D’Angelo, FCMLS
Luigina Mollica, MD, FRCP(C), PhD
Josée Hébert, MD, FRCP(C)
Lambert Busque, MD, FRCP(C)

Introduction OBJECTIVES
Epidemiology—Etiology— At the end of this chapter, the learner should be able to:
Pathogenesis
MDS: Clonal Proliferative 1. Provide an overview of the clonal nature and stem cell origin of myelodysplastic
Diseases syndromes (MDS).
Ineffective Hematopoiesis . Master the criteria of the French-American—British and World Health Organization
iw)
Genetic Anomalies (WHO) classifications.
Biologic Characteristics of
Disease Progression 3. Recognize the morphologic features of MDS on examination of blood and bone
marrow smears.
Morphological
Characteristics of Blood 4. Differentiate the different subtypes of MDS based on their characteristic laboratory
and Bone Marrow features.
Definitions of Specific
5. Distinguish MDS from acute myeloid leukemia on examination of blood and bone
Morphological
marrow smears.
Characteristics
Lineage Dysplasias 6. Know the ringed sideroblast values in MDS.
Classification of MDS 7. Differentiate between primary and secondary MDS.
Subtypes
8. Know the International Prognostic Scoring System (IPSS) and the value of Wilms
FAB Classification Tumor gene (W717) expression.
Refractory Anemia
Refractory Anemia with 9. Recognize a myelodysplastic/myeloproliferative disorder.
Ringed Sideroblasts 10. Describe the therapeutic regimen that is most likely to achieve long-term survival.
Refractory Anemia with
Excess Blasts
Refractory Anemia with
Excess Blasts in
Transformation
Chronic Myelomonocytic
Leukemia
WHO Classification
Laboratory Features
Cytochemistry
Bone Marrow Histology
Cytogenetics and Molecular
Abnormalities
Immunology

412
 Chapter 19 Myelodysplastic Syndromes 413

Evaluation of Progenitor Cell

Growth in Semisolid
Media
Cellular Dysfunction
Secondary Myelodysplastic
Syndromes
Myelodysplastic Syndromes
in Children
Clinical Features
Evolution and Prognosis
Diagnostic Problems in MDS
MDS with Hypoplastic
Marrow
MDS with Severe
Myelofibrosis
MDS with Thrombocytosis -
MDS with Features of
Chronic Myelogenous
Leukemia
MDS versus Acute Erythroid
Leukemia
Treatment
Supportive Care and
Hematopoiesis-Improving
Therapies
Therapies Oriented Toward
Improving Survival
Case Study

Introduction at correcting some of the defective pathways involved in the

pathogenesis of MDS may offer new hope for certain patients.
Myelodysplastic syndromes (MDS) are a heterogeneous This chapter summarizes the current knowledge of the patho-
group of clonal hematological malignancies characterized by genesis, Clinical features, and treatment of MDS, emphasizing
peripheral blood cytopenias, dysplastic blood cells, and a the role of the laboratory investigation in the diagnosis of this
propensity to transform into acute leukemia. MDS were first important hematological disorder.
described in the 1930s as preleukemic anemia. In 1982, the
French—American—British (FAB) Morphology Cooperative Epidemiology—Etiology—Pathogenesis
Group classified these syndromes into five distinct entities
according to specific morphological criteria.'* The World MDS affect predominantly the elderly, with a median age at
Health Organization (WHO) recently modified this classifica- onset of 70 years old and an incidence close to 45 per 10° in that
tion, incorporating biologic and cytogenetic features that help age group.* MDS occur at a lower frequency in younger adults
better categorize these disorders.* The FAB and WHO classi- and in children. Men are slightly more frequently affected than
fications are presented in detail later in this chapter. are women, with the exception of the Sq— syndrome.
The clinical outcome of patients with MDS is variable, Little is known about the cause of de novo MDS. Several
ranging from relatively benign conditions necessitating no risk factors have been associated with the development of
therapy to rapidly progressive diseases associated with a dis- MDS, such as exposure to environmental and occupational
mal vital prognosis. Although MDS may show variable clini- products, including ammonia, petrochemicals, and low-dose
cal severity, they are almost always fatal. There is no curative irradiation.° There is a recent report of a sideroblastic anemia
treatment for MDS other than allogeneic stem cell transplan- (a subtype of MDS) after bariatric surgery which has been
tation, a therapeutic modality available to a minority of corrected after replacement of copper and pyridoxine.° How-
patients. The mainstay of treatment has historically been ever, the relative risk after exposure to most environmental
supportive care, but novel pharmacologic molecules aimed and occupational products is low and, in some cases, the
414. Chapter 19 Myelodysplastic Syndromes

associations have not been clearly established. In contrast, Pluripotent stem

exposure to benzene has been convincingly associated with
the development of MDS. Benzene, which is processed in Cell most
vivo to hydroquinone, induces damage to hematopoietic probably v
progenitor cells that may lead to MDS.’ A recent case control mutated CFU-GEMM

study found smoking and a family history of cancer to “= Lymphoid

progenitor
be associated with an increased risk of MDS.* MDS can
also occur as a late complication in patients treated with
chemotherapy or radiation therapy; in this case, it is referred BFU-E CFU-Meg CFU-GM
to as secondary MDS (sMDS).
The precise mechanisms that lead to the development of
MDS have not yet been fully elucidated. It is generally
accepted that the pathogenesis is complex and involves the
Red cells
!
Platelets
!
Granulocytes Lymphocytes
accumulation of different genetic alterations in a hematopoi- Monocytes
etic progenitor cell. Recent advances in molecular biology
have allowed the characterization of some of the fundamental Figure 19-1 m@ Schematic representation of marrow stem cell hierar-
chy showing the stem cell theoretically involved in the pathogenesis
defects present in MDS.
of myelodysplastic syndromes (MDS), according to cytogenetic and
fluorescence in situ hybridization (FISH) analyses (see text). CFU-
GEMM = colony-forming unit-granulocyte-erythrocyte—
MDS: Clonal Proliferative Diseases megakaryocyte-macrophage; BFU-E = burst-forming unit-erythroid;
CFU-Meg = colony-forming unit-megakaryocyte; CFU-GM =
Monoclonal (clonal) derivation of cells is the hallmark of can- colony-forming unit-granulocyte-macrophage.
cer. A sqmatic mutation giving rise to a growth advantage of the
mutated cell leads to a clinically significant disorder. The clonal
nature of MDS has been firmly established by the documenta- lymphoid phenotype be demonstrated.'* Cytogenetic analysis
tion of clonal cytogenetic anomalies in a significant proportion of marrow metaphases and fluorescence in situ hybridization
of patients with MDS.’ X-inactivation assays have also con- (FISH) performed on interphase nuclei have consistently
firmed the monoclonal nature of the MDS.'° These assays are found cytogenetic anomalies in myeloid cells, but not in lym-
based on the principle of X inactivation in females, whereby phoid cells.'*'° The controversy has been fueled by the obser-
early in embryogenesis one of the two X chromosomes is ran- vation that a significant proportion (30% to 40%) of female
domly inactivated by methylation in each cell. Each subsequent MDS patients had results compatible with a clonal derivation
daughter cell will inactivate the same X chromosome as its pre- of cells in all lineages, including T and B lymphocytes, by the
cursor. Thus, most females are balanced mosaics at the cellular X-inactivation assay.'° This was interpreted as evidence that,
level, with half their cells in any given tissue carrying the pater- in some cases, MDS originated from a stem cell with the abil-
nal X in the active state and the other half with the maternal X ity to differentiate into all cell lineages. These data have been
in the active state. The documentation that blood cells of female subjected to reanalysis in the light of recent documentation that
patients with MDS have a preponderance of the same X chro- close to 40% of normal women older than 60 years of age
mosome in the active state instead of the predicted random demonstrate an abnormal pattern of X-inactivation of blood
X-inactivation pattern confirms the clonal derivation of the cells that mimics clonal derivation of cells, known as
disease. This suggests that a somatic mutation conferring a “acquired skewing.”'’ As patients with MDS are usually older
selective growth advantage has occurred in a progenitor cell than 60 years of age, it is possible that the skewing found in T
with repopulating ability." and B lymphocytes reflects the normal change in clonality pat-
terns with age and may not be interpreted as proof positive of
CELL OF ORIGIN OF THE NEOPLASTIC CLONE primitive hematopoietic stem cell involvement in these patients.
The precise cellular origin of the neoplastic clone is central to In summary, most studies aimed at determining the cell of
the understanding of the pathogenesis of MDS. In which origin of MDS point to a defect arising in a repopulating stem
hematopoietic progenitor cell does the genetic defect causing cell already committed to myeloid differentiation. Whether
MDS occur? This question has given rise to controversy in the molecular events not detectable by the present technology are
past several years. Although some authors argue that the cel- present in the omnipotent stem cell remains to be shown.
lular origin of MDS is an omnipotent stem cell with the abil-
ity to differentiate into all cell lineages, including lymphoid
Ineffective Hematopoiesis
cells, others favor a pluripotent stem cell already committed
to myeloid differentiation (Fig. 19-1). The phenomenon by which each cell is able to trigger its own
This question can be addressed first by looking at the death is called apoptosis or programmed cell death. Apopto-
nature of the blast cell population in patients undergoing Sis is essential to tissue homeostasis. An abnormal decrease in
leukemic transformation during the course of their disease. the rate of natural apoptosis will lead to cell accumulation,
In the vast majority of cases, blasts are of myeloid or whereas an increase in the rate of apoptosis will lead to
myelomonocytic phenotype.'* Only extremely rarely can a decreased cellular output.
 Chapter 19 Myelodysplastic Syndromes 415

Apoptosis is characterized by three sequential phases: factor, GM-CSF) are diminished in MDS. Therefore, the
(1) initiation, (2) commitment, and (3) execution.'* A variety balance between growth-stimulatory and growth-inhibitory
of factors can initiate apoptosis, such as chemotherapy, radio- cytokines may favor apoptosis in MDS. As Fas antigen and
therapy, corticosteroids, and growth factor deprivation. It may Fas-ligand are also abnormally elevated on the surface of MDS
also occur as a direct effect of cytotoxic T-cell activity. Apop- cells, the Fas apoptotic pathway may also be important in the
tosis can be initiated by inhibitory cytokines such as tumor pathogenesis of MDS.
necrosis factor-alpha (TNF-a), transforming growth factor- Although increased apoptosis is a key biologic feature of
beta (TGF-B), and by the action of Fas-ligand on its receptor MDS, it is still not clear if it is central to the pathogenesis of this
FAS/CD95 (Fig. 19-2). Proteins belonging to the Bcl-2 family disease or merely an indication of ineffective hematopoiesis
are involved in the regulation of the commitment phase. Some caused by other genetic anomalies of the involved clone. The
of these are pro-apoptotic (Bax, Bak, Bad, Bcl-XS, Bik), investigation of the cause and mechanisms of apoptosis in MDS
whereas others are anti-apoptotic (Bel-2, Bcl-XL, Mcl-1). If may provide insight into its pathogenesis and lead to novel
the balance of Bcl-2 family protein activities favors apopto- therapeutic approaches.
sis, a family of cysteine proteases called caspases will be
activated, leading to cell death after a series of molecular
cleavages affecting the cytoskeleton, cytosol, and nuclear Genetic Anomalies
proteins. This will lead to a pathognomonic phenotype char- The most common genetic anomaly associated with MDS is
acterized by chromatin condensation, oligodimerization of the loss of genetic material. The cytogenetic analysis of
DNA, and nuclear disintegration. © patients with MDS is characterized mainly by chromosomal
Yoshida first proposed that the ineffective hematopoiesis deletions, involving part of or a whole chromosome (see the
seen in MDS was caused by an abnormally high rate of later discussion of cytogenetics). This is in sharp contrast to
intramedullary apoptosis.'” This hypothesis was tested by sev- the acute leukemias, where chromosomal translocations pre-
eral groups using different methods (such as in situ end labeling dominate. The acute leukemias appear to follow the model of
(ISEL), nick-end labeling (TUNEL), and flow cytometry), all of oncogene activation, resulting in the transformation of the
which confirmed an elevated apoptotic rate in MDS patients cell of origin, whereas the deletional pattern found in MDS
compared to normal controls. Apoptosis seems to affect CD34+ favors a tumor suppressor gene model. In this model, the lack
progenitors as well as maturing cells. The degree of apoptosis is of both copies (one from each parental chromosome) is nec-
more pronounced in the early phases of the disease, compared essary to lead to carcinogenesis. One allele may be mutated
to more advanced or proliferative stages. Several lines of and the other deleted.*' The best described examples of this
evidence suggest a major role for inhibitory cytokines in the model are found in retinoblastoma and the Li-Fraumeni syn-
initiation of apoptosis in MDS. TNF-a is elevated in the bone drome (p53).” It is also possible that haploinsufficiency (the
marrow of MDS patients, and correlates with the degree of presence of only one active allele of a gene) alone may be suf-
apoptosis.”? TGF-B has also been shown to be elevated in MDS, ficient to cause a malignant hematological process in some
and may also play an important initiating role.*° Certain growth- cases. Hereditary haploinsufficiency for the gene RUNX/
stimulatory cytokines endowed with anti-apoptotic activity (AMLI) has been recently found to be associated with the devel-
(such as granulocyte/macrophage-monocyte colony-stimulating opment of acute leukemia in a family.* These data suggest
that MDS patients are missing key genes essential for maintain-
Fas and Fas ligand up-regulation ing normal hematopoiesis and that, with time, this leads to the
T lymphocyte
accumulation of more severe and numerous genetic anomalies
associated with disease progression and deterioration of the
hematological phenotype. To date, no single gene has been
found to be commonly deleted in MDS patients, but the identi-
fication of such genes is the focus of research of several groups.

Biologic Characteristics of Disease

Macrophage
Progression
The MDS phenotype changes as the disease progresses toward
leukemia. The number of blast cells increases in the marrow,
peripheral blood leukocytosis may replace leukopenia, and
hepatosplenomegaly can occur. MDS then evolves into a pro-
Apoptotic progenitor cell
liferative peripheral blood disorder. Acceleration of MDS has
Figure 19-2 ® Apoptosis of progenitor cells in bone marrow of been associated with a number of biologic phenomena, such as
patient with MDS. There is an increased production of inhibitory decrease in medullary apoptosis, decrease in Fas expression,
cytokines such as TNF-a and TGF-8 in MDS patients. This may con- and mutations at the p53 (tumor suppressor) and RAS (proto-
tribute to upregulation of Fas and co-expression of Fas-ligand (FL). oncogene) loci. The p/5ink4b gene, involved in the control
This is one of the several pathways that could activate programmed
of the cell cycle, becomes more heavily methylated and, there-
cell death. Cysteine proteases (caspases) will be activated and will
execute the cell. fore less active to block abnormal cells in G phase, facilitating
416 Chapter 19 Myelodysplastic Syndromes

the proliferation of abnormal cells.*? MDS is thus one of the best specific and nonspecific esterases may be used in the investiga-
examples of the multistep pathogenesis of neoplasia for hema- tion of MDS. The review of these materials allows the distinc-
tological cancers. The accumulation of different genetic alter- tion between MDS subtypes based on established criteria. MDS
ations changes the phenotype of the cell from normal to clonal are most often suspected in patients presenting with anemia
and dysplastic, and further changes confer a full-blown malig- (usually macrocytic or normochromic), with or without addi-
nant phenotype, leading to the development of acute leukemia. tional cytopenias. A vast array of laboratory tools is available
to help the physician to establish the diagnosis of each type
Morphological Characteristics of Blood ofLMDS 2:
and Bone Marrow BLASTS
Definitions of Specific Morphological Some types of myelodysplastic syndromes are diagnosed
Characteristics according to the number of blasts in the bone marrow at
initial diagnosis. There are two types of blast, according to
At the laboratory level, the diagnosis of a MDS is made by the the FAB classification: type I refers to a myeloblast of
careful morphological study of blood and bone marrow smears variable size, without any azurophilic granules or Auer rods
using the Wright-Giemsa stain, and of the bone marrow biopsy (Fig. 19-3); type II refers to a myeloblast that is slightly
specimen using hematoxylin and eosin (H & E) and reticulin larger and contains few azurophilic granules (1 to 20)
stains (Table 19-1). The iron content of both marrow aspirate (Fig. 19-4); Goasguen and associates*® have proposed a type
and biopsy specimens is revealed by the use of Prussian blue II myeloblast, containing more than 20 azurophilic granules
(Perl’s stain).*® Other special stains, such as myeloperoxidase with a basophilic cytoplasm and absence of the Golgi zone
(MPO), Sudan black B (SBB), periodic acid—Schiff (PAS), and similar type I and II myeloblasts.

‘apie 19-1 Hallmark of Morphologic Findings in MDS —

eed hb ole ahha Sees SEN eal eae

Erythroid/Dyserythropoiesis

Peripheral Blood Bone Marrow Aspirate

Macrocytosis Megaloblastic changes
Anisopoikilocytosis Erythroblastic hyperplasia
Oval macrocytes Occasionally erythroblastopenia
Dimorphic population Multinuclearity
Dacryocytes Nuclear/cytoplasmic asynchrony
Acanthocytes Karyorrhexis
Basophilic stippling Internuclear bridging
Pappenheimer bodies Nuclear fragments and budding
Howell—Jolly bodies Pseudovacuoles
Decreased polychromatophilic cells Ringed sideroblasts (some types)
Nucleated red blood cells PAS positive erythroblasts (some cases)

Myeloid/Dysgranulopoiesis

Peripheral Blood Bone Marrow Aspirate

Atypical neutrophil segmentation and hypercondensed Increased cellularity
chromatin
Pseudo—Pelger—Huet Myeloblasts, immature granulocytes
Occasional hypersegmentation Occasional increase of monocytes and basophils
Hypogranularity and/or large granules Nuclear/cytoplasm maturation
Immature granulocytes and monocytes Asynchrony and maturation arrest
Occasionally myeloblasts Hypogranularity and/or large granules
Dohle bodies Auer rods

Thrombopoiesis/Dysmegakaryopoiesis

Peripheral Blood Bone Marrow Aspirate

Large or giant platelets Normal or increased megakaryocytes occasionally decreased
Hypogranular platelets Micromegakaryocytes mononuclear
Abnormal granules and vacuoles Hypo and monolobulation
Micromegakaryocytes and megakaryocyte fragments Multiple separated nuclei “Botryoids or Pawn Ball”
hypogranularity

Bone Marrow Biopsy

Cellularity and evaluation of dysplasia of cell lineages
and abnormal localization of immature precursors (ALIP)
Cluster of megakaryocytes and degree of myelofibrosis
 Chapter 19 Myelodysplastic Syndromes 417

Figure 19-3 m RAEB-t (bone marrow): blast cells (A), immature

Figure 19-5 m 5q— syndrome (peripheral blood): anisomacrocytosis
granulocytes with some degree of hypogranularity and vacuolization
and thrombocytosis.
(B), and Pelgeroid hypogranular neutrophil (©.

SIDEROBLASTS
A type I sideroblast refers to a normal sideroblast containing
one to four cytoplasmic iron-containing granules, normally
accounting for 15% to 50% of bone marrow erythroblasts.
A type II sideroblast is considered abnormal, harboring 5 to
10 granules, scattered throughout the cytoplasm. Type III is
synonymous with a ringed sideroblast, having more than
10 granules that cover at least one-third of the nuclear rim or
forming a complete ring around the nucleus."

Lineage Dysplasias
DYSERYTHROPOIESIS
PERIPHERAL BLOOD Anemia is present in at least 90% of
cases and is most often macrocytic or normocytic with a
decreased reticulocyte number. Erythrocyte morphological on i ie 4
characteristics that may be encountered are macrocytosis, Figure 19-6 m RAEB (peripheral blood): anisomacrocytosis, neu-
anisopoikilocytosis, basophilic stippling, a dual red blood cell trophil and macrothrombocyte (arrow).
population (normochromic, hypochromic), Pappenheimer bod-
ies, dacryocytes (teardrop cells), fragmented cells, elliptocytes,
Howell—Jolly bodies, and acanthocytes (Figs. 19-5 to 19-8).

Figure 19-7 @ RARS (peripheral blood): dimorphic macrocytosis.

igure 19-4 wi RAEB-t (bone marrow): two blasts with azurophilic Note the small lymphocyte in the center which can be used as a
rods, blast with an Auer rod (arrow). micrometer.
418 — Chapter 19 Myelodysplastic Syndromes

ee

Figure 19-8 mAn erythrocyte with basophilic stippling (A), one Figure 19-10 @An erythroblast with basophilic stippling (A), and
with a Howell-Jolly body (B), and two erythroblasts connected by two erythroblasts connected through internuclear bridging (bone
internuclear bridging (peripheral blood) (©). marrow) (8B).

BONE MARROW Erythroblasts may harbor megaloblastoid

changes, with dense or fine chromatin with asynchronous
cytoplasm maturation, internuclear bridging, and broad-based
nuclear budding (Figs. 19-9 to 19-12). Cytoplasmic abnor-
malities may include intense basophilia, Howell—Jolly bodies,
and ghost cells. With iron stains, abnormal sideroblasts of
type I and type III may be found (Figs. 19-13 and 19-14).

DYSGRANULOCYTOPOIESIS
PERIPHERAL BLOOD Neutropenia is found in almost 60% of
patients, although neutrophilia may be encountered occasion-
ally. Granulocytes show variable degrees of hyposegmentation
with bilobulation (pseudo-Pelger—Huet anomaly) or monolobu-
lation (pseudo-Stodtmeister anomaly),** abnormal chromatin,
or, rarely, atypical hypersegmentation (Figs. 19-15 to 19-17). In
the cytoplasm, one may find variable degrees of hypogranula-
tion, persistent basophilic zones (pseudo-Dohle bodies), or,
more rarely, hypergranulation with larger granules than usual
(Fig. 19-18). Combined nuclear and cytoplasmic dysplasia is
detected in more than 90% of cases.

Figure 19-]2 @ RAEB (bone marrow). Note the pronormoblast

Figure 19-9 @ RAEB-t (bone marrow): dysplastic multinucleated (arrow) and the dyserythropoiesis and dysgranulopoiesis with nuclear
erythroblasts with asynchronous maturation. hyposegmentation and hypogranulation.
 Chapter 19 Myelodysplastic Syndromes 419

Inna

a i

igure 19-16 @ Bilobulated and monolobulated neutrophil (periph-

eral blood).

toa igure 19-17 @ RAEB (peripheral blood): anisomacrocytosis and

the ringed sideroblasts. Pelgeroid neutrophil.

‘Figure 19-18 l@ RAEB-t (peripheral blood): pseudo-Pelger—Huét

anomaly, mononuclear Stodtmeister type. and Stodtmeister neutrophils.
420 Chapter 19 Myelodysplastic Syndromes

BONE MARROW The nuclei of neutrophils often show vari-

able degrees of hyposegmentation (pseudo-Pelger—Huet or
Stodtmeister anomalies) (see Figs. 19-17 and 19-18). Other
nuclear abnormalities include hypersegmentation, ring for-
mation, chromatin clumping, and formation of chromatin
sticks. According to the specific type of MDS, there may be
numerous immature myeloid cells presenting asynchronous
nuclear-cytoplasmic maturation. The cytoplasm may be
hypogranular or agranular, with persistent basophilia at the
rim of the cell. One can also observe hybrid myelomonocytic
cells with specific staining properties for granulocytes
(chloroesterase) and monocytes (a-naphthol acetate or
butyrate esterase). Vacuolated monocytes or myeloid precur-
sors, or both, have been encountered in a few cases.

Figure 19-20 m RAEB (bone marrow): large megakaryocyte with

DYSMEGAKARYOCYTOPOIESIS
multilobulated nuclei, some of them distinctly detached and small
PERIPHERAL BLOOD Thrombocytopenia is present in in size (botryoid, “pawn-ball” lz shape).
almost 60% of patients, occasionally being severe (less than
20 X 10°/L). Thrombocytosis is of rare occurrence and is usu-
ally associated with the S5q— syndrome (see Fig. 19-15). One
may find variable morphological abnormalities such as
platelet gigantism, ballooning, and more or less severe
hypogranulation.* wi
BONE MARROW There are two characteristic morphologi-
&
cal abnormalities of megakaryocytes in the bone marrow: the
presence of micromegakaryocytes (dwarf or mononuclear —
megakaryocytes) and megakaryocytes with multiple small
nuclei detached from one another or separated by a thin strand
of nuclear material and cytoplasmic hypogranularity (botry-
oids, “pawn-ball” shape) (Figs. 19-19 to 19-24). In rare
cases, megakaryocytes may show large cytoplasmic vacuoles
(Fig. 19-25).

Classification of MDS Subtypes

Figure 19-21 ™ Sq- syndrome (bone marrow): monolobulated
MDS classification has evolved considerably since the first
micromegakaryocytes.
introduction of the term preleukemia or refractory anemia
several decades ago. The French—American—British (FAB)

Figure 19-19 @ RAEB (bone marrow): monolobular (dwarf) Figure 19-22 ™ Sq- syndrome (bone marrow): monolobulated
megakaryocyte. micromegakaryocytes.
 Chapter 19 Myelodysplastic Syndromes 421

classification introduced in 1982 has been instrumental in

helping investigators and clinicians to categorize patients in
more homogeneous subsets, help predict outcome, and select
therapeutical options for patients.** It is still widely used
worldwide and served as the basis for the new WHO classifi-
cation for MDS introduced in 1997. The WHO classification
is an upgraded FAB classification that takes into account
novel information on diagnostics, cytogenetics, and signifi-
cant biologic features of MDS.

FAB Classification
MDS are classified into five distinct subtypes. The distinction
between these subtypes of MDS relies essentially on the study
Figure 19-25 @ RA (bone marrow): hypogranular megakaryocyte. of the bone marrow aspirate stained with Wright—Giemsa and
Prussian blue. The distinctive hematologic features of each
MDS subtype are summarized in Table 19-2.

Refractory Anemia
Refractory anemia (RA) is the most difficult MDS to recog-
nize and diagnose. The diagnosis rests mainly on the sub-
jective identification of qualitative abnormalities involving
one or more bone marrow hematopoietic cell lineages, in a
patient suspected of having RA. Anemia is present in more
than 90% of patients. It is usually normochromic and
macrocytic, but in some cases, erythrocytes may be normo-
cytic, with a variable degree of morphological abnormali-
ties, usually more subtle than those found in other
subgroups. Cytopenias are common, with a white blood cell
count less than 3.9 * 10°/L and a platelet count below
130 X 10°/L. Morphological abnormalities such as dys-
granulopoiesis or dysmegakaryopoiesis are more subtle
than in other types of MDS. The bone marrow is usually
normocellular or hypercellular. Infrequently, it may be
Figure |9-24 @ RA (bone marrow): detached nuclei megakaryocyte. slightly hypocellular or may present a variable degree of
fibrosis. Blast cells, type I and type II, represent less than
5% of all nucleated cells. Erythroid hyperplasia is relatively
common, with mild dyserythropoiesis and occasional
ringed sideroblasts (less than 15% of nucleated red blood
cells). Cytogenetic studies are crucial in the diagnosis of
this particular subtype of MDS in view of the absence
of quantitative criteria for its diagnosis. Indeed, the finding
of a clonal chromosomal abnormality in such a setting con-
firms the diagnosis. Approximately 50% of patients are
found to have clonal abnormalities. The diagnosis may also
be supported by abnormal bone marrow culture results or
an X-inactivation clonality assay showing a clonal pattern
in myeloid cells in the presence of a normal somatic tissue
control. In the absence of a clonal chromosomal abnormal-
ity, the establishment of this diagnosis based solely on
single lineage dysplasia should be made with great caution.
Isolated dyserythropoiesis on blood or bone marrow smears
may be a feature shared by a variety of hematological
disorders, including relatively benign diseases with no
potential to progress to acute leukemia and bearing no rela-
Figure 19-25 mRA (bone marrow): vacuolated immature
megakaryocyte. tionship to the MDS.
422 Chapter 19 Myelodysplastic Syndromes

Peripheral Blood Bone Marrow Aspirate

Blasts <1% Blasts < 5%

Cytopenia Ringed sideroblasts <15%
Blasts <1% Blasts <5%
Cytopenia Ringed sideroblasts >15%
Blasts <5% Blasts 5%-—20%, no Auer rods
Cytopenia Ringed sideroblasts, variable
Blasts >5% Blasts 20%-30%, or blasts
Cytopenia 5%-20% with Auer rods
Blasts <5% Blasts 1%-20%
Monocytosis > 1 x 10°/L Monocytes, promonocytes 220%
WBC <13 or 13 X 10°/L Ringed sideroblasts, variable

Refractory Anemia with Ringed

Sideroblasts
Refractory anemia with ringed sideroblasts (RARS) shares
almost all the morphological features of RA, although gen-
erally to a lesser extent. Fortunately, the diagnosis rests on
quantitative criteria: the presence of at least 15% ringed
sideroblasts in the bone marrow (varying from 15% to
90% among nucleated red blood cells) (see Fig. 19-14).
Ringed sideroblasts are pathologic sideroblasts in which the
usual migration of mitochondria from the nucleus toward
the cytoplasm does not occur. As a result, they remain
frozen around the nucleus, where insoluble iron conglomer-
ates and further contributes to cell destruction. This feature, ie,
combined with lineage dysplasia as mentioned earlier, is
satisfactory to establish the diagnosis. Red blood cells Figure 19-26 @ RAEB (bone marrow): myeloblast (A) and Pelgeroid
are usually macrocytic or normocytic with dimorphic fea- neutrophils (B).
tures, i.e., dual population of hypochromic and_nor-
mochromic red blood cells (see Fig. 19-7). Leukopenia
and thrombocytopenia are relatively infrequent (10% to
15% of cases).

Refractory Anemia with Excess Blasts

In refractory anemia with excess blasts (RAEB), the degree
of lineage dysplasia is more significant. The anemia is
macrocytic and normochromic, with important morpholog-
ical abnormalities such as ovalocytosis and the presence of
dacryocytes related to bone marrow dyserythropoiesis. On
rare occasions, one can find a peripheral blood sample with
an increased number of immature granulocytes. Although
occasional blasts may be seen in the peripheral smear, the
count is always below 5% (Fig. 19-26). Dysgranulopoiesis
with nuclear hyposegmentation and hypogranulation is fre-
quent. Thrombocytopenia is a common feature, with giant
size and hypogranular platelets. Bone marrow cellularity is
normal or increased, with myeloblasts of type I and type II
ranging between 5% and 20% (Fig. 19-27). Dysmegakary-
ocytopoiesis and dyserythropoiesis (with variable number Figure 19-27 ™ RAEB (bone marrow): myeloblasts, mitosis, and
of ringed sideroblasts) can be significant. Pelgeroid neutrophil.
 Chapter 19 Myelodysplastic Syndromes 423

Refractory Anemia with Excess Blasts

in Transformation
Refractory anemia with excess blasts in transformation
(RAEB-t) is closely related to RAEB. The major difference
lies in the number of blasts found in the bone marrow at ini-
tial diagnosis (Figs. 19-28 and 19-29). In RAEB-t, the num-
ber of blasts ranges from 20% to 30%. In the peripheral
blood, blasts frequently comprise more than 5% and may con-
tain Auer rods. Their presence is diagnostic of RAEB-t, even
if the number of blasts in the bone marrow is less than 20%.
Acute myeloblastic leukemia, type M2, can mimic RAEB-t
when the blast count is between 25% and 30%. The degree of
dysplasia in the bone marrow and the presence of a t(8;21),
typical for a small proportion of AML type M2, helps in dif-
ferentiating between these two entities.*° Figure 19-29 m@ RAEB-t (bone marrow): myeloblasts, megaloblastoid
erythroblast with Howell-Jolly body (arrow).

Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is controversial
in regard to its classification as a myelodysplastic syndrome.
In the blood and bone marrow, the same morphological
abnormalities as those found in the other types of MDS may
occur. On clinical grounds, this type of MDS is more closely
related to the chronic myeloproliferative disorders, particu-
larly when the white,blood cell (WBC) count is higher than
13 x 10°%/L. Patients often present with splenomegaly or
hepatomegaly. Peripheral WBC counts may be as high as
75 X* 10%7/L, a picture resembling chronic myelogenous
leukemia (CML) (Fig. 19-30). The bone marrow is usually
hypercellular, with monocytosis and a blast cell count between
1% and 20% (Fig. 19-31). In some cases, cytogenetics may be
the only means of distinguishing CMML from CML. CML has
a specific clonal marker, the Philadelphia chromosome
t(9;22)(q34q11.2).* In contrast, about 20% to 30% of CMML
cases have nonspecific cytogenetic abnormalities including tri-
Figure 19-30 @ CMML (peripheral blood): promonocytes—
somy 8, monosomy 7, del(11q), and del(20q). Rare specific monocytes and Pelgeroid neutrophil (arrow).
chromosomal translocations have been described in CMML,
such as the t(5;12)(q33;p13) translocation*’’ (see Cytogenetics
and Molecular Abnormalities section). The diagnosis rests

Figure 19-31 ® CMML (bone marrow): myeloblasts, monocytes,

Figure 19-28 m RAEB-t (bone marrow): myeloblast with Auer rods. and promonocytes.
424 Chapter 19 Myelodysplastic Syndromes

mainly on the presence of more than | X 10°/L monocytes in the The most significant amendments of the WHO classifi-
peripheral blood and the findings of lineage dysplasia in the cation (relative to the FAB classification) are the following:
peripheral blood or bone marrow, or both, as in other MDS.
1. RA is now subdivided according to the presence or absence
of multilineage dysplasia. The presence of dysplasia in at
WHO Classification**® least 10% of the progeny from two or more myeloid cell
lines defines a new subtype of MDS called RCMD. This
The FAB classification of MDS was revisited by WHO in con- subtype of MDS has less than 5% blasts in marrow, no
junction with the European Association of Haematopatholo- Auer rods, and no peripheral monocytosis. The subdivision
gists and the Society for Haematopathology.* The WHO of RA according to level of lineage dysplasia is based on
classification preserves many of the concepts and definitions the worse prognosis of patients in which more than the ery-
of the FAB classification, but incorporates other data to throid progenitors are affected.
improve clinical relevance.** Several studies have validated the tO. RARS is also subdivided according to the presence or absence

improved clinical significance of the WHO classification of of multilineage dysplasia. The presence of multilineage dys-
MDS.***° The WHO classification defines eight subtypes of plasia defines another subtype of MDS called RCMD-RS.
MDS: refractory anemia (RA), refractory anemia with ringed 3. The 5q— syndrome becomes a specific category with the 5q
sideroblasts (RARS), refractory cytopenia with multilineage deletion (q21—q32) as the sole cytogenetic anomaly, fewer
dysplasia (RCMD), refractory cytopenia with multilineage dys- than 5% blasts in the marrow, no Auer rods, and normal or
plasia and ringed sideroblasts (RCMD-RS), refractory anemia elevated platelet counts.
with excess blasts-1 (RAEB-1), refractory anemia with excess 4. RAEB is subdivided into RAEB-1 for patients with 5% to
blasts-2_ (RAEB-2), myelodysplastic syndrome-—unclassified 9% bone marrow blasts, and RAEB-2 for patients with
(MDS-u), and MDS associated with isolated del (5q). The dis- 10% to 19% bone marrow blasts.
tinctive hematologic features of each MDS subtype are summa- 5. RAEB-t is eliminated and all patients are considered to
rized in Table 19-3. have acute leukemia.

Disease Blood Findings Bone Marrow Findings

Refractory anemia (RA) Anemia Erythroid dysplasia only

No or rare blasts <5% blasts
<15% ringed sideroblasts
Refractory anemia with ringed Anemia Erythroid dysplasia only
sideroblasts (RARS) No blasts =15% ringed sideroblasts
<5% blasts
Refractory cytopenia with Cytopenias (bicytopenia or Dysplasia in =10% of cells in two or
multilineage dysplasia (RCMD) pancytopenia) more myeloid cell lines
No or rare blasts <5% blasts in marrow
No Auer rods No Auer rods
< | x 10°L monocytes <15% ringed sideroblasts
Refractory cytopenia with Cytopenias (bicytopenia or Dysplasia in = 10% of cells in two or
multilineage dysplasia and ringed pancytopenia) more myeloid cell lines
sideroblasts (RCMD-RS) No or rare blasts =15% ringed sideroblasts
No Auer rods < 5% blasts
< 1 x 10°L monocytes No Auer rods
Refractory anemia with excess Cytopenias Unilineage or multilineage dysplasia
blasts-1 (RAEB-1) < 5% blasts 5%-9% blasts
No Auer rods No Auer rods
< 1 x 10°L monocytes
Refractory anemia with excess Cytopenias Unilineage or multilineage dysplasia
blasts-2 (RAEB-2) 5% to 9% blasts 10% to 19% blasts
With or without Auer rods With or without Auer rods
< 1 x 10°L monocytes
Myelodysplastic syndrome, Cytopenias Unilineage dysplasia in granulocytes
unclassified (MDS-U) No or rare blasts or megakaryocytes
No Auer rods < 5% blasts
No Auer rods
MDS associated with isolated del(5q) Anemia Normal to increased megakaryocytes
< 5% blasts with hypolobulated nuclei
Platelets normal or increased < 5% blasts
No Auer rods
Isolated del(5q)
 Chapter 19 Myelodysplastic Syndromes 425

¢ Chronic myelomonocytic leukemia

* Atypical chronic myeloid leukemia
¢ Juvenile myelomonocytic leukemia
¢ Myelodysplastic/myeloproliferative disease, unclassifiable

6. CMML is reclassified under the subgroup of Myelodys-

plastic/Myeloproliferative Diseases (Table 19-4). More-
over, CMML is divided into two subtypes (Table 19-5).
The level of leukocytes is not taken into account but the
Figure 19-32 @ RA (peripheral blood, peroxidase stain): neutrophil,
percentage of blasts defines the CMML subtype. CMML-1 positive reaction.
has fewer than 5% blasts in blood, and fewer than 10% in
marrow. CMML-2 has 5% to 19% blasts in blood or 10%
to 19% in marrow.
precursors; PAS positivity in erythroblasts; dual esterase in
7. There is a new category for unclassifiable MDS. One of the
myeloid and monocytoid cells; and decreased Sudan black B
entities included in this category is RARS associated with (SBB) staining are often found in MDS (Figs. 19-32 to
marked thrombocytosis.* In the latter, the marrow presents 19-34). An increase in the number of erythrocytes with HbF
morphologic features of RARS coupled with megakary- (positive Kleihauer—Betke acid elution test) is a common
ocytic proliferation, and there is a markedly elevated observation (Fig. 19-35). Cytochemistry is of less diagnostic
platelet count (>600 x 10°/L) in the peripheral blood. value in MDS with major qualitative and quantitative abnor-
e
malities such as RARS, RAEB, RAEB-t, and CMML than in
Laboratory Features RA, where cytochemical studies of blood and bone marrow
cells can lend credence to the diagnosis.
Cytochemistry Following is a summary of the frequency of cytochemical
anomalies in RA patients, based on our observations and those
Decreased enzymatic activity of myeloperoxidase, chloroesterase, of others. For leukocytes, in 60% to 78% of cases, neutrophil
and alkaline phosphatase; abnormal positivity in myeloid peroxidase shows reduced activity in most of the neutrophils
(polymorphonuclear neutrophils, PMNs) or an absence of activ-
ity in a small percentage of cells; chloroesterase and leukocyte
alkaline phosphatase (LAP) staining show decreased activity in
mature neutrophils, but LAP activity may be increased in neu-
trophil precursors. In the erythrocyte lineage, 18% to 25% of
erythroblasts stain positive for PAS, with basophilic erythrob-
lasts and proerythroblasts showing granular and droplet

Persistent peripheral blood monocytosis greater than | x 10°/L

No Philadelphia chromosome or BCR-ABL fusion gene
Fewer than 20% blasts* in the blood or bone marrow
Dysplasia in one or more myeloid lineages. If myelodysplasia
is absent or minimal, the diagnosis of CMML may still be
made if the other requirements are present and an acquired,
clonal cytogenetic abnormality is present in the marrow
cells, or the monocytosis has been persistent for at least
3 months and all other causes of monocytosis have been
excluded.
Diagnose CMML-1 when blasts fewer than 5% in blood and
fewer than 10% in bone marrow
Diagnose CMML-2 when blasts are 5%—19% in blood, or
10% to 19% in marrow, or if Auer rods are present and
blasts are fewer than 20% in blood or marrow
Diagnose CMML-1 or CMML-2 with eosinophilia when the
criteria above are present and when the eosinophil count
in the peripheral blood is greater than 1.5 10°/L

*In this classification of CMML, blasts include myeloblasts,

monoblasts, and promonocytes. Figure 19-55 m RA (bone marrow, peroxidase stain): pseudo-
Pelger—Huét-negative cells.
426 Chapter 19 Myelodysplastic Syndromes

bone marrow biopsy specimen may be of great help in reveal-

ing typical histological changes such as the disruption of normal
hematopoietic architecture, with displacement of granu-
lopoiesis, erythropoiesis, and megakaryocytopoiesis from their
usual sites. Abnormal localization of immature precursors
(ALIP),*? clusters or aggregates of myeloblasts and promyelo-
cytes of usually three or more cells distant from the bone mar-
row trabeculae, is also a feature of some MDS. This finding has
been considered by some authors as an indicator of early
leukemic transformation. Bone marrow biopsy is especially
relevant in the rare cases of MDS with severe myelofibrosis
(10%)*** (Fig. 19-36) or associated with bone marrow
hypoplasia.*°*” Although dyserythropoiesis and dysgranu-
lopoiesis are difficult to assess on bone marrow biopsy speci-
mens, dysmegakaryocytopoiesis can be fairly estimated.
Figure 19-34 ™ RAEB (bone marrow, PAS stain): erythroblasts,
positive granules.
Cytogenetics and Molecular Abnormalities
reactions whereas more mature erythroblasts (polychro- A full description of the terminology and abbreviations used
matophilic and orthochromatic) have diffuse cytoplasmic in describing chromosomes and their abnormalities is beyond
reactivity. Occasionally, some erythrocytes stain positive, the scope of this chapter. For detailed guidelines, the reader is
with a more homogeneous or diffuse pattern. Although no sin- referred to a specialty textbook.***” A brief review adapted for
gle cytoenzyme anomaly can be considered a specific early the comprehension of this section is presented in Table 19-6.
marker for leukemic transformation, a progressive deteriora- Since the introduction of the FAB classification of MDS
tion of cytochemical enzymatic activities associated with in 1982, cytogenetics has become one of the most informative
morphological abnormalities can become indicators of an laboratory tools in their investigation and management. The
impending leukemic phase. These changes in enzymatic nature and complexity of clonal chromosomal abnormalities
activity must, however, be interpreted with caution because have proved to be essential in the diagnosis and establishment
infections, toxic bone marrow damage, and some congenital of the prognosis. Specific chromosomal abnormalities, when
defects of hematopoiesis may present similar cytochemical documented, contribute independent prognostic factors, and
abnormalities.“°? thus provide the opportunity to individualize therapy.
Clonal cytogenetic abnormalities are detected in 30% to
Bone Marrow Histology 60% of de novo MDS cases” and the incidence rises to close
to 90% in secondary MDS.°*! Structural and numerical anom-
In patients with MDS, discrepancies between the cellularity alies are found, but deletions are by far the most frequent and
estimates from aspirate smears and biopsy specimens occur in characteristic cytogenetic events observed. The most frequent
up to 20% of cases. It is well accepted that the most accurate anomalies include partial deletions of chromosome arms 5q,
estimate of bone marrow cellularity is made from an adequate 20g, 11q, or 7g, loss of chromosome 7 or chromosome Y, tri-
biopsy specimen. In addition to the cellularity evaluation, the somy 8, and anomalies of the short arm of chromosome 17

si

Figure 19-35 @ RA (peripheral blood, Kleihauer—Betke staining): Figure 19-36 @ RAEB (bone marrow, reticulin stain, magnification
positive RBCs, HbF. 400): severe fibrosis.
 Chapter 19 Myelodysplastic Syndromes 427

are found in about 15% to 30% of de novo MDS and in up to

50% of therapy-related MDS. Certain cytogenetic anomalies
are associated with morphological or clinical particularities.
Monosomy 7, especially in children, is associated with a
greater risk of bacterial infection independent of the neu-
trophil count.*° Anomalies involving the short arm of chromo-
some 17 (17p) where the p53 gene resides, are associated with
Abbreviation Significance hypolobulated granulocytes with small cytoplasmic vac-
uoles.*° Involvement of the 3q26 band (EVI/) is associated
Short arm of a chromosome with thrombocytosis and dysplastic megakaryocytes.°’ Spe-
Long arm of a chromosome
cific chromosomal translocations involving the EV//, MLL,
Loss of chromosome material to long
arm NUP98, and TEL genes have been described in MDS but with
Deletion of chromosome material a low frequency. The t(5;12)(q13;p13) translocation is a
Translocation of chromosome material recurrent anomaly associated with the ETV6—PDGF® fusion
(DNA exchange between 2 chro- gene. It is found in a rare subgroup of diseases with features
mosomes)
of both myeloproliferative disorder and MDS and with
Marker chromosome that is not fully
characterized eosinophilia (Fig. 19-38). This translocation is important to
Addition of a chromosome detect because these patients can benefit from therapy with
Loss of a chromosome tyrosine kinase inhibitors.**
Hypodiploid Cells having fewer than 46 chromo- Cytogenetic analysis provides an important prognostic
somes
tool for the risk of transformation to acute myeloid leukemia
Hyperdiploid Cells having more than 46 chromo-
somes (AML) and for survival. Patients with monosomy 7 and com-
Complex karyotype The presence of 3 or more chromoso- plex karyotypes have a short survival and a high rate of pro-
mal abnormalities in the same cell gression to AML. Patients with —Y, del(20q), or del(5q) as a
sole anomaly have a more favorable clinical outcome, with a

«
tendency for a long leukemia-free survival. The remaining
(Fig. 19-37). The cytogenetic anomalies reported in sec- patients have an intermediate prognosis.”
ondary MDS are related to the type of therapy that patients
THE S5q- SYNDROME
have previously received. More than 90% of patients treated
with an alkylating agent show anomalies of chromosome 5 or In the mid-1970s, van den Berghe® described patients with an
7, or both.*' Patients treated with agents of the epipodophyl- acquired interstitial deletion of the long arm of chromosome 5
lotoxins class demonstrate anomalies (mainly translocations) (Fig. 19-39). These patients had specific clinical features that
involving the MLL gene located at chromosome band 1|1q23.°? included a macrocytic anemia, normal or elevated platelet
count, mild leukopenia, and, in the bone marrow, monolobular
Most chromosome abnormalities are not specifically associ-
ated with any FAB subgroup; however, the 5q— anomaly or dwarf megakaryocytes and erythroid hypoplasia. Patients
is documented more frequently in RA,» as is del(20q). Mono- are usually classified as having RA according to the FAB clas-
sification and are, in contrast to other MDS patients, predomi-
somy 7 is rarely seen in RA.* Complex karyotypes (three
nantly females. The 5q— syndrome is associated with a good
or more chromosomal abnormalities within the same cell)
prognosis with a median survival of approximately 8 years.
The deleted DNA segment lies between bands 5q13 and 5q33
e % ~~ ”
4 & 1 4 13 a a ee hy * % % 4 te
tamy ; 4 4 :
o- eb4 —<.§ a
v 2
8
g
i
to
ee
23
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O84
7
5 eA. 2 $8 eG LFed
1 2 34 emo : 1 2 3 4 5
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f ais 4f oF os ees # i & & & 4 $ a? ra £ et 2 a gt

6 7 8 9 iL): ee is
x
7 $2 af «Ee OKs 4
6 i 8 9 10 11 12
A » 4
a * i s+
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13 14 65 ¢? 16 17 18
J & 4 ae <a
NS 13 14 15 16 1 1
a
; a ¥ 21 22 y;
19 20 Soy
ne ee a8 ae § g
19 20 21 22 X V4
Figure 19-57 @ Karyotype GTG banding, resolution 450 bands,
showing complex clonal abnormalities involving at least 8 chromo- Figure 19-38 @ Karyotype (GTG banding) showing the
somes within the same cell, including del(5)(q13q33), —7, t(5;12)(q33;p13) translocation and a loss of chromosome 7 in a
del(20)(q11.2). case of CMML.
428 Chapter 19 Myelodysplastic Syndromes

treatment,® confirming the particular status of this MDS

ES
Be entity.
5s
4
IL4 THE FUTURE OF CYTOGENETICS
3 IL5
2
1 IRF1 One limitation of traditional cytogenetic analysis is the neces-
aap
IL3 sity to obtain cell metaphases through marrow culture, which
GM-CSF is at times unsuccessful or leads to suboptimal chromosome
le
2 identification. The use of gene- or chromosome-specific
3 probes in the fluorescent in situ hybridization (FISH) tech-
4
nique allows the analysis of the interphase nuclei. FISH is
Ss
particularly useful for numerical anomalies and for some dele-
tions (Fig. 19-41). New moiecular cytogenetic techniques
using 24 fluorescent labeled chromosome painting probes such
as spectral karyotyping (SKY) and multiplex-FISH (M-FISH)
allow a better identification of chromosomal abnormalities,
ILO
CD14 especially for MDS with complex karyotypes (Fig. 19-42).
Moreover, these methods may reveal cryptic chromosomal
rearrangements not detected by GTG banding.°° Compara-
tive genomic hybridization (CGH array) is another tool
to identify gene deletions and amplifications in MDS.°
CHROMOSOME 5 Recently, these techniques have allowed the identification of
Figure 19-39 m The 5q- syndrome. Several genes related to
new genetic alterations in MDS such as the duplication or
hematopoiesis are localized on 5q. A. The interstitial deletion may amplification of chromosome band 11q23 including the MLL
comprise any region located between band 5q13 and 5q33. B. The gene in therapy-related MDS.°* These new techniques rein-
band 5q31 is consistently deleted in most patients. force the predominant role of cytogenetics in the investigation
of MDS.
(Fig. 19-40). Although there may be variable breakpoints
within this region, band 5q31 seems to be consistently Immunology
deleted.°' Interestingly, several genes that encode cytokines
and their receptors are located in the involved region, but no Surface antigens of cells from peripheral blood, bone marrow,
gene has been shown to be clearly involved in the pathogene- or biopsy specimens, can be studied by flow cytometry or
sis of MDS. It is important to note that a 5q anomaly found immunocytochemistry using alkaline phosphatase antialkaline
in conjunction with other cytogenetic anomalies, or in a phosphatase methodology (APAAP) and other techniques. An
patient with a history of treatment with an alkylating agent, is increase in the percentage of CD34+ cells (stem cell progeni-
not associated with this clinical syndrome and 1s not associated tors) in the bone marrow is of prognostic significance and
with a good prognosis. The 5q— syndrome has now been usually correlates with impending leukemic transformation.”
recognized as a special entity by the WHO classification.* Blasts from patients evolving toward acceleration or transfor-
Patients with the 5q— syndrome also demonstrate a high hema- mation in any type of MDS may carry antigens characteristic of
tological and cytogenetic response rate to Lenalidomide other cell lineages, mostly myeloid, monocytic, and lymphoid.

Fy ‘ ¥
é ¥, 4 ¥ 44 a
x eS ¥ i
‘ & om

2 3 4 5

~ * a¢
. 4 wo, % ¥ Ge ¥ ®.
‘ 4 id mS ss 3% wo Ff
6 7 8 9 10 11 12 X
x -e RZ
3 * A . $ «@ $

13 14 15 16 ily 18
ne eh 8s

19 20 21 22 NV.

Figure 19-40 @ Karyotype with GTG banding, 450 bands resolution,

showing the interstitial deletion of chromosome 5 > 46,XY,del(5) Figure 19-41 @ Fluorescence in situ hybridization (FISH), +8 in
(q13q33) characteristically found in the Sq— syndrome. neutrophil.
 Chapter 19 Myelodysplastic Syndromes 429

6. @-: LJ & | Bo
$3 10 4

=~ 17 ~-22
W -16
16 17

eC Ret Meer wel Bel)

Figure 19-42 m RAEB-2, spectral karyotyping (SKY): complex karyotype with numerous chromosomal translocations and trisomy 22.

In the rare cases in which a lymphoblastic proliferation is Marrow cells pass through all stages of maturation to become
found, the immunophenotypic pattern is consistently of the morphologically recognizable mature cells. In vitro cultures
early B type (CD19+, CD10—, TdT+). RAEB and RAEB-t of bone marrow cells from individuals with MDS have pro-
show a higher frequency of myeloblastic and megakaryoblastic vided information about the nature of the pathophysiologic
immunologic markers, whereas in RA, RARS, and CMML, a defect. In general, the ability of hematopoietic progenitor
granulomonocytic phenotype predominates. Hybrid lymphoid- cells to form colonies is found to be either reduced or
myeloid cells or cells coexpressing granulomegakaryocytic absent.’° Aberrant growth patterns are mostly observed in the
antigens are also frequently observed. At the molecular level, more severe subgroups of MDS, such as RAEB and RAEB-t.
rearrangements of the immunoglobulin H ({gH) and the T-cell A notable exception is CMML, which is distinguished by
antigen receptor gamma (TCR-y) and -delta (TCR-5) genes increased colony growth.’° Investigators have tried to corre-
can be found. late these growth patterns with the propensity to evolve to
Patients with type I and II blasts expressing the H blood acute leukemia, dividing them into leukemic and non-
group antigen” have a poor prognosis, as opposed to those leukemic types. Given the lack of standardization and results
expressing a purely myeloid phenotype. Expression of surface that are often conflicting, no firm conclusions can be drawn
antigens from monocytes and granulocytes may vary consider- about growth patterns with respect to either prognosis or
ably, with either loss or gain of specific or nonspecific antigens. leukemic potential in individual patients with MDS at this
Lymphopenia is frequently observed, with a decrease of CD4+ time. In vitro bone marrow culture from patients with MDS is
cells and a slight increase of CD8+ cells.”' It has been reported a valuable complementary tool to be considered together with
that an increased number of CD8+ cells at initial diagnosis in cytogenetics and other laboratory tests.
RA and RARS is associated with a decreased incidence of acute
non-lymphoblastic leukemia (ANLL) transformation. Although
natural killer lymphocytes are decreased, antibody-dependent Cellular Dysfunction
cellular cytotoxicity is conserved. Genetic mutations or other abnormalities such as chromoso-
Fewer B-lymphocyte abnormalities have been reported. mal deletions, translocations, or gene amplification are
B-cell Epstein-Barr virus (EBV) receptors are decreased in responsible for a vast array of functional abnormalities within
number.’”? Immunoglobulin synthesis may be abnormally reg- specific cell lineages.
ulated. Polyclonal hypergammaglobulinemia is observed in
approximately 30% of patients with MDS; 12% have a mono- RED BLOOD CELLS
clonal gammopathy, whereas 13% are hypogammaglobuline- Several metabolic abnormalities may be encountered, the
mic. Polyclonal hypergammaglobulinemia is observed in most frequent being acquired pyruvate kinase deficiency and
more than 60% of patients with CMML.” Autoantibodies have increased level of HbF. Acquired hemoglobin H, increased
been reported in 22% of all patients with MDS, but they are a expression of the i antigen, loss of ABO blood group antigens,
more frequent occurrence (present in more than 50% patients) unmasking of the Tn antigen, and increased sensitivity to
in CMML. Platelet antibodies are present in 55% of the cases complement (positive serum acid or sugar water test) have
studied, and erythrocyte autoantibodies in 46%. The direct also been reported.””*° At times, loss of erythrocyte mem-
antiglobulin test has been found positive in 8% of the cases brane proteins such as decay accelerating factor (DAF)
where IgG! and C3d were detected on the erythrocytes.” CD55, and glycosyl phosphatidyl] inositol (GPI)-linked anti-
gen CD59, may also be observed. Concomitantly, such loss
Evaluation of Progenitor Cell Growth may be observed on neutrophils and other cells, thereby over-
lapping with paroxysmal nocturnal hemoglobinuria.
in Semisolid Media
Several investigators have studied in vitro growth patterns WHITE BLOOD CELLS
from MDS patients. There is a specific pattern of colony Abnormal granulocyte function is frequently observed
clusters that grow in culture from normal progenitor cells. with decreased cytoplasmic granules and myeloperoxidase
430 Chapter 19 Myelodysplastic Syndromes

deficiency. Alkaline phosphatase and a-naphthol acetate these patients have been previously exposed to epipodophyllo-
esterase isoenzyme activities may also be decreased in granu- toxins and anthracyclines, which target DNA topoisomerase if
locytes. Chemotaxis, adhesion, phagocytosis, and microbicidal often in combination with other drugs including alkylating
capacity may be impaired.*'** These functional abnormalities agents. DNA topoisomerases are a class of enzymes important
are not necessarily related to cellular hypogranulation. Abnor- in various DNA transactions such as replication, transcription,
mal phagocytosis, chemotaxis, or bactericidal activities in and recombination.?! Most of the balanced translocations
neutrophils have been closely associated with chromosome involve 11q23 and different partners such as chromosomes 4, 6,
7 anomalies—monosomy 7 and del(7q). These specific dys- 9, or 19. Usually the MLL gene (located at 11q23) is rearranged.
functions are associated with a high frequency of bacterial Balanced translocations involving the gene RUNX1I(AMLI) at
infections. In these situations, the GP130 molecule, produced 21q22 are also reported in this setting. Patients with these
by a gene located on chromosome 7, is secreted in inadequate translocations usually have a short latency between exposure
amounts, and is responsible for the major defect in chemotaxis and development of sMDS, and tend to present with acute
and phagocytosis.*? leukemia with monocytic features. The sMDS caused by topoi-
somerase II inhibitors respond better to treatment than those
caused by alkylating agents.
PLATELETS
Platelet aggregation, adhesion, and other platelet functions
are also frequently impaired in MDS.™ Other laboratory find- Myelodysplastic Syndromes in Children
ings that may be found in MDS include abnormal vitamin B,,
and folate levels, increased serum ferritin and transferrin sat- MDS occurs rarely in children, and most cases are of the
uration, and increased muramidase (in serum and urine of RAEB and RAEB-t subtypes. Children are most often symp-
patients with CMML). tomatic with fever, pallor, and asthenia.”* Unlike adults, chil-
dren more commonly present normocytic, normochromic
anemia rather than macrocytic anemia. Several syndromes
Secondary Myelodysplastic Syndromes such as Fanconi’s anemia, Down’s syndrome, Kostmann’s
agranulocytosis, and Shwachman’s syndrome are associated
MDS are said to be secondary when they occur after signifi-
with an increased risk of development of acute myeloblastic
cant exposure to chemotherapy, radiotherapy, or other toxic
leukemia in children and should be investigated.”*°* Mono-
agents.°!*°8° The term “secondary myelodysplastic syn-
somy 7 syndrome and juvenile chronic myeloid leukemia
dromes” (SMDS) seems more appropriate than the often-seen
usually present with leukocytosis and hepatosplenomegaly
“therapy-related MDS” because exposure to toxic agents is
and are considered myeloproliferative disorders rather
also responsible for sMDS. The laboratory findings, clinical
than MDS.”
manifestations, and evolution resemble those of de novo MDS,
but sMDS show a higher frequency of clonal chromosomal
abnormalities and a greater tendency to early leukemic transfor- Clinical Features
mation, particularly if a complex karyotype is present. The prog-
nosis is generally poorer in sSMDS than in de novo MDS. Most commonly, the presenting symptoms are those attributable
There has been an increase in the number of patients diag- to progressive bone marrow failure. Clinical manifestations
nosed with sMDS in recent years, which is likely attributable develop in relationship to the degree of anemia, neutropenia, or
to the increased usage of intensive chemotherapy protocols. thrombocytopenia. Elderly patients with anemia may present
Between 5% and 10% of patients submitted to chemotherapy symptoms of cardiac failure, such as dyspnea on exertion.
(with or without radiotherapy) as preparative regimen for autol- Fatigue and weakness may also be noted, particularly in patients
ogous stem cell transplantation will develop sMDS.*’*’ Most with RAEB and RAEB-t. Symptoms related to infection and
cases are causally related to previous therapy with alkylating hemorrhage may be encountered in patients with MDS and
agents, almost all of which have been shown to be leuke- severe bone marrow failure. Patients without excess numbers of
mogenic. The leukemogenic potential of alkylating agents is blasts in the bone marrow may remain asymptomatic for a long
enhanced further by the use of radiotherapy. The period of period of time. Rarely, patients may present systemic symptoms
latency between exposure to alkylating agents and development such as infection, arthralgias, weight loss, fever, and cutaneous
of sMDS is usually 6 to 8 years, and patients usually have a poor vasculitis.
response to therapy with the exception of allogeneic bone Abnormal physical findings are neither prominent nor
marrow transplantation. Recently, the use of the epipodophyllo- specific. The spleen may be palpable in 20% of patients, and
toxins etoposide and tenoposide has been shown to be leuke- the liver may be enlarged in approximately 10%. Enlarged
mogenic if administered in combination with cisplatinum, lymph nodes are not usually present. Compared to the other
doxorubicin, or alkylating agents.”° In patients with MDS and a types of MDS, in CMML, splenomegaly, hepatomegaly, lym-
history of previous exposure to alkylating agents, cytogenetic phadenopathy, and nodular cutaneous leukemic infiltrates are
studies usually show unbalanced cytogenetic aberrations, pri- more common.”°*’ The overall clinical picture of CMML is
marily —7, —5, 5q—, and 7q—. Recently, an increasing number of more closely related to the myeloproliferative disorders such
patients with sMDS have been observed with balanced chromo- as CML, polycythemia vera, essential thrombocytosis, and
somal translocations. Studies have revealed that the majority of primary myelofibrosis.
 Chapter 19 Myelodysplastic Syndromes 431

Evolution and Prognosis Diagnostic Problems in MDS

Although the overall survival of patients with MDS is less Some MDS patients carry features shared with other hemato-
than 2 years, MDS are a heterogeneous group of bone marrow logical disorders (Fig. 19-44). These clinical situations may
disorders with survival that can vary from weeks to several lead to confusion concerning the appropriate diagnosis for the
decades after the initial diagnosis. The causes of mortality are patient. The most common of these difficult diagnostic quan-
also not uniform. One third of patients die of leukemic trans- daries are presented next.
formation whereas close to 40% suffer complications related
to bone marrow failure, such as infection or bleeding. Almost
one third of patients with MDS die of conditions unrelated to MDS with Hypoplastic Marrow
their disease.**””
Between 8% and 22% of MDS patients have a marrow cellu-
There is a need for a classification system that allows the
larity inferior to 25%.*’ It may be difficult to differentiate
prospective categorization of patients at diagnosis into differ-
such cases from aplastic anemia, which also presents with
ent prognostic groups to select appropriate therapy for an
peripheral blood cytopenias and a hypocellular marrow. The
individual patient. The FAB classification has been useful in
single most important test that may allow the differentiation
this regard, although it requires further refinements. It incor-
between these two entities is the demonstration of a clonal
porates the single most important prognostic factor: the blast
cytogenetic anomaly that will confirm the diagnosis of MDS.
count. The survival and leukemic transformation rates accord-
If the karyotype is normal, careful analysis of the bone mar-
ing to FAB subtype are shown in Figure 19-43.
row smear for dysplastic changes may provide some clues to
Several other prognostic risk factors have been identi- the correct diagnostic category. In some cases, it may not be
fied, including:
possible to differentiate between the two.
1. The nature and number of chromosome abnormalities
2. The severity of pancytopenia at diagnosis
3. The age of the patient
MDS with Severe Myelofibrosis
4 The presence of ALIP.*?1© Close to 15% of MDS patients show significant bone marrow
Different prognostic scoring systems based on the combina- fibrosis on biopsy.'*® This is more frequently seen in the setting
tion of these factors have been proposed. These include the of sMDS. Patients may have a dry tap on bone marrow aspira-
Bournemouth,”* Dusseldorf,'°' Sanz,'? and Goasguen'®? scor- tion. They often have trilineage dysplasia, profound cytopenias,
ing systems. Although each of these scoring systems may aid and no organomegaly. The differential diagnosis includes acute
the prognostic evaluation of MDS patients, they have been myelofibrosis (AML, M7) and a myeloproliferative disorder
recently supplanted by the International Prognostic Scoring such as myeloid metaplasia. The absence of organomegaly and
System (IPSS).' The latter has been thoroughly validated the presence of characteristic cytogenetic anomalies support
and offers a more precise estimate of overall survival than the the diagnosis of MDS.
previously published systems. The IPSS is based on the num-
ber of peripheral blood cytopenias, the bone marrow blast cell MDS with Thrombocytosis
count, and cytogenetic results (Table 19-7). In addition, the
quantitative assessment of Wilms’ tumor gene (W771) expres- Cytogenetic analysis is mandatory to establish the diagnosis,
sion by real-time quantitative polymerase chain reaction as this presentation is associated with either the Sq— syndrome
(RQ-PCR) has recently proved to be a useful molecular or a translocation or other anomaly involving chromosome
marker for risk assessment in MDS.'” 3q. Essential thrombocytosis should be considered, and the

RARS
RA
RAEB
CMML
RAEB-t

10 20 30 40 50 60 0 10 20 30 40 50 60
Survival in months MDS subtypes Leukemic progression in %

Figure 19-45 m Survival and leukemic progression for each subgroup of MDS. Data pooled from several series with a total of 3554 patients.
432 Chapter 19 Myelodysplastic Syndromes

§ tapie 19-7 International Prognostic Scoring System (IPSS) for N 1:

Percentage of Bone Marrow Blasts, Karyotype, and BI
Score 0 0.5

Blasts <5) Sy Ko)

Karyotype Good Intermediate
Cytopenias O/1 2/3

Karyotype:
“Good” = normal, —y, del (5q) only, del (20q) only
“Poor” = complex (= 3 abnormalities) or chromosome 7 anomalies
“Intermediate” = other abnormalities
Cytopenias = Hb < 100 g/L; neutrophils < 1.5 X 10°/L; platelets < 100 x 10°/L
*This group is recognized as AML in the WHO classification.

Median Survival by IPSS Score According to Age Group

Overall Score/Risk < 60 years > 60 years > 70 years

Score 0 = low risk 11.8 4.8 9

Score 0S:0n 1:0}
Intermediate risk | Ze, 4.4
SCOLes On 2: 0k
Intermediate risk 2 13
Score = 2.5 = high risk 0.4

Sources: From Greenberg, P, et al: International scoring system for evaluating prognosis in myelodysplastic syndromes.
Blood 89:2079, 1997; and Heany, ML, and Golde, DW: Myelodysplasia (review article). N Engl J Med 340:1649, 1999.

documentation of the JAK2V617F mutation will orient the t(9;22) chromosome abnormality by standard cytogenetics,
diagnosis toward a myeloproliferative disorder.'’ molecular studies by FISH or PCR techniques may be neces-
sary. CMML with the t(5;12)(q33;p13) and other chromoso-
mal translocations involving tyrosine kinase genes give rise to
MDS with Features of Chronic a clinical picture that is closely related to CML.'°* According
Myelogenous Leukemia to the WHO, MDS falling into this category should be classi-
fied as myelodysplastic/myeloproliferative diseases.
Because CMML may present with a very high WBC count
and a significant number of immature myeloid cells in the
peripheral blood, this disease may mimic CML. Cytogenetic MDS versus Acute Erythroid Leukemia
studies are of major importance to distinguish between these
two entities. The finding of t(9;22) is diagnostic of CML. MDS are often difficult to differentiate from acute erythroid
Because 5% to 10% of patients with CML may not exhibit the leukemia when the total population of marrow erythroblasts
exceeds 50% of all nucleated cells. In that situation, the num-
AML FAB M2 with less
ber of blasts may not exceed the minimal requirement of 20%
than 30% blasts (WHO) or 30% (FAB) of all nucleated cells. FAB and WHO
AML FAB M6 when
have suggested that to establish the correct diagnosis, the
MDS with PNH features erythroblasts are >50% number of blasts should be calculated as a percentage of non-
erythroid cells (NEC). If the percentage exceeds 20% (WHO)
or 30% (FAB) of NEC, the diagnosis is AML of acute ery-
throid leukemia (WHO) or AML M6 (FAB) rather than MDS
(Fig. 19-45).

Treatment
Allogeneic stem cell transplantation is the only therapeutic
MDS with hypocellular marrow MDS with fibrosis modality with the potential to cure MDS. However, as most
MDS with thrombocytosis patients with MDS are elderly, they are not amenable to
transplantation and are therefore incurable. As there is signif-
Figure 19-44 @ Overlap between MDS and other hematologic
disorders. AA = aplastic anemia; MPD = myeloproliferative icant prognostic heterogeneity among MDS patients, the
disorder; PNH = paroxysmal nocturnal hemoglobinuria; AML = IPSS is an invaluable tool that helps tailor therapy to the pre-
acute myeloid leukemia. dicted prognosis. In general, elderly patients in the IPSS low
 Chapter 19 Myelodysplastic Syndromes 433

CELLULAR BONE MARROW ASPIRATION Supportive Care and Hematopoiesis-

Improving Therapies
ERYTHROBLASTS % ANC
For elderly patients with good prognosis MDS, the main-
stay of treatment consists of supportive measures. Transfu-
<50% erythroblasts 250% erythroblasts sion of red cell concentrates may alleviate symptoms
related to anemia. Chelation therapy with desferrioxamine
COCR E ERS SEN RRA DTS aq
or novel oral chelators may be indicated for heavily trans-
Blasts
% ANC | Blasts
% NEC |
PERV iRy Tareas Sea

fused patients with otherwise favorable prognosis, to pre-

vent secondary hemochromatosis.'"” Some groups begin
chelation when patients have received 20 to 25 units of
BL 230%* BL <30%* BL <30%* BL 230%*
BL 220%** BL <20%** BL <20%** BL 220%** red cells. Platelets can also be transfused to patients
who bleed.
Hematinics such as vitamin B,,, folic acid, and pyridox-
ine are notoriously ineffective in the absence of a documented
deficiency. Steroid or androgen therapy has not been associ-
ated with satisfactory responses.
ANC = All Nucleated B.M. Cells AML = Acute Myeloid Leukemia The use of growth factors has been widely studied in
NEC = Non-Erythroid Cells MDS = Myelodysplastic Syndrome MDS. Erythropoietin (Epo) significantly increases the
BL = Blast cells “FAB = French American British
“WHO = World Health Organization hemoglobin level in a minority of patients.''? Good respon-
ders are usually not transfusion dependent, have an inap-
Figure 19-45 m Algorithm to distinguish MDS from different types propriately low endogenous level of Epo, and a diagnosis of
of acute myeloid leukemia (AML) according to FAB criteria.
RA. The addition of granulocyte colony-stimulating factor
(G-CSF) is associated with an improved erythroid response
or intermediate-1 risk category should receive treatments to Epo.''! G-CSF may also be used to restore and maintain
that aim to improve their hematopoiesis. On the other hand, neutrophil counts in severely neutropenic MDS patients.
in younger patients’ or in patients belonging to the IPSS However, given the prohibitively high cost of long-term
intermediate-2 or high-risk category, every effort should be G-CSF treatment, it is generally employed on a short-term
made to try to improve survival. Treatment options are sum- basis, mainly during episodes of severe infection. Other
marized according to risk category in Figure 19-46. cytokines (such as IL-3) or combinations of cytokines
have also been evaluated, but proved to be of only modest
benefit.
DETERMINE IPSS SCORE Immunosuppressive therapy using antithymocyte globu-
lin (ATG) has initially been associated with clinically interest-
ing results in patients with a normocellular or hypocellular
marrow, no ringed sideroblasts, and a normal karyotype.''”
More recent studies have shown that the best predictors of
response are (1) age younger than 70 years and (2) HLA DR
15 allele. Indeed, 60% of these patients may become transfu-
sion independent for several years.''? Treatment with
cyclosporine A has also been associated with hematological
improvement in patients with hypoplastic marrows.''* Other
immune response modifiers such as interferon have not given
adequate clinical responses.
Differentiating agents such as retinoids have proved
Lenalinomide
(5q-, normal Hypomethylating unsuccessful. Low-dose cytarabine therapy has led to a pos-
karyotype) agents itive outcome in 10% to 25% of patients so treated, but no
survival advantage could be demonstrated over supportive
ATG Induction therapy.''°
(HLA DR15, chemotherapy The most significant pharmacologic development for
<70 years old) (young, good
cytogenetics) low-grade MDS patients has been the introduction of
lenalidomide, a derivative of thalidomide. Thalidomide has
Transplantation
(if progression,
shown modest beneficial effects in MDS patients, but also
Investigational
life-threatening protocols significant side effects.''® Lenalidomide is a structural analog
cytopenias) of thalidomide that lacks teratogenicity and most neurological
side effects of the parent drug. The exact mechanism of action
Figure 19-46 ® Treatment options for MDS patients according to of lenalidomide is not fully characterized, but the molecule
IPSS. exhibits immunoregulatory and antiangiogenic effects, which
434 Chapter 19 Myelodysplastic Syndromes

are likely to contribute to its clinical efficacy. The erythroid

response to lenalidomide is very promising, with 85% of
patients with the Sq- syndrome experiencing transfusion
Case Study
independence and at least a 20 g/L increase in hemoglobin
A 72-year-old man was admitted to the hospital with
level. Although 57% of low-grade MDS patients with a nor-
complaints of increasing weakness. Physical examination
mal karyotype obtained a similar response, only 12% of those revealed pallor but no organomegaly. His previous medical
with an abnormal karyotype responded significantly.* The history was unremarkable.
most striking effect was the cytogenetic response in 75% of The laboratory results were as follows:
patients with the 5q— syndrome.
CBC
WBC 2907
RBC ZAB X10
Therapies Oriented Toward Improving Hgb 91 g/L
Survival Het 0.27 g/L
MCV WL
Patients with advanced MDS should be considered for allo- MCH 37.4 pg
geneic stem cell transplantation, the only curative treatment MCHC 337 g/L
available for MDS. However, stem cell transplantation is usu- RDW 19.0%
Rio 90 X 10°/L
ally limited to patients younger than 60 years of age with a
PDW 17.4%
matched sibling donor, or to patients younger than 55 years of
age if the donor is matched but unrelated to the recipient. The Blood Smear Differential and Morphology
overall survival is between 55% and 60% for patients with Blasts 2%
Promyelocytes 0%
low-risk MDS, 40% for intermediate-risk MDS, and only
Myelocytes 1%
20% for high-risk MDS.''’ The risk of relapse following Metamyelocytes 0%
bone marrow transplantation is less than 10% for low- and Bands 3%
intermediate-risk patients, but 30% for high-risk patients. Segmented neutrophils 69%
Bone marrow transplantation may become more widely Eosinophils 1%
Basophils 1%
applicable in the future with prophylactic and therapeutic
Lymphocytes 20%
improvements in the areas of graft-versus-host disease and Monocytes 3%
infection, and with the introduction of nonmyeloablative
preparative regimens (mini-transplantation), associated with
fewer toxic side effects than standard regimen transplantation. Additional findings at the blood smear: occasional
pseudo-Pelger—Huét neutrophils with hypogranulation,
High-dose chemotherapy (without transplantation) with
occasional giant platelets, erythrocyte anisomacrocytosis
regimens similar to those used to induce remission in acute
with occasional basophilic stippling.
leukemia has met with disappointing results. Although a
hematological response can be achieved in 20% to 50% of
Bone Marrow Smear
patients, these responses are short lived, with fewer than 20% Hypercellular
of patients still in remission 2 years after such therapy." Blast cells (type I and type IL): 23%; a few blasts with
Younger age, presence of Auer rods, and normal karyotype Auer rods
tend to be associated with a better response rate. Consolida- Myeloid dysplasia with immature cells with asynchronous
nuclear-cytoplasmic maturation
tion with autologous transplantation in patients achieving a
Pelgeroid cells with hypogranulation
complete response is being investigated in some centers. Erythroblasts (35%) with megaloblastoid changes
Differentiation therapy with agents acting on the reactiva- Micromegakaryocytes (mononuclear)
tion of genes at the epigenetic level has shown promising results Prussian blue stain showed 10% ringed sideroblasts
for patients with advanced MDS not amenable to transplant. (type III)
Targeting genes that have become hypermethylated (and thus Cytogenetics
underexpressed) during disease progression with hypomethyla- Complex karyotype, including monosomy 7
tion agents such as 5-azacytidine (Vidaza) or decitabine (Daco- Diagnosis: RAEB-t (FAB); acute myeloid leukemia
gen) has been associated with hematological response and (WHO)
improved time to progression to leukemia.''*:'”° Larger, random- Treatment: Supportive therapy (transfusion)
ized clinical trials are underway to evaluate further the therapeu-
tic benefit of these treatments.
Recent advances in the understanding of the pathogen-
esis of MDS have paved the way to the development of
novel drugs that may prove promising to improve the qual-
ity of life and potentially the survival of MDS patients,
expanding therapeutic options for MDS and generating new
hope for patients.
 Chapter 19 Myelodysplastic Syndromes 435

Ques tions
1.Which type of anemia is most common in MDS? : Which of the following agents may lead to secondary
~ a. Microcytic, hypochromic MDS (sMDS)?
b. Normocytic, hypochromic a. Hydrocortisone
c. Macrocytic, normochromic b. Alkylating agents
d. Dimorphic c. Irradiated blood components
2. Which of the following morphological abnormalities are d. Folic acid
helpful in the diagnosis of MDS? . Which of the following 1s most likely to affect prognosis
CO

a. Pseudo-Pelger—Huét and hypogranulation in MDS?

b. Ringed sideroblasts a. Leukopenia
c. Micromegakaryocytes, monolobular megakaryocytes b. Increased bone marrow myeloblasts
d. All of the above c. Erythroid hyperplasia
3. Which of the following criteria will differentiate RAEB-t d. Thrombocytosis
from AML of the M2 type? uh In female patients with RA and elevated platelet counts,
a. Thrombocytopenia which chromosome abnormality is most often found?
b. Anemia a. Sq-
c. The finding of t(8;21) b. t(8;21)
d. Blasts less than 5% c. 7q-
4. Which of these cell surface antigens is of prognostic sig- Gatley
si7))
nificance in MDS? 10. Which of the following biologic or genetic anomalies are
anc Dio important in the pathogenesis of MDS?
b. CD HLA-DR a. Chromosomal translocations
Ones b. Chromosomal deletions
+, €D33 ‘ c. Hemolysis
5. Which one of these MDS subgroups has the most favor- d. Increased intramedullary apoptosis
able prognosis? . Which of the MDS subtypes is more closely related to
a. RAEB the chronic myeloproliferative disorders?
b. CMML a. RAEB
c. RA b. CMML with WBC > 13 X 10°/L
d. RARS c. CMML with WBC < 13 X 10°/L
6. Which type of treatment may offer a cure in patients d. RARS
with MDS? . Which MDS scoring system is most often used today?
a. Chemoradiotherapy a. FAB
b. Allogeneic bone marrow transplantation b. IPSS
c. Autologous bone marrow transplantation c. Sanz
d. Growth factors such as G-CSF d. Bournemouth

See answers at the back of this book.

436 Chapter 19 Myelodysplastic Syndromes

w RARS shares all the morphological features of RA, with

~'? SUMMARY CF the exception of more than 15% ringed sideroblasts in the
bone marrow. Cytopenias are relatively infrequent.
a Myelodysplastic syndromes (MDS) are clonal hematologi-
cal malignancies characterized by peripheral blood w RAEB is associated with significant dysplasia. Immature
cytopenias, dysplastic cells in peripheral blood and bone granulocytes and occasional blasts (5%) may be seen in
marrow, and a propensity to transform into acute leukemia. the peripheral blood. Hyposegmented and hypogranulated
granulocytes are frequent. Thrombocytopenia with giant
g Diagnosis of MDS is based on the morphological study of
and hypogranular platelets is a common finding. The
blood and bone marrow smears.
number of bone marrow blasts increases to between 5%
a MDS is characterized by multilineage morphological and 20%. Dysmegakaryopoiesis and dyserythropoiesis
abnormalities including dyserythropoiesis, dysgranu- with ringed sideroblasts are variable.
lopoiesis, and dysmegakaryopoiesis. = RAEB-t is closely related to RAEB. The number of blasts
# Anemia is present in 90% of cases and is often macrocytic increases to between 20% and 30% in the bone marrow
or normocytic with decreased reticulocyte count. and to more than 5% in the peripheral blood.
g The erythrocyte features that may be found in the periph- mg CMML exhibits the same morphological abnormalities as
eral smear are macrocytosis, a dual red cell population, red the other types of MDS. Patients often present with
cell fragments, dacryocytes, ovalocytes, acanthocytes, Pap- hepatosplenomegaly. White blood cell counts are high,
penheimer bodies, Howell—Jolly bodies, and basophilic with an increase in monocytes. The bone marrow is usu-
stippling. ally hypercellular, with monocytosis and a blast cell count
mw Neutropenia is found in 60% of patients with MDS. of 1% to 20%.
Nuclear and cytoplasmic dysplasias are found in more m=The WHO classification of MDS consists of eight
than 90% of cases. Granulocytes show variable degrees of subtypes:
hyposegmentation. Granulocyte hypergranulation is a rare
¢ Refractory anemia (RA)
finding.
¢ Refractory anemia with ringed sideroblasts (RARS)
m= Thrombocytopenia is present in almost 60% of
patients. Platelet morphology includes gigantism and ¢ Refractory cytopenia with multilineage dysplasia (RCMD)
hypogranulation. Refractory cytopenia with multilineage dysplasia and
m The bone marrow smear shows erythroid hyperplasia with ringed sideroblasts (RCMD-RS)
megaloblastic changes such as asynchronous cytoplasmic Refractory anemia with excess blasts-1 (RAEB-1)
maturation, internuclear bridging, nuclear budding, and
Refractory anemia with excess blasts-2 (RAEB-2)
Howell—Jolly bodies. Iron staining may reveal abnormal
sideroblasts. Megakaryocyte abnormalities may include ¢ Myelodysplastic syndrome-unclassified (MDS-u)
micromegakaryocytes, mononuclear megakaryocytes, or ¢ MDS associated with isolated del (Sq)
megakaryocytes with multiple small nuclei detached from
w The morphologic abnormalities are as described in the FAB
one another by a thin strand of nuclear materials.
classification. The WHO proposal has refined the subtypes
m The FAB classification of MDS consists of five subtypes: of the original FAB classification, in particular by distin-
Refractory anemia (RA) guishing refractory cytopenia with multilineage dysplasia
(RCMD) from refractory anemia (RA), and by separating
Refractory anemia with ringed sideroblasts (RARS)
out patients with an isolated 5q-. The RAEB-t subtype is
Refractory anemia with excess of blasts (RAEB) eliminated, and replaced by acute myeloid leukemia. The
Refractory anemia with excess of blasts in transforma- CMML subtype is also eliminated, and placed in the group
tion (RAEB-t) of myelodysplastic/myeloproliferative disorders.

Chronic myelomonocytic leukemia (CMML) m Cytochemical abnormalities in MDS include decreased

or abnormal positivity of enzymatic activities of myeloper-
¢ The distinction between each subtype is made on
oxidase, chloroesterase, alkaline phosphatase, Sudan black
examination of bone marrow aspirate stained with
B, and dual esterase in myeloid and monocytic cells.
Wright—Giemsa and Prussian blue.
= Bone marrow biopsy is helpful to estimate cellularity, his-
@ RA is characterized by macrocytic or normocytic anemia
tological changes, abnormal localization of immature pre-
with variable degrees of morphological abnormalities.
cursors (ALIP), and myelofibrosis.
Leukopenia and thrombocytopenia are common findings.
Dysgranulopoiesis and dysmegakaryopoiesis are less # Clonal cytogenetic abnormalities are detected in 30% to
common. The bone marrow is usually normocellular or 60% of patients with MDS. The most frequent abnormali-
hypocellular with erythroid hyperplasia and a mild ties are deletion of long arm of chromosome 5 (Sq—), mono-
dyserythropoiesis. There are fewer than 5% blasts and somy 7 (—7), trisomy 8 (8+), and deletion of part of
fewer than 15% ringed sideroblasts in the bone marrow.
continued
 Chapter 19 Myelodysplastic Syndromes 437

chromosomes 11, 12, and 20. There are no chromosomal @ Secondary myelodysplastic syndromes (SMDS) occur after
abnormalities specifically associated with any given sub- significant exposure to chemotherapy, radiotherapy, or other
group of MDS. However, the Sq— anomaly is more frequent
toxic agents. The laboratory findings, clinical manifesta-
in RA. tions, and evolution resemble those of de novo MDS, except
for a higher frequency of chromosomal abnormalities and a
m The 5q— syndrome has specific clinical features that greater tendency to early leukemic transformation.
include macrocytic anemia, normal or elevated platelet
@ MDS occurs rarely in children. RAEB and RAEB-t
count, mild leukopenia, and in the bone marrow,
(AML) are the most frequent.
monolobular or dwarf megakaryocytes and erythroid
hypoplasia. Patients are predominantly female. w Clinical manifestations of patients with MDS are related to
the degree of anemia, neutropenia, or thrombocytopenia.
m Cell surface antigens of peripheral blood cells, bone mar-
row, or biopsy specimens can be studied by flow cytome- # Progenitor cell growth in semisolid media is dramatically
try or immunocytochemistry via alkaline phosphatase decreased in MDS, with the exception of CMML.
antialkaline phosphatase (APAAP) and other techniques. # Several factors influence the prognosis in MDS. The most
w Generally, the ability of hematopoietic progenitor cells to significant are cytogenetic abnormalities, severity of
form colonies in vitro is decreased or absent. Aberrant cytopenias, age of the patient, and percentage of blasts.
growth patterns are mostly observed in RAEB and @ The International Prognosis Scoring System (IPSS) helps
RAEB-t. In CMML, increased colony growth is noted. determine the prognosis of individuals with MDS.
# Functional abnormalities of red blood cells, white blood mAt the present time, allogenic hematopoietic stem cell
cells, and platelets are frequently observed in MDS. transplantation is the only curative treatment for MDS.

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. Miller, JS, et al: Myelodysplastic syn- and the International Prognostic Scoring improves patient outcomes in
drome after autologous bone marrow System Score in patients with myelodys- myelodysplastic syndromes: Results of
transplantation: An additional late com- plastic syndromes. J Clin Oncol 21:1988, a phase III randomized study. Cancer
plication of curative cancer therapy. 2003. 106:1794, 2006.
Blood 83:3780, 1994. 106. Pagliuca, A, et al: Myelofibrosis in pri-
89. Darrington, DL, et al: Incidence and char- mary myelodysplastic syndromes: A
acterization of secondary myelodysplastic clinico-morphological study of 10
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leukemia following high-dose chemora- 107. Tefferi, A, and Pardanani, A: Mutation
diotherapy and autologous stem-cell screening for JAK2V6/7F: When to
 Chapter

Chronic Lymphocytic
Leukemia and Related
Lymphoproliferative
Disorders
Laurel D. Holmer, MEd, MT(ASCP)SH
Carlos E. Bueso-Ramos, MD, PhD

Introduction OBJECTIVES
Chronic Lymphocytic At the end of this chapter, the learner should be able to:
Leukemia/Small
Lymphocytic Lymphoma 1. List general features of chronic lymphocytic leukemia.
Etiology and 2. Name laboratory methods used to study lymphocytes in lymphoproliferative disorders.
Pathophysiology
Immunologic Features and 3. List diagnostic criteria of chronic lymphocytic leukemia.
Methods for Studying 4, Describe treatment for chronic lymphocytic leukemia.
Lymphocytes
Clinical Features 5. Explain differential diagnostic criteria that are used to characterize lymphoproliferative
Laboratory Features disorders.
Chromosomal Abnormalities
Clinical Course, Prognostic
Factors, and Staging
Treatment
Differential Diagnosis
Case Study 1
Case Study 2

Introduction prolonged cell survival, and a transformation phase, with the

frequent occurrence of extramedullary proliferation and an
Chronic lymphoproliferative disorders are clonal proliferations increase in large, immature cells.
of morphologically and immunophenotypically mature B_ or In recent years, considerable progress has been made in
T lymphocytes. Although the morphological and cytologic fea- our ability to diagnose and classify hematopoietic malignan-
tures of chronic lymphoproliferative disorders are critical to the cies accurately. Through cytogenetics and molecular biology,
subclassification of these disorders, the final diagnosis repre- it has been shown that many hematopoietic neoplasms are
sents a synthesis of morphological, immunologic, cytogenetic, associated with a unique genotypic profile. These genetic
molecular, and clinical features. Disorders affecting bone mar- lesions often have a direct bearing on the pathogenesis of the
row and peripheral blood are regarded as leukemias, whereas disease and its clinical behavior. Similarly, the development
diseases affecting lymph nodes and other extramedullary sites of widely available monoclonal antibodies has allowed the
are lymphomas. The chronic lymphoid leukemias may proceed identification of a unique immunophenotypic profile for most
through different phases: an early phase in which tumor cells are leukemias and lymphomas. The use of these techniques
predominantly small in size, with a low proliferation rate and enhances both diagnostic accuracy and reproducibility,

440
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 44]

Chronic Lymphocytic Leukemia/Small

Lymphocytic Lymphoma
Chronic lymphocytic leukemia (CLL)/small lymphocytic
lymphoma is included in a general category of conditions
known as the lymphoproliferative disorders (Table 20-1).
Chronic lymphocytic leukemia is the most common type of
leukemia in older adults and is most frequently a neoplasm of
B lymphocytes (B-CLL). A malignant proliferation of T lym-
phocytes can occur, but this is less common. The hematologic
abnormalities of CLL are characterized by a peripheral blood
and bone marrow lymphocytosis. In contrast, small lympho-
cytic lymphoma involves mainly lymph nodes and, to a lesser
extent, peripheral blood and bone marrow. Morphologically,
the lymphocytes are small or slightly larger than a normal
lymphocyte and have a hypercondensed, almost “soccer Figure 20-1 ™ Photomicrograph of peripheral blood smear from a
ball”-appearing nuclear chromatin pattern. Bare nuclei called patient with chronic lymphocytic leukemia (CLL). Note the character-
istic mature-appearing lymphocyte morphology with hypercondensed
smudge cells are common (Fig. 20-1). Morphological hetero- nuclear chromatin creating a “soccer-ball” pattern. Two smudge cells
geneity in CLL does exist and has been addressed by the are also seen. Note the lack of platelets in this thrombocytopenic
French—American—British (FAB) group.' Some are indistin- patient. A, denotes lymphoblasts; B, lymphocytes; and C, smudge cells.
guishable from normal mature lymphocytes when examined
with Wright stain. Alternatively, CLL cells may show the fol-
The consequences of the accumulating lymphocyte mass
lowing: 1) increased prolymphocytes (greater than 10%), or
in CLL include neutropenia, anemia, and thrombocytopenia.
2) features of activated, proliferating lymphocytes, which are
Lymphadenopathy, splenomegaly, or both may be present.
larger in size (greater than two red blood cells), have exagger-
Altered humoral immunity in patients with CLL results from
ated nuclear irregularities, lobulations or deep nuclear folds
suppression of all classes of immunoglobulin (Ig), leading to
(greater than 15% cleaved, “reider cells”), open chromatin,
hypogammaglobulinemia and a subsequent increase in sus-
one or two nucleoli, moderate amounts of cytoplasm, or
ceptibility to infections. Another important complication of
3) increased lymphoplasmacytoid cells (designated Mixed
altered immunity that can develop in the patient with CLL is
type in the FAB classification). This subset of activated, in
autoimmune disease. The production of autoantibodies may
cycle CLL cells feed the pool of small G,/G, CLL cells.*
lead to idiopathic thrombocytopenic purpura (see Chap. 25),
or autoimmune hemolytic anemia (see Chap. 13) to further
compromise the patient’s hematologic status.
Patients with CLL are typically older than 50 years at the
time of diagnosis and frequently have a prolonged survival.
Although some patients may die within a few years of diag-
Mature B-Cell Nessie nosis, more often they succumb to an unrelated disorder asso-
Chronic lymphocytic leukemia/small lymphocytic lymphoma ciated with the elderly. Generally, 50% of patients with CLL
B-Cell chronic prolymphocytic leukemia are living 5 years after diagnosis, whereas 30% of patients can
Lymphoplasmacytic lymphoma/immunocytonia
expect a survival of 10 years or more.
Mantle cell leukemia/lymphoma
Follicle center lymphoma, follicular lymphoma
Nodal marginal zone B-cell lymphoma
Extranodal marginal zone B-cell lymphoma Etiology and Pathophysiology
Splenic marginal zone lymphoma (+ villous lymphocytes)
No specific etiologic agent or cause of CLL is currently known.
Hairy-cell leukemia
A possible viral etiology has been investigated ever since the
T-Cell and Putative NK-Cell Neoplasms isolation of human T-lymphotropic virus type | (HTLV-1), a
T-cell prolymphocytic leukemia type C retrovirus from the leukemic cells of patients with T-cell
T-cell large granular lymphocytic leukemia
malignancies.*° The B-CLL cells from some patients appear to
T-cell type
NkK-cell type be a malignant transformation of an antigen-committed B cell
Aggressive NK-cell leukemia responding to the HTLV-1 infection, suggesting an indirect role
Mycosis fungoides/Sézary syndrome for this retrovirus in leukemogenesis. Infection with HTLV-1
Peripheral T-cell lymphoma, unspecified has preceded the development of CLL in some patients.°
Adult T-cell lymphoma/leukemia
In its classic form, this neoplastic disorder is character-
NK = natural killer. ized by the gradual accumulation of small mature B cells
Source: Jaffe ES, et al (Eds): World Organization Classification of (most of which are in G,/G, phase of the cell cycle), which are
Tumors. Pathology and Genetics of Tumors of Haematopoietic and
Lymphoid Tissues. IARC Press, Lyon, France, 2001.
long-lived and immunologically dysfunctional lymphocytes
in the peripheral blood and bone marrow.’ B-CLL cells show
442 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

failed programmed cell death (PCD), also called apoptosis.* accompanied by a large fraction of CLL cells in S/G,, phase
In essentially all self-renewing tissues, new cell production is of the cell cycle.
normally offset by a commensurate amount of cell destruction Expression of some of these cell cycle regulatory fac-
through PCD. Imbalances in the activities of opposing genes tors, e.g., cdk4, cyclin E, and cyclin D2 as well as other cell-
that either promote or block physiologic apoptosis can, there- cycle dependent factors such as nucleoside diphosphate
fore, slow or halt the rate of cell turnover, creating a selective kinase NM23-F2 in CLL lymphocytes led to the hypothesis
survival advantage for a particular clone that permits expan- that CLL cells are not quiescent but are recruited to the cell
sion at the expense of its normal neighbors.* cycle and blocked in its early G, phase. These observations
Most B-CLL cells have been reported to contain high show that the accumulation of CLL lymphocytes is due not
levels of the antiapoptotic protein BCL2.? The mechanisms only to defective apoptosis but also to an abnormality of the
responsible for the high amounts of BCL2 observed in more regulation of cell proliferation.
than 80% of B-CLL cells remain unknown, but only rarely do Additional infiltration of the lymph nodes and spleen by
they involve rearrangements of the BCL2 gene as a result of the malignant lymphocytes occurs in 50% of patients,
chromosomal translocations.'° whereas cutaneous invasion occurs in 5% of patients.'° As the
Although the pathogenesis of the disease remains largely bone marrow becomes more extensively infiltrated by the
unknown, previous investigations have focused on the defec- leukemic clone, marrow replacement results in anemia,
tive apoptosis of the malignant cells that seems to play an thrombocytopenia, and neutropenia (Fig. 20-2). Organ infil-
important role in disease progression and chemotherapy resis- tration can lead to massive adenopathy with splenomegaly,
tance.'' There is additional evidence that the deregulation of hypersplenism, and subsequent peripheral cytopenias. An
cell cycle regulatory genes may contribute to the expansion of increased tendency for hemorrhage further contributes to ane-
the malignant clone. CLL consists not only of resting lympho- mia and compromises hemostasis.
cytes, but also of proliferating leukemia cells, which have Patients with CLL have significantly impaired immuno-
been des¢ribed in proliferation centers in the lymph node and logic activity. Hypogammaglobulinemia is found in approxi-
the bone marrow. This proliferative compartment is thought mately 50% of patients with CLL. The deficiency in Ig leads to
to be important for disease progression.'*!? infections with a variety of agents. Bacterial infections, espe-
Cell cycle progression in mammalian cells is governed cially of the respiratory tract, urinary tract, and skin, as well as
by interactions between cyclins, cyclin-dependent kinases viral infections such as herpes zoster and herpes simplex are
(CDKs) and cyclin-dependent kinase inhibitors. Individual common and dramatically contribute to patient morbidity and
cyclins act at different phases of the cell cycle by binding to mortality (Fig. 20-3). Autoimmunity is a phenomenon that is
suitable cdks and allowing the subsequent activation of the frequently seen in CLL, with 15% to 35% of patients develop-
cyclin-cdk complexes. The D-type cyclins (D1, D2, and D3) ing autoimmune hemolytic anemia at some time during the
regulate the G, phase progression by binding to and stimulat- course of the disease.'® Antibodies produced against red blood
ing the activities of their catalytic partners: cdk4 and cdk6. cells and detected with the direct antiglobulin (Coombs) test
Kinases cdk4 and cdk6 phosphorylate pRB protein. This may precede, simultaneously occur with, or follow the devel-
process leads to inactivation of pRB and release of a tran- opment of CLL. Red cell aplasia is a rare occurrence.'’ Autoan-
scription factor E2F, which activates transcription of the tibodies to platelets and neutrophils may also develop and lead
components of the DNA replication machinery, thereby com- to immune thrombocytopenic purpura (ITP) and neutropenia.
mitting the cell to S phase of the cell cycle. A family of pro- The production of autoantibodies coupled with marrow crowd-
teins termed cdk inhibitors exerts the inhibitory effect on the ing and hypersplenism can lead to low peripheral platelet and
cdks or cyclin—cdk complexes. Two classes of these inhibitors
have been described so far. The first one (p15, p16, and p19)
is specific for pRB kinases, 1.e., cdk4 and cdk6, the second
one (p21, p27, and p57) has a broader range of activity and
inhibits most of the known cdk-cyclin complexes. D-type
cyclins regulate early G, progression and are thought to be
growth factor sensors linking extracellular signals to the cell
cycle. In contrast to normal B-cells, cyclin D2 mRNA but not
cyclin D3 mRNA is overexpressed in CLL cells.'* Expression
of D-type cyclins is needed to activate cyclin-dependent
kinases and mediate phosphorylation of target proteins.
Recent research has indicated that cyclin D2 and D3 are
both key regulatory cyclins in CLL cells with cdk4 as
the corresponding catalytic partner.'° Phosphorylation of the
retinoblastoma (RB) protein identifies the restriction point in
G, of the cell cycle. The expression of RB protein is altered
in CLL. However, when an appropriate second signal is pro- Figure 20-2 @ Photomicrograph of bone marrow aspirate smear
vided, the proliferative hyporesponsiveness of CLL cells can from a patient with CLL. Note monotonous appearance of mature-
be overcome and rapid phosphorylation of the RB protein is appearing lymphocytes with condensed nuclear chromatin.
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 443

Immune paresis in some patients may include the production of

paraproteins. Bence—Jones paraproteinemia has been reported
in up to 51% of patients with B-CLL," and heavy-chain para-
proteins, either IgM or IgG, can be detected. The pathophysiol-
ogy of CLL is summarized in Figure 20-4.

Immunologic Features and Methods

for Studying Lymphocytes
In normal adult peripheral blood, up to 20% of the circulating
lymphocytes have surface Ig (sIg) and are B cells, 61% to 89%
are T cells, and up to 22.3% are natural killer (NK) cells. On the
basis of morphological features alone, it is not possible to dis-
tinguish B cells from T cells. When a lymphoproliferative
Figure 20-5 ™ Severe generalized herpes zoster with a varicelli- process exists, it is important to be able to characterize the
form rash in a patient with CLL. (From Henderson, ES: Diagnosis of
leukemia. In Gunz, FW, and Henderson, ES [eds]: Leukemia, ed 4.
nature of the lymphocytes involved. A number of methods are
Grune & Stratton, New York, 1983, p 409, with permission.) available to study lymphocytes in lymphoproliferative disor-
ders such as CLL and are listed in Table 20-2.
For the most part, CLL can be diagnosed morphologi-
neutrophil counts. Although the leukemic clonal B cells from cally. An immunophenotypic characterization of the neoplasm
CLL patients often are the source of pathogenic autoantibodies as either B cell or T cell using the monoclonal antibodies
that may react with antigens present on red blood cells or (mAb) available for detecting differentiation antigens (cluster
platelets, or both, it is the nonmalignant polyclonal bystander B differentiation CD antigens), as shown in Table 20—3, confirms
cells that have been shown to produce autoantibodies that con- the diagnosis. As lymphocytes mature from pluripotent stem
tribute even more significantly to autoimmune disease.'* Inter- cells and migrate to lymphoid tissue, they acquire a variety of
estingly, the relatives of patients with CLL have also been developmental markers that are helpful in identifying lympho-
shown to have an increased risk of autoimmune diseases. cyte subpopulations.

Massive adenopathy

Infiltration of lymph nodes

XexotUN TOW-Valo)\Ke)ilWo)\(em WI) ip )y-\\[0)

POORLY FUNCTIONING LYMPHOCYTES

Impaired Splenomegaly
immunologic
activity

Hypersplenism

esi
Hemorrhage

Hypogammaglobulinemia

|
Infection
Thrombocytopenia
Neutropenia

Autoimmune hemolytic anemia (AIHA)

ITP
Immune neutropenia

Figure 20-4 m The pathophysiology of CLL. The three major processes that typically interact are marrow replacement by long-lived lympho-
cytes, hypersplenism, and autoimmunity. ITP = immune thrombocytopenic purpura.
444 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

Table 20-2 Methods Used to Study Lymphocytes in Lymphoproliferative Disorders ne

Method Marker Detected or Feature Demonstrated

Immunoperoxidase and flow cytometry Vator Piticrentiation antigens on B cells siaionT ak usingit anes antibodies
(see Table 20-3)
Molecular probes Rearrangements of the B-cell Ig and T-cell receptor genes
Cytogenetics Consistent chromosomal abnormalities such as t(8;14)-B-ALL, Burkitt’s lymphoma;
t(14;18)—follicular lymphoma; trisomy 12—CLL, SLL; t(11;14)—mantle cell
lymphoma; t(4;11)-ALL-L2
Cytochemistry Tartrate-resistant isoenzyme 5 of acid phosphatase in hairy cells
Localized alpha-naphthol acetate esterase positivity in Golgi area of T cells, i.e., T-ALL,
Sézary cell, T-CLL
Electron microscopy Nuclear and cytoplasmic ultrastructure such as nuclear whorls in Sézary cells and
_Tibosomal lamellar cytoplasmic aggregates iin hairy cells

T-ALL= eanT- acute

& sip eCeLeE fenkema: B-ALL= B-lineage acute lymphoblastic leukemia; SLL= feu
lymphocytic leukemia

Immunophenotypic marker expression and gene differentiation. Studies have shown, however, that under certain
rearrangement during normal B-cell ontogeny is shown in in vitro conditions such as stimulation with phorbol ester, typi-
Figure 20-5. The malignant B lymphocytes of CLL do not cal CLL cells can undergo transformation to more mature levels
progress normally to the final stages of B-cell development, of B-cell development.*??' Genetic recombination in pre-B cells
namely the formation of plasma cells, but rather appear to be in the bone marrow generates an immature B cell that expresses
developmentally arrested at an earlier B-lymphocyte stage of a functional Ig on the surface, IgM or IgM and IgD, which then

“Table 20-3 Cluster Differentiation (CD) Markers and Clinical Application ate a
Markers (CD Designation) — Monoclonal Antibodies _ Clinical Application —

B Cells
CD5 Ole eent B-CLL, some NHL, T lymphomas
CD9 BA-2 (p24) Pre-B
CD10 J5, BA-3 (CALLA) Lymph progenitor, CALL, some NHL
CD19 B4, Leul2 CALEB Clik, Bapebat Cle
CD20 B1, Leul6 CALL, B-CLIE B-PLIc Hel
@p22) B3, Leul4 Late B cells, hairy cells
CD23 CD23.1 (BU38), Leu20 B-CLL, activated B cell
CD24 BA-1 Most B cells
@D25 TAC (IL-2 receptor) HEE
CD43 Leu22 B-CLL, mantle cell lymphoma
CD79 CD79 B cells
CD103 HML-1 Hairy cells

T Cells
CD1 T6 some T-€LIE, 1-PLL EALD
GD? T11, LeuS, 9.6 (E rosette) T-CLL, T-PLL, Sézary cells, LGL, ATLL
Gps eed! T-CELL, T-PLL, Sézary cells, IM
CD4 T4, Leu3 T-PLL, Sézary cells, IM, ATLL
CD5 JUL, ieewmil, MOA T-CLL, T-PLL, Sézary cells, ATLL
CD7 3A1, Leu9 T-PLL
CD8 T8, Leu2 T-CLL, some LGL, IM
CD25 Tac (IL-2 receptor) ATLL
CD57 Leu7 (HNK1) LGL

Other Cells
CD38 T10, Leul7 Plasma cells, germinal center B cells,
cortical thymocytes
CD52 _CAMPATH- 1H All lymphoid cellsand monocytes
B-CLL= B-Giee chronicfoipraplion teleukemia; NHL = non-Hodgkin’sEe iyniphonaneCALL = common acute
lymphocytic leukemia; B-PLL= B-lineage prolymphocytic leukemia; HCL= hairy-cell leukemia; T-ALL= T- lineage
acute lymphoblastic leukemia; LGL= large granular lymphocytosis (T-gamma lymphocytosis): ATLL = adult T-cell
leukemia/lymphoma; IM = infectious mononucleosis; HNK = human natural killer.
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 445

B-CELL ONTOGENY exits the bone marrow as a virgin (mature) B cell (antigen-
independent phase) and migrates to the secondary lymphoid tis-
sues. The normal B cell may undergo stimulation by antigen
(antigen-dependent phase) and enter the lymphoid follicle. At
p rearrangement this point in differentiation, the somatic hypermutation mecha-
nism is activated, introducing point mutations into the variable
K rearrangement
(V) region genes. In the presence of limiting antigen, there will
i rearrangement be subsequent selection of V gene sequences that provide
Cytoplasmic immunoglobin increased affinity for antigen. After antigen-driven B-cell prolif-
eration, B cells can initiate two pathways of maturation. Some
Surface immunoglobin
B cells revert back to small lymphocytes to generate memory
HLA-DR B cells, whereas others undergo terminal differentiation into
CD19 long-lived plasma cells.**** The postulated normal B-cell devel-
CD24 opment is shown in Figure 20-6.
The level of somatic mutation in CLL is low.* This has
CD10
led to the conclusion that the cell of origin has not encoun-
CD20 tered antigen. The subset that remains unmutated, perhaps
CD21 derived from naive B cells, appears prone to accumulate the
CD22
trisomy 12 abnormality. In contrast, the mutated subset of
CLL, perhaps derived from more mature B cells, appears
CD23
more prone to abnormalities of 13q14.**
Figure 20-5 @ Hypothetical scheme of marker expression and The characteristic immunophenotype for B-CLL, is
gene rearrangement during normal B-cell ontogeny. (From Pui, CH, expression of faint or low-density slg with kappa (Kk) or
et al: Clinical and biologic relevance of immmunologic marker stud- lambda (\) light-chain restriction and expression of B-cell-
ies in childhood acute lymphoblastic leukemia. Blood 82[2]:344, associated antigens (CD19, CD20, CD79a), CD5, CD23,
1993, with permission.)
CD43, and faint CD11c (Table 20-4). A typical example of a
«
B-CLL immunophenotype is shown in Figure 20-7.

PREFOLLICULAR FOLLICULAR :

Lymphoblasts Mature
“Virgin”
B cell

Centrocytes_
2aved cell
SPs s)
tes

Memory B
cells

Lymph Node

Figure 20-6 ™ Postulated B-lymphocyte development scheme. Lymphoblasts undergo several maturation steps in the microenvironment of
the bone marrow (BM), giving rise to mature (virgin or naive) lymphocytes. Naive B lymphocytes enter blood vessels (BV) to reach lymph
nodes and populate the mantle zone of the lymphoid follicles. These cells differentiate into centroblasts (large, noncleaved follicular center
cells) in the dark zone of the germinal center. Centroblasts undergo rearranged-immunoglobulin gene hypermutation and develop into
centrocytes (cleaved follicular center cells). Centrocytes that bind antigen on the surface of the follicular dendritic cells survive, whereas those
that fail to bind die by apoptosis. Surviving centrocytes in the light zone of the germinal center may differentiate into monocytoid B cells,
which populate the marginal zone of the lymphoid follicle, and plasma cells, which reenter the bone marrow and memory cells. Memory cells
actively recirculate between the blood, lymph, and lymphoid organs.
446 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

Table 20-4 Immunophenotypic Features andsGenetic Abnormalities of

B-Lymphoproliferative Disorders
Common Comments/ Associated Genetic
Disorder: Epenolypes » Yaniabons Abnormalities”

Ghrontc ivannacyae EDIOT CD20+, CDs+, ~ cp20+ ae Abhomnane: ar 134,

leukemia/small ED22G/2)) 2C2377, 14q, 11q
lymphocytic lymphoma CDIOG);GDilleS Er CD43.— BCL2 overexpressed Trisomy 12, 17p
clonal sIgM and sIgD weak Bright slg, CD20, FMC7
Prolymphocytic leukemia CDF CD20a-@D> (Gas) CD20 and FMC7 bright No consistent alteration
CD22, CD23); CD10); Negative Cyclin D1
bright clonal sIg
Mantle cell lymphoma CDI9 CD20st CD Saas Cyclin D1 (BCL J) t(11;14)
CD22-7 D235 GC DlLOGe): overexpressed
CD43+, moderate clonal sIg
(IgM > IgD)
Follicular lymphoma CDIOT CD20 CDs GC 225-. CD10 negative < 20% t(14;18)
CDI0O7; CDi lc) -eDBE);
CD23(—/+), bright clonal slg
BCL2 overexpressed
Clonal evolution del(17p)
Marginal zone and CD95 CD20 CDs (Ga), TRAP weak positive Trisomy 3
associated lymphomas CD77 CD23) CDI),
CDitica- CD25);
# CD103(—), moderate clonal slg
Hairy-cell leukemia CD19 CD20 CDSG): Bright CD22, CD22, and No consistent alteration
CD22. €D23(@) ;CDlLOG): CD1 1c, and CD103
CDililicCD25cE CDIOsce TRAP strong positive
moderate clonal slg
Plasma cell dyscrasias DRG epi Ga) Cb20 (Gea); Bright CD38, CD138 and Add(14) (q32), total loss
CD27GE SCW33 5 CD 5Occ/as dim CD45-— sensitive chromosome 13, 3q27,
_CD138+, clonal lg _marker 17q24-25, and 20q11

ye = ee moreennapositive;eee = ae more often negative; ie ) = negative; DR = HLA-DR; slg= surface Ig;

clg = cytoplasmicIg, BCL2 gene expression, TRAP = tartrate resistant acid phosphatase.

Of particular interest is the unique expression of CD5 in are necessary. Aberrant loss of T-cell-associated antigens, such
B-CLL. Expression of CD5 is normally seen on T lympho- as CD3, CD5, and CD7, or the predominance of specific T-cell
cytes and on the majority of B cells of early ontogeny such as subsets, either CD3+CD4+, CD3+CD8+, or CD31CD4+
cord blood cells; however, normally less than 20% of B cells CD8-+, in blood or bone marrow suggests the need for T-cell
in adult human peripheral blood express CD5. Although receptor molecular studies. A typical example of a T-CLL/
CD5-negative B-CLL does exist,*’° it accounts for fewer T-PLL immunophenotype is shown in Figure 20-9.
than 10% of all B-CLL cases. The precise functional role of When the conventional immunophenotypic techniques
the CD5 molecule is unknown. CDS has been shown to phys- fail to reveal the nature of the lymphoid neoplasm, molecular
ically associate with the antigen-specific receptor complex probe technology using DNA probes can often contribute to
present on both T and B lymphocytes.” Studies are ongoing the diagnosis and classification of malignancy by detecting
to determine whether differences in antigen expression and gene rearrangements that occur in lymphocytes. On a molec-
gene rearrangement are associated with variation in clinical ular level, Ig heavy and light chains are rearranged. The
course.*°?*? As mentioned earlier, T-CLL/T prolymphocytic rearrangement of heavy-chain Ig genes occurs first and is
leukemia is rare but can be distinguished from B-CLL on the followed by rearrangement of light-chain genes. The order of
basis of differentiation antigens. Immunophenotypic marker this rearrangement proceeds from mu () to k to \ and is the
expression and gene rearrangement during normal T-cell earliest detectable commitment to B-cell development.*!
ontogeny is shown in Figure 20-8. Monoclonality rather than polyclonality (the presence of a
The demonstration of slg in B-CLL was mentioned earlier mixture of k- and \-bearing B lymphocytes) is a feature of
and is, in fact, the classic marker for B cells. The detection of a many malignancies, including CLL; however, it is not, per se,
predominance of either k or \ light chains by the B cells indi- indicative of malignancy. Analogous to the detection of Ig
cates monoclonality’? In contrast, no immunophenotypic gene rearrangements in B cells, is the ability of molecular
markers for T-cell clonality are available, and molecular studies probes to detect rearrangement patterns of the genes coding
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 447

1118YW.001 x 1118YW.003 for the T-cell receptor (TCR), the antigen-specific surface
1000 SAE i Alen reas
molecule characteristic of T cells.** With the use of Ig and
= TCR gene probes to detect gene rearrangements, the unusual
:
a

oO
;
Ww

a
case of CLL that cannot be diagnosed and classified by mor-
phology and cell markers can now be characterized. It must
wo oO
wn
be stressed that it is extremely important that the final diagno-
oO sis of any lymphoproliferative disorder be made as a result of
40° ae10' 10° 10° 104 10° 10' 10% 10% 104 composite information from clinical data in addition to mor-
CD45 PerCP CD19 FITC phological, histologic, and immunologic analysis.
4° 1118YW.008 =° 1118YW.004 Although CLL is generally characterized by an elevated
~
white blood cell count in which there are abundant lympho-
cytes to analyze, if the number of neoplastic cells is low, a
os on

on a technique for amplifying a specific segment of deoxyribonu-

Qo
[a=]
2 %S
a cleic acid (DNA), called the polymerase chain reaction
om om
iS [o) (PCR), can be applied to improve sensitivity. The PCR
a —e L method involves denaturation, primer annealing, and poly-
10. 10" “407 fo" 40° oe ba Re Ce merization to yield millions of copies of the original scarce
FMC7 FITC KAPPA FITC sequence of DNA.** This technique is valuable for detecting
minimal residual disease in patients who have previously
been treated for CLL (or other leukemias or lymphomas) but
who currently lack histopathologic evidence of relapse.*>°°
The purpose of using PCR is to identify as early as possible
those patients who will subsequently have a relapse but who
PE
CD19
currently have only 1% to 5% malignant cells present or have
gene rearrangements present in malignant clones that require
amplification in order to be detected.
io 10° 107 Jo° 10
LAMBDA FITC
Figure 20-7 @ Flow cytometric analysis of chronic lymphocytic Clinical Features
leukemia. Leukemia bone marrow lymphocytes are gated by
CD45-scattered analysis. The plot of CD5 versus CD19 is used to CLL occurs mainly in older adults, with 90% of all cases
demonstrate dual-positive neoplastic lymphocytes with weak CD19 occurring in persons older than 50 years. In patients younger
fluorescence intensity. The plot of CD23 versus FMC7 illustrates
than 40 years of age CLL is rare; however, CLL has been
positive staining of the cells for CD23 but no staining of FMC7.
The cells are then plotted for both CD19 versus «k and CD19 described in young adults.*’ As with most other leukemias and
versus A, showing « light-chain clonality with a 15:1 ratio of k to A myeloproliferative disorders, CLL is found in twice as many
for the dual CD19+, CD5+ cells.

T-CELL ONTOGENY

TCR 6 rearrangement

TCRy rearrangement

TCRB rearrangement

TCR @ rearrangement

CD7
CD5
CD2
CD1
CD4
CD8
CD3
TCR (af or y 8) Cytoplasmic |=iFay

Figure 20-8 @ Hypothetical scheme of marker expression and gene rearrangement during T-cell ontogeny. TCR = T-cell receptor.
(From Pui, CH, et al: Clinical and biologic relevance of immmunologic marker studies in childhood acute lymphoblastic leukemia. Blood
82[2]:344, 1993, with permission.)
448 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

1068BR.001 1060BR.003
1000

800

=o
as
Dn oO

= 10°
9g
OSs
CD5
PE
a
Oo o
N oe

a
10° 10! 10° 10° 10 CD19 FITC
CD45 PerCP

Be 4060ADD.002 1060ADD.003
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w
a
oO
Q
oO PE
CD3

10° 10°
CD4 FITC CD8 FITC

1060ADD.004 1060ADD.005

PE
CD3 PE
CD4

10° 10 40° 10
CD7 FITC CD7 FITC

Figure 20-9 @ Flow cytometric analysis of T-prolymphocytic leukemia (T-PLL). Leukemic peripheral blood lymphocytes are gated by CD45-side
scattered analysis. Comparison of CD5 versus CD19 shows a predominance of CD5-positive cells (upper panel). Comparisons of CD3 versus CD4
and CD3 versus CD8 demonstrate a predominance of CD4+ T lymphocytes (middle panel). CD4+ T lymphocytes also express CD7 antigen
(lower panel).

males as females. Unlike acute leukemia, the signs and symp- gastrointestinal tract, prostate, and gonads.** CLL has also
toms of CLL develop gradually, and the onset of the disease been reported to occur simultaneously with acute myeloblastic
is difficult to pinpoint. In fact, it is not unusual for the leukemia (AML).*°
disease to be accidentally discovered during the course of a
routine visit to a physician. The duration of a relatively asymp-
tomatic phase of CLL is extremely variable. Unexplained
Laboratory Features
absolute and persistent lymphocytosis; cervical, supraclavicu-
lar, or axillary lymphadenopathy; and splenomegaly are the The requirements for the diagnosis of CLL have undergone
earliest signs of CLL. The clinical course is indolent, but as the revision since earlier criteria were established by Rai and col-
disease progresses, chronic fatigue, recurrent or persistent leagues*” in 1975 and Binet and colleagues in 1981.*!4? The
infections, and easy bruising are the consequences of anemia, International Workshop on Chronic Lymphocytic Leukemia‘
neutropenia, B-cell immunologic dysfunction, and throm- recommends a minimum peripheral blood B-cell lymphocytosis
bocytopenia. Hepatomegaly may accompany splenomegaly. of 5000 cells/uL (5 x 10° cells/L) along with a 30% lymphocy-
Dermatologic manifestations such as nodular and diffuse tosis of the bone marrow, consisting of morphologically mature-
skin infiltrations, erythroderma, exfoliative dermatitis, and appearing lymphocytes. This guideline is similar to criteria
secondary skin infections may occur. Leukemic lymphocytes established by the National Cancer Institute-sponsored working
may invade unusual locations such as the scalp, orbits, subcon- group. A new classification and terminology for the lympho-
junctivae, gums, pharynx, pleura and lung parenchyma, cytic neoplasms, including CLL, has been proposed.*®
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 449

Anemia, when it occurs, is usually normochromic and

normocytic, with a normal or low reticulocyte count. Autoim-
mune hemolytic anemia may precede, accompany, or follow
the development of CLL and be characterized by a secondary
reticulocytosis, a positive direct antiglobulin test, and an
elevated indirect serum bilirubin level. A decreased platelet
count is not uncommon in CLL and is related to bone marrow
replacement by leukemic cells, hypersplenism, and platelet
antibodies.
The lymphocytes of CLL may be morphologically indis-
tinguishable from normal mature lymphocytes when examined
with Wright’s stain. Alternatively, the leukemic lymphocytes
may have exaggerated nuclear chromatin clumping with
numerous dark-staining chromatin aggregates separated by
light-staining areas of parachromatin. The staining pattern that
results from the contrast between the nuclear chromatin and
parachromatin resembles the surface pattern of a soccer ball,
which may be a helpful image to recall in distinguishing the
lymphocytes of CLL from those of other lymphoproliferative
disorders. The morphology of peripheral blood lymphocytes in
CLL is duplicated in the bone marrow aspiration and biopsy
specimens (see Fig. 20-2). The extent of marrow infiltration
varies from patchy accumulations of lymphocytes to diffuse
sheets that involve the entire marrow space. The pattern of
bone marrow involvement is categorized into three types:
nodular, interstitial, and diffuse, but combinations of these
types also occur.**47 The nodular pattern (Fig. 20-10) is char-
acterized by distinct, randomly distributed aggregates of small
lymphocytes. In the interstitial pattern, the lymphocytes
infiltrate the interstitium to a greater or lesser degree with-
out displacement of fat cells. The nodular and _ interstitial
patterns are usually accompanied by preservation of normal Figure 20-1] | © A. Photomicrograph of bone marrow biopsy
hematopoiesis. In the diffuse pattern (Fig. 20-11), the entire section showing involvement by CLL, diffuse pattern. The entire
bone marrow space between bone trabeculae is replaced by bone marrow space between bone trabeculae is replaced by small
small lymphocytes. The life expectancy is significantly longer lymphocytes. 8. Immunohistochemistry ZAP-70 expression in CLL
cells indicates poor prognosis. Flow cytometry can also be used to
in patients with a nodular or an interstitial pattern than in those
detect ZAP-70 expression.
with a diffuse pattern.

TUMOR AND MICROENVIRONMENT

Angiogenesis, the growth of new capillaries from preexist-
ing blood vessels, has increasingly been recognized as an
important process in the growth of various hematologic
malignancies.*® Studies in CLL revealed bone marrow
microvessel area correlated with disease stage** and a
marker of the risk of progression in early disease. Other
reports suggest serum and cellular levels of vascular
endothelial growth factor (VEGF) pro-angiogenic factor
are increased in CLL.*”*° These results suggest that angio-
genesis may be an important process in the pathogenesis of
CLL and could provide important early clues about patients
destined to have more aggressive disease.
Although the morphological characteristics of the lym-
phocytes involved in most cases of CLL are quite distinctive,
the membrane phenotype of the neoplastic cells must be
Figure 20-10 m Photomicrograph of bone marrow biopsy section
determined for a definitive diagnosis. Immunologic features
showing involvement by CLL, nodular pattern, characterized by dis-
tinct, randomly distributed aggregates of small lymphocytes. and methods used to characterize lymphocytes as B cells or
450. Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

T cells were discussed earlier in the section entitled Immuno- Clinical Course, Prognostic Factors,
logic Features and Methods for Studying Lymphocytes. and Staging
Immune dysfunction within the proliferating B cells is
indicated by the presence of hypogammaglobulinemia or The overall median survival for CLL is currently 4 to 5 years;
hypergammaglobulinemia and monoclonal gammopathy. The 50% of patients are living 5 years after diagnosis, whereas
frequent expression of autoantibodies in CLL contributes to a 30% have a 10-year survival. CLL can be an indolent disease
variety of autoimmune phenomena, including those leading to with an asymptomatic presentation and may not require any
anemia and thrombocytopenia. treatment until progressive lymphocytosis of the peripheral
blood and marrow, lymphadenopathy, splenomegaly, anemia,
neutropenia, thrombocytopenia, autoimmune phenomena,
and infection develop. This may be as late as 10 to 15 years
Chromosomal Abnormalities
after the initial diagnosis. In contrast to those with an indolent
The most common chromosomal abnormality in B-CLL is course of disease, approximately 20% of patients with CLL
an extra chromosome 12, called trisomy 12 (in 15% to 20% have a very aggressive clinical course that progress rapidly
of cases),°!°* which may occur alone or together with dele- from initial diagnosis and results in death within | to 2 years.
tions or translocations of chromosome 13q14 (Gin 25% of The wide variation seen among patients is not fully under-
cases). The 13q14 deletions represent early clonal aberra- stood, but clinical and pathologic data have been used to try
tions and suggest the presence of a tumor suppressor gene to predict the CLL patient’s prognosis and identify various
whose loss or inactivation may be crucial to development stages and risk groups.
of CLL. Most of the 13q14 deletions are undetectable at The Rai system,*° the Binet system,*' and the International
the cytogenetic level but are detectable in more than 50% Workshop on CLL system* are the three major staging systems
of the CLL cases with the use of molecular probes for the developed for CLL; however, only the Rai system is widely
13q14 région.** used in the United States. The Rai and Binet staging systems,
Genetic abnormalities, including the most common along with median survival for each system by stage, are shown
chromosomal abnormalities and gene rearrangements, are in Table 20-5. Staging systems for CLL do not consistently pre-
listed in Table 20-4.°'° The presence of multiple chromoso- dict whether a patient’s clinical course is more likely to be indo-
mal abnormalities in B-CLL has been implicated as a poor lent or progressive. The most reliable predicting factors for
prognostic indicator. None of the proto-oncogenes involved indolent CLL are blood lymphocyte doubling time (LDT)
in the pathogenesis of other mature B-cell malignancies, greater than 12 months*’ and a nondiffuse pattern of bone mar-
including BCL2, BCL6, PAX5, and c-MYC, show primary row lymphocyte infiltration,*® along with a Rai stage of 0, I, or
alterations in CLL.°° II. A short LDT (less than 12 months), a diffuse lymphocyte

“Table 20-5 Staging Systems for Chronic Lymphocytic Leukemia sane

Stage Clinical Features Median Survival (Years)

Original Rai Modified Rai

System System
0 Low Lymphocytosis in PB and BM (25 x 10° SWS
lymphs/L in PB, = 30% lymphs in BM)
Intermediate Lymphocytosis + enlarged lymph nodes 8.5
Il Intermediate Lymphocytosis, + enlarged spleen 6
or liver, +/— lymphadenopathy
Ill High Lymphocytosis + anemia (Hgb < 11 g/dL) LES,
+/— enlarged nodes, spleen, liver
IV High Lymphocytosis + thrombocytopenia IRS
(platelets < 100 x 10°/L) +/— anemia
+/— organomegaly

Binet System
A Two or fewer node-bearing regions, no it
anemia or thrombocytopenia (Hgb > 10 g/dL,
platelets > 100 x 10°/L)
B Three or more node-bearing regions + no 5
anemia or thrombocytopenia
GE Anemia and/or thrombocytopenia 2
independent of regions involved

*Cervical, axillary, inguinal, palpable spleen and liver.

‘Survival equivalent to that of age-and sex-matched (French) population.
PB = peripheral blood; BM = bone marrow; Hgb = hemoglobin.
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 45]

Parameter, ae

hee in eee, ll( fee or7 (7pay Déhner et alt

Lack of somatic mutations in the expressed Hamblin et al'*°; Damle et al'*°
immunoglobulin VH-genes
Expression of cytoplasmic ZAP-70 Rassenti et al'*’; Crespo et al'”*;
Orchard et al!”°
Short lymphocyte doubling time Montserrat et al*”
(less than 12 months)
Elevated serum levels of B2-microglobulin Keating et al'*°; Hallek et al'*!
Elevated serum levels of soluble CD23 Reinisch et al!**; Sarfati et al'?
Elevated serum thymidine kinase activity Hallek et al’?!
Leukemia cell-‘surface expression of OD 38 _Damle et all, Tbrahim etalg

Source: Bink JL, et al: Perspective on the use of new DAienoene oe in the treatment ofeee oes here
Blood 107:859, 2006.

infiltration of the bone marrow (see Fig. 20-11A), a Rai stage of the original CLL or an independent disease; these include
III or IV, an elevated level of serum 8,-microglobulin, elevated cytogenetic analysis, immunoglobulin gene rearrangement by
level of soluble CD23 in serum, and the presence of chromoso- Southern blot analysis, and anti-idiotypic antibodies. Mole-
mal abnormalities, are associated with a more progressive clin- cular studies have shown that the development of Richter’s
ical course and shorter survival duration.®*“ In previous, mostly syndrome in CLL may represent either the identical clone of
retrospective analyses, the parameters shown in Table 20-6 cells present in the preceding CLL or a different malignant
have shown promising results. The first four parameters of clone.” In addition, p53, a tumor suppressor gene that is fre-
Table 20-6 have proven prognostic value independent of the quently mutated in a variety of human cancers, has been dis-
clinical stage in several studies. However, the methods for mea- covered in 15% of CLL patients and in 40% of patients with
suring some of these parameters (ZAP-70, Fig. 20-11B, serum Richter’s syndrome.’ The close association of p53 with
markers, cytogenetics, and mutational status of immunoglobu- transformation of CLL into a very aggressive lymphoma may
lins) have yet to be fully standardized and/or may not be readily also be a prognostic indicator for resistance to chemotherapy
feasible in most clinical laboratories. Further, the prognostic by interference with normal programmed cell death
value of some of the more easily accessible markers (apoptotic) pathways in tumor cells.*.”°
(e.g., B.-microglobulin) has not been as well established as that
of immunoglobulin mutation status or certain cytogenetic Treatment
abnormalities (e.g., 20p—).
Several types of transformation in B-CLL have been Some patients diagnosed with CLL do not require immediate
described: prolymphocytoid transformation, which is rela- treatment; however, when the signs and symptoms of pro-
tively low grade and slowly progressive; Richter’s syndrome gressive disease appear, it is time to begin therapeutic inter-
(diffuse large-cell lymphoma), which is rapidly progressive vention. Major physical and clinical signs and symptoms
and accounts for about 5% of all deaths in CLL®*’; and acute identify advancing disease. These include progressive mar-
leukemia. Transformation to acute Jeukeinia is unusual in row failure with resulting anemia; thrombocytopenia and
CLL. Most patients with CLL die with residual leukemia and neutropenia; progressive lymphocytosis; progressive lym-
usually succumb to infection or a cause totally unrelated to phadenopathy; enlarging spleen; autoimmunity (autoimmune
their CLL, such as cardiovascular disease.®* The onset of the hemolytic anemia or ITP); and increased susceptibility to
terminal transformation of CLL is suggested when there is a infection and persistent constitutional symptoms such as
proliferation of a new population of lymphoid cells, namely, night sweats, fever, and weight loss.
larger cells with immature-appearing morphological features, Conventional treatment for CLL is chemotherapy. Com-
a finer nuclear chromatin pattern, and a prominent nucleolus. binations of chemotherapeutic agents are used for patients
This morphological transformation is often accompanied by with disease that is refractory to conventional therapy or those
the appearance of complex chromosomal changes that were with advanced disease.°'”*’? The initial management of
not present earlier or are in addition to the commonly present patients with CLL according to Rai staging groups is summa-
trisomy 12. The proliferation of a more malignant clone of rized in Table 20-7.
cells is accompanied by an increasing resistance to therapy Radiation therapy is an alternative or adjunctive therapeu-
and an exceptionally poor prognosis.°* tic approach in treating CLL.’* Leukemic masses in enlarged
A variety of techniques is available to help determine lymph nodes and in the spleen may respond to focused, local
whether the transformation represents a clonal evolution of irradiation to relieve discomfort or eliminate obstruction. As
452 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

Table 20-7 Treatment Options forLymphocyticc Leukemia

Rai System Risk Group Treatment Options —

Low (stages 0-I) Observation only if Ro isniomane every 13 joonthe

Intermediate (stage II) Observation only if asymptomatic; fludarabine, cytoxan, prednisone, chlorambucil, or
rituximab if symptomatic
High (stages III and IV) Combination chemotherapy: fludarabine, prednisone with chlorambucil or
Intensive regimens: CHOP (cyclophosphamide, doxorubicin, oncovin, vincristine,
prednisone); anti-CD20, anti-CD52 antibody therapy, other nucleoside analogs
(experimental)

with chemotherapy, irradiation also has its detrimental effects, between 5 X 10° and 10 X 10° cells/L, the presence of both
especially in terms of causing life-threatening neutropenia. bone marrow infiltration of more than 30% lymphocytes and
Splenectomy is recommended in patients with massive B-CLL immunophenotype (dim CD20+, CD19+/CD5+,
splenomegaly and autoimmune hemolytic anemia or autoim- CD23+, FMC7-, and weak surface intensity Ig) is necessary
mune thrombocytopenia resulting from splenic pooling that is for the diagnosis of CLL.
uncontrollable by chemotherapy.” The distinction between CLL and ALL is easily made in
In addition to chemotherapeutic intervention, the use most instances based on morphological differences of the pro-
of high-dose intravenous gammaglobulin therapy prevents liferating cell population (see Fig. 20-12A to L). The differ-
major bacterial infections,*’*' and the immunosuppressant ence between the smoother nuclear chromatin pattern of the
cyclosporine, a fungal metabolite, aids in the prevention or lymphoblast in ALL and the heavy condensation of nuclear
treatment of red cell aplasia, both of which can be manage- chromatin in the CLL lymphocyte is readily appreciated when
ment problems in patients with CLL. examining an appropriate monolayer area or feather-like
Experimental therapies are also being studied. They edge of a well-stained blood smear. Lymphoblasts show
include not only new drugs but also the use of the monoclonal positive expression of the nuclear enzyme terminal deoxynu-
antibodies.°****° cleotidyl transferase (TdT). B-CLL cells are TdT-negative.
Bone marrow transplantation (autologous and _allo- B-Prolymphocytic leukemia (B-PLL) is characterized by a
geneic) is being explored as a possible curative therapy for predominance of circulating prolymphocytes (greater than
patients with aggressive CLL, especially in patients younger 55%, usually greater than 70%). Prolymphocytes are larger,
than 50 years of age. 87°? 9° less mature-appearing cells than the typical lymphocytes seen
in CLL, with moderately condensed nuclear chromatin and a
Differential Diagnosis prominent vesicular nucleolus (see Fig. 20—-12C). The clinical
and laboratory features that make PLL a distinct lymphopro-
As previously outlined in Table 20—1, CLL is a lymphoprolifer- liferative disorder include extreme leukocytosis (often greater
ative disorder that must be differentiated from other malignant than 100 X 10° cells/L) and prominent splenomegaly without
or reactive lymphoid proliferations. The differential diagnosis lymphadenopathy.**°°°* As in all disorders accompanied by
includes: acute lymphoblastic leukemia (ALL); prolymphocytic leukocytosis, morphological detail of the predominating
leukemia (PLL)**°°; non-Hodgkin’s lymphomas in leukemic cell may not be appreciated unless appropriate areas of
phase, especially mantle cell lymphoma (MCL); small cleaved- well-stained blood and bone marrow smears are examined.
cell lymphoma (SCCL); hairy cell leukemia (HCL); Sézary syn- Prolymphocytes may be seen in patients with CLL but
drome; T-cell large granular lymphocytic leukemia; and reactive account for less than 10% of the circulating cells (see
lymphocytosis (Fig. 20—12A). The morphological and immuno- Fig. 20-12D). When 11% to 55% prolymphocytes are pre-
logic characteristics of these lymphoproliferative disorders are sent, a mixed-cell type of CLL, designated CLL/PLL is diag-
shown in Table 20-8. In addition to these disorders, other hema- nosed.” This category includes patients with prolymphoid
tologic malignancies may be confused with CLL. These include transformation. Most cases of PLL are B cell in nature
adult T-cell leukemia/lymphoma (ATLL) and Waldenstrom’s (B-PLL) as demonstrated by strong CD20 and sIg (in contrast
macroglobulinemia.”” to weak CD20/sIg in CLL), and reactivity with the B-cell
The diagnosis of CLL requires a sustained absolute lym- markers CD19 and CD22.'
phocytosis of mature-appearing lymphocytes in the absence For cases of T-cell CLL or PLL, in addition to
of other causes. The diagnosis of CLL is established when the immunophenotype two cytochemical techniques are helpful
peripheral blood lymphocyte count is 10 X 10° or more in distinguishing T cells from B cells, namely, the e-naphthol
cells/L (which is typically the case), lymphocyte infiltration acetate esterase (ANAE) stain and the acid phosphatase (AP)
of the bone marrow is more than 30% lymphocytes of all reaction.'*' The typical T-lymphocyte pattern with ANAE and
nucleated cells, and the circulating lymphocytes have a AP is one large, localized dot of reaction product positivity
B-CLL immunophenotype. When lymphocyte counts are (Fig. 20-13).
 A. Chronic lymphocytic leukemia (CLL). B. Acute lym-
Figure 20-12 ™ Periph eral blood smears of various lymphoproliferative disorders.
prolymphocyte. E. Small lymphocytic lymphoma (SLL) in
phoblastic leukemia (ALL). C. Prolymphocytic leukemia (PLL). D. CLL with occasional
G. Hairy-cell leukemia (HCL). H. Sézary syndrome. |. Adult T-cell leukemia/lymphoma.
leukemic phase. F. Small cleaved-cell lymphoma (SCCL).
mononucleosis with atypical lymphocytes. L. Plasma cell dyscrasia.
J. T-gamma lymphocytosis with large granular lymphocytes. K. Infectious continued
454 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

Figure 20-12 ™ Cont'd

Small lymphocytic lymphoma (SLL) is the nodal counter- lymphoma is shown in Tables 20-4 and 20-8. In contrast to
part of B-CLL.* It is a diffuse non-Hodgkin’s lymphoma B-CLL, the cells in MCL lack expression of the CD23 surface
characterized by neoplastic transformation of small B lym- antigen.*°!° A typical example of the MCL immunopheno-
phocytes (Fig. 20-14). type is shown in Figure 20-15. The bright slg expression and
Mantle cell lymphoma arises from the centrocytes of the positivity for CD19, CD20, CDS, and FMC7 are characteris-
mantle zone of lymphoid follicles (see Fig. 20-6). In the tic. This immunophenotype also distinguishes MCL from
lymph node, MCL usually exhibits a diffuse pattern with follicular lymphoma, which are CD10+ and CD5— (see
complete effacement of lymph node architecture.** It can also Table 20-4). In most cases, a distinctive chromosomal
form a nodular or mantle zone pattern. MCL is composed of translocation t(11;14)(q13;q32) may occur.'°?'® It involves
a homogeneous population of small- to intermediate-sized the Ig heavy-chain locus on chromosome 14 and the BCL/
neoplastic lymphoid cells, usually slightly larger than normal locus on the long arm of chromosome 11. The hybrid gene is
lymphocytes. The nuclear characteristics and cytology of the associated with overexpression of the cyclin DI (PRAD /)
cells are variable: round to clefted nuclear contour, and mRNA and protein. This chromosomal translocation can be
clumped to disperse and blastoid chromatin. detected in MCL (70%) by Southern blot analysis or (30% to
MCL involves the peripheral blood in up to 25% of 45%) by PCR. Unlike most other B-cell neoplasms, MCL is
cases, and the peripheral blood count may exceed 200 x 10° cyclin D1+ (72% to 100%) as demonstrated using immuno-
cells/L. Under these circumstances, morphological recogni- histochemical staining on sections of formalin-fixed tissue.!°°
tion of MCL and its distinction from CLL/SLL are difficult. Small cleaved-cell lymphoma is also a non-Hodgkin’s
Identification of these two entities 1s important because MCL lymphoma consisting of B lymphocytes that may be nodular
is considered to have a poor prognosis, with a median survival (follicular) or diffuse in distribution*s (Fig. 20-16) and can
of 3 to 5 years. Bone marrow involvement may also occur progress to a leukemic phase.'”’ The circulating cells of SCCL
with or without peripheral lymphadenopathy. MCL shows are morphologically characterized by nuclei that are irregular
diffuse or focal bone marrow involvement (paratrabecular in shape and that demonstrate irregular clefts, notches, or
and nonparatrabecular). folds that may traverse the entire width of the nucleus (see Fig.
Neoplastic lymphoid cells in MCL cells are exclusively 20-12F). These abnormal lymphocytes typically have very
of B-cell lineage. The common immunophenotype of this scanty cytoplasm and were formerly called “lymphosarcoma
oe

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456 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

polyacrylamide gel electrophoresis.'” Immunologic markers

that support the diagnosis of HCL are reactivity with B-cell—
associated antigens (CD19, CD20, CD22, CD79a), CD1Ic,
CD25 (the monoclonal antibody that recognizes the
interleukin-2 receptor, Tac), FMC7, and CD103.**'!°° CD103 is
the most useful marker for distinguishing HCL from other
B-cell leukemias. A typical example of the HCL immunopheno-
type is shown in Figure 20-19.
A variant of HCL with splenomegaly and leukocytosis
has been described,''? as well as a splenic form of non-
Hodgkin’s lymphoma that resembles HCL, called splenic
lymphoma with villous lymphocytes (SLVL).**'"' SLVL has a
distinct immunologic profile, underscoring the importance of
using immunophenotyping to differentiate SLVL from CLL,
HCL, and other lymphoproliferative disorders.'”!°
Figure 20-15 © a-Naphthol acetate esterase (ANAE) stain showing Sézary syndrome is the leukemic phase of the most
localized “dotlike” positivity in two T lymphocytes. common cutaneous T-cell lymphoma, mycosis fungoides,
and is characterized by abnormal circulating lymphocytes,
called Sézary cells.''? A Sézary cell is typically the size
cells,”'°* a term that is no longer used. Compared with
of a small lymphocyte and has a dark-staining and a hyper-
immunologic markers in B-CLL, SCCL shows strong slg,
chromatic nuclear chromatin pattern with numerous
CD22 positivity, CD5 negativity, and often CD10 positiv-
folds and grooves that is referred to as cerebriform (see
ity (gee Chap. 21).
Fig. 20-12H). Nuclear folding is best appreciated at the
Hairy-cell leukemia is another form of B lymphocyte—
ultrastructural level using electron microscopy. A less com-
derived chronic leukemia and so named because of the fine,
mon large cell variant of the Sézary cell is larger than a neu-
hairlike, irregular cytoplasmic projections that typify the disease
trophil and often larger than a monocyte, but has the same
(see Fig. 20-12G). Pancytopenia is common in HCL (unlike the
grooved nuclear chromatin pattern as the smaller Sézary
other lymphoid disorders discussed in this chapter), along with
cell. The bone marrow is infrequently involved. The diag-
splenomegaly and marrow fibrosis. A bone marrow aspirate 1s
nosis of Sézary syndrome is dependent on the primary
often difficult to obtain because of associated fibrosis (the
diagnosis of the cutaneous T-cell lymphoma mycosis fun-
so-called dry tap). Nevertheless bone marrow biopsy and biopsy
goides in the skin. The skin biopsy shows infiltration in the
touch imprints are essential for diagnosis. The bone marrow
epidermis with an accumulation of atypical convoluted
biopsy shows a _ loose interstitial lymphocytic _ infiltrate
lymphocytes forming structures called Pautrier microab-
surrounded by a clear cytoplasm that separates one cell from
scesses (Fig. 20-20). The nuclear folding of a Sézary cell
another, creating a “‘fried-egg” appearance (Fig. 20-17). The
may at first glance suggest a monocytic cell line; however,
most characteristic cytochemical feature of HCL is a strong acid
a monocyte gives a diffuse pattern of cytoplasmic positiv-
phosphatase reaction that is not inhibited by tartaric acid, known
ity with the nonspecific esterase stain ANAE (mentioned
as the tartrate-resistant acid phosphatase stain (Fig. 20-18); this
earlier) as compared with a localized dotlike positivity
enzyme corresponds to the isoenzyme 5, as demonstrated by
pattern that identifies T cells. Immunologic marker studies
of Sézary cells show a mature T-lymphocyte phenotype
with reactivity for CD2, CD3, and CD4 (the monoclonal
antibody that recognizes the helper/inducer subset of
T lymphocytes). The detection and amplification of mark-
ers of T-cell clonality at the TCR and alpha-beta (af)
and gamma-delta (y5) loci by PCR can significantly
improve the sensitivity in detecting a clonal T-lymphoid
population.''?
Adult T-cell leukemia/l_ymphoma, common in Japan and
the Caribbean, is caused by human T-cell leukemia/
lymphoma virus-1 (HTLV-1). Although there is a large
heterogeneity in the clinical manifestations of the disease,
characteristic clinical features include generalized lym-
phadenopathy, hypercalcemia, bone and skin lesions, and
10% to 80% abnormal lymphoid cells in the blood and bone
marrow.'>''* The most outstanding feature of these abnormal
lymphocytes is the highly convoluted nuclear shape, which
Figure 20-14 © Small lymphocytic lymphoma (SLL), lymph node.
often is “cloverleaf’ in appearance (see Fig. 20-12/). There is
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 457

1365WH.008 1365WH.008

wi
oO
= Lo
a
a (6)
o
t
oO
”
iri

CD19 FITC

0 200 400 600 800 1000

SSC-Height

1365WH.011 1365WH.015

Ww Ww
o a
o oO
N
a N
ra
oO (6)

10° 4
. FMC-7 FITC CD10 FITC

= 1365WH.012 1365WH.013
iS

Ww Ww
o a
2 QD
Q a
S) oO

10
KAPPA FITC LAMBDA FITC

Figure 20-15 @ Flow cytometric analysis of mantle cell lymphoma in leukemia phase. Leukemic marrow lymphocytes are gated by CD45-
scattered analysis. The plot of CD5 versus CD19 demonstrates dual positive neoplastic lymphocytes. In contrast to CLL, the neoplastic lympho-
cytes in mantle cell lymphoma show positive staining for FMC7 but no staining of CD23. Cyclin D1 immunohistochemical stain was positive.

marked variation in the size of the cells, ranging from that of Chronic T-cell large granular lymphocytic leukemia
a small lymphocyte to that of a large monocyte. Nucleoli are (LGL) has the morphological distinction of persistent circu-
typically inconspicuous but, when present, may be prominent lating lymphocytes that have abundant pale blue cytoplasm
and cause confusion with prolymphocytes. The clinical with azurophilic granules (see Fig. 20-12/). These granular
course of ATLL can be acute, with a high white cell count and lymphocytes usually constitute 50% to 95% of the circulating
survival less than | year; chronic, with a lower white cell white cells. Large granular lymphocytic leukemia was first
count and survival of more than | year; or “smoldering,” with described in patients with CLL of T-cell origin''® then subse-
a normal white cell count and low numbers of abnormal T quently in patients with chronic neutropenia.''’ Three distinct
lymphocytes. As seen in T-PLL and Sézary syndrome, ATLL clinical syndromes are now described in patients with an
cells show reactivity with T-cell-associated antigens (CD2, increased number of circulating LGL cells.''’ When LGL car-
CD3, and CD5); most are CD4+, CD25+ but usually lack ries a phenotype of T-LGL leukemia (a clonal proliferation of
CD7. Rare CD8+ cases have been reported. Mutation in the CD3+ LGL), then chronic neutropenia and autoimmunity,
tumor suppressor gene p53 is seen in 30% to 50% of patients especially rheumatoid arthritis, are characteristic.!!-'?!
with ATLL.'» Natural killer LGL leukemia is characterized by a clonal
—_
458 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

a Se ee CD3-— LGL proliferation with an aggressive clinical course

$45
and multiorgan involvement.'”? The majority of patients with
21 % ba increased numbers of CD3— cells do not have features of
3," NK-LGL leukemia but rather demonstrate a more indolent
clinical course.'?! Because quantitative abnormalities of LGL
ee"
eat are fairly common and their presence in peripheral blood may
represent a transient reactive phenomenon associated with
viral infections, it is important to perform immunophenotyp-
ing and molecular studies, and to correlate these data with the
clinical picture. Oral low-dose methotrexate has been shown
to be an effective treatment for some patients with LGL.'*°
Reactive (atypical) lymphocytosis is self-limiting, rarely
exceeds 5 X 10° cells/L, and is most commonly caused by a
viral infection such as infectious mononucleosis, viral hepati-
tis, and cytomegalovirus in adults and Bordetella pertussis in
Figure 20-16 @ Small cleaved-cell lymphoma (SCCL), lymph node. children.' The large reactive lymphocytes that characterize
viremia are polyclonal and T cell in origin. Abundant cyto-
plasm that may vary in degree of basophilia from very pale to
deep blue is the most prominent feature of the reactive lym-
phocyte. These cells often have an irregular nuclear outline
resembling a monocyte, and the nuclear chromatin is mostly
coarse (see Fig. 20—k2K). Reactive B-cell lymphocytosis is
rare. See Chapter 15, which discusses infectious mononucle-
osis and other causes of reactive lymphocytosis.
Plasma cell dyscrasias, namely Waldenstrom’s
macroglobulinemia,’’ multiple myeloma, and plasma cell
leukemia, may be associated with the presence of abnormal
circulating plasma cells (see Fig. 20-12L). Plasma cells are
characterized by abundant basophilic cytoplasm, an eccentric
nucleus with clumped nuclear chromatin, and a prominent
perinuclear clear zone. Plasma cells are end-stage B lympho-
cytes with the aforementioned characteristic morphology and
distinctive immunologic markers, namely, the presence of
monoclonal cytoplasmic Ig and expression of CD38. Plasma
cell disorders are covered extensively in Chapter 22.
Figure 20-17 ® Hairy-cell leukemia (HCL), bone marrow aspirate. The significant clinical, morphological, and immunophe-
notypic features of CLL, as well as treatment, prognosis, and
differential diagnosis, are shown in Table 20-9.
In summary, B-cell small lymphocytic disorders are
clonal diseases that usually present in an indolent fashion in
older patients. The diagnosis is often made incidentally from
a persistent lymphocytosis. B-cell CLL is a clonal accumula-
tion of mature-appearing B lymphocytes caused by failed
apoptosis, exhibiting monoclonal sIg. An unusual feature is
expression of the CD5 membrane antigen seen on mature T
lymphocytes. The B lymphocytes accumulate slowly in the
bone marrow, blood, spleen, liver, and lymph nodes. About
half of the patients with B-CLL exhibit hypogammaglobu-
linemia and an increased susceptibility to infection. Red cell
aplasia, immune-mediated anemia, and thrombocytopenia are
also common.
The course of CLL disease depends on the leukemia
burden, which can be assessed by clinical staging systems.
Lymphoproliferative disorders caused by T lymphocytes
are rare. T-prolymphocytic and ATLL are rapidly progressive
Figure 20-18 @ Tartrate-resistant acid phosphatase (TRAP) stain of diseases with more aggressive clinical courses. Morphologi-
peripheral blood showing positivity in hairy cell and no staining in cal and immunophenotypic studies, and positive HTLV-1
neutrophilic. serology, are helpful in characterizing these entities.
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 459

t2 1414WH.012 =iS

lop)
2
Ww
W aq
o See
= a
Q oO
) 5
©

oa oO -
5 if 3 4 P 10° 10! 102 103 ©6104
10 10 10 10 10 CD103 FITC
CD22 FITC
= 1414WH.015 a 1414WH.013
[o) ‘
So
el ‘
=
ae Ae

~
2

ae a
2a = a
oO
oO

o, (00) ee10. 10> eee10> ie

= 440 a AS ang ee LAMBDA FITC
KAPPA FITC
4 1414WH.008

3 ©
o (49)

a
oe 3°
2°
o
a
oO
© —

B g =

200

CD25 FITC

10° 10! 107

CD45 PerCP

Figure 20-19 m Flow cytometric analysis of hairy-cell leukemia. Large mononuclear cells in leukemic bone marrow are gated by CD45-side
scattered analysis (left lower panel). The plots of CD22 versus CD11¢ and CD20 versus CD103 demonstrate predominance of dual positive B cells
(upper panel). The plots for both CD20 and 2X show X light-chain clonality (middle panel). The B cells are also reactive with anti-CD25
(anti-interleukin-2 receptor) (lower right-sided histogram).
460 Chapter 20. Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

Figure 20-20 @ Infiltration of the epidermis and upper dermis by

lymphocytes, many with convoluted (cerebriform) nuclei, histiocytes,
and formation of Pautrier microabscesses, characteristic of the cuta-
neous T-cell lymphoma mycosis fungoides.

Clinical features Patient >50 years of age, lymphadenopathy, lymphocytosis

Morphology Mature-appearing lymphocytes in blood and marrow, often showing hyperclumped nuclear
chromatin pattern; smudge cells
Immunophenotype Positive slg, CD19, CD20, CD5, CD23, CD43
Treatment Varies from no treatment to use of single or combination chemotherapeutic agents with or
without radiation therapy
Prognosis 50% 5-year survival; 30% = 10-year survival
Differential diagnosis ALL, PLL, SLL, SCCL, HCL, Sézary syndrome, LGL, ATLL, Waldenstrom’s macroglobu-
linemia, viral infection

ALL = acute lymphoblastic leukemia; PLL = prolymphocytic leukemia; SLL = small lymphocytic lymphoma; SCCL =
small cleaved-cell lymphoma; HCL = hairy-cell leukemia; LGL = large granular lymphocytosis; ATLL = adult T-cell
leukemia/lymphoma.

be clinically septic and was started on clindamycin, gentam-

Case Study 1 icin, and ampicillin therapy. Supportive care included fluid,
blood products, sodium bicarbonate, and pressor agents.
A 74-year-old black woman was admitted to the hospital for Blood and urine cultures were negative. X-ray exam showed
confusion. Her past medical history revealed chronic obstruc- an abdominal ileus, increased congestion in the lungs, and left
tive pulmonary disease, hypertension, renal insufficiency, lower lobe atelectasis. The patient had an episode of brady-
sickle-cell trait, and CLL. The CLL had been diagnosed cardia and expired.
13 years ago and treated with chlorambucil, prednisone, vin- Autopsy findings were remarkable for a monomorphic lym-
cristine, and bleomycin. A recent follow-up bone marrow phocytic infiltrate of the lymph nodes, liver, and spleen, which
analysis showed no lymphoid infiltrates. On the day before also showed the presence of sickled red blood cells. The pyloric
admission she was confused, minimally communicative, and region of the stomach showed a large ulcer, which contained
weak. In the emergency department, she became gradually adenocarcinoma, necrotic tissue, chronic inflammation, and
more unresponsive and appeared to be guarding her abdomen. colonization with fungus resembling Candida. Fungus was also
Blood was found rectally and in the nasogastric tube that was found along the tracheal epithelium and invading the tracheal
placed in the patient during the initial examination. She was mucosa. Gomori methenamine silver stain showed no septation
also found to be markedly acidotic, with a pH of 6.7. Her and branching of the hyphae consistent with Candida.
lactic acid level was 15.8 mg/dL with no acetone or ketones Note: This case illustrates how susceptibility to oppor-
present, and her glucose level was less than 10 mg/dL. Coag- tunistic infections and immunologic deficiency can con-
ulation studies revealed fibrinogen of 48 mg/dL, positive tribute to morbidity and mortality in a patient with CLL.
fibrinogen degradation products, and prolonged prothrombin Although patients with CLL frequently have a prolonged
and partial thromboplastin times. The patient was thought to survival and succumb to an unrelated disorder, the course of
continued CLL varies widely in different patients.
 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders 461]

Gees Sthdy 2-2

prrrecementonsnmanteennsneramnmmmn nitoeenart

ie
examination, however, disclosed retroperitoneal lym-
phadenopathy. Laboratory workup showed marked leuko-
cytosis (182 x 10° cells/L), thrombocytopenia (71 xX
A 64-year-old man with no significant past medical 10° cells/L), and anemia (hemoglobin, 8.1 g/dL). Periph-
history was admitted to the hospital with mild upper eral blood and bone marrow aspirate smears were per-
abdominal pain, malaise, anorexia, weight loss, and formed. More than 60% of the leukocytes counted in the
low-grade fever of 1-month’s duration. He denied having peripheral blood and 85% of all nucleated cells counted in
a cough, dyspnea, nausea, vomiting, jaundice, or recent the bone marrow specimen were described as “medium to
viral infection. His vital signs were essentially unremark- large atypical cells with distinct rim of cytoplasm, round
able except for a mild fever of 37.8° C. Physical nuclear contour, dispersed nuclear chromatin, and a single
examination revealed marked splenomegaly with no prominent nucleolus.”
hepatomegaly or peripheral lymphadenopathy. Radiologic
continued

©), estions
1. The ceils of CLL are morphologically identical to those of: ¢. The neoplastic lymphocytes in the blood or bone mar-
a. ALL Z = row are large with fine chromatin and prominent
b. Small lymphocytic lymphoma nucleoli.
c. Infectious mononucleosis Directions: Each item below contains one or more correct
d. Sézary syndrome an swers. Use the following letters to answer questions
. Surface immunoglobulin is the most reliable surface
i) 7 through 9.
marker for: a. If 1, 2, and 3 are correct
a. T lymphocytes , b. If 1 and 3 are correct
b. Plasma cells c. If 2 and 4 are correct
c. B lymphocytes d. If 4 is correct
d. Histiocytes e. If 1, 2, 3, and 4 are correct

Uo . Cells that demonstrate a positive reaction with the tar- . Which of the following findings would point to a diag-
trate-resistant acid phosphatase (TRAP) stain are most nosis of CLL?
likely: 1. Clonal proliferations of B lymphocytes
a. T lymphoblasts of ALL PACDIOT CDsicer CD23=
b. Atypical lymphocytes of a viral infection SR CDI9F ECMailee eD23en
c. Large granular lymphocytes of T-gamma lymphopro- 4, Ph chromosome—positive
liferative disorder . Which of the following clinical and laboratory manifesta-
d. Hairy cells of hairy-cell leukemia tions would point to a diagnosis of hairy-cell leukemia?
4, A mutated tumor suppressor gene found in a variety of 1. Pancytopenia
human cancers, including CLL is: 2. Splenomegaly
a, p53 3, D200 CD102Cm2a-
b. MDM2 4. Prominent generalized lymphadenopathy
c. HLA-DR . The poorest prognosis for patients with CLL is associ-
d. CD4 ated with which of the following features?
5. The immunophenotype that best describes mantle cell 1. Anemia
lymphoma is: 2. Splenomegaly
[email protected], FMC7 +, cyclin DI+ 3. Thrombocytopenia
bHGDS PaCD194— CD23-—, EMC] +, cyclin DI— 4. White cell count greater than 15,000 cells/uL
Peel) se eto tC D237, hMC/—, cyclin DI+ Directions: Choose the one best answer:
GeO DSee eC DIO smc 2am eMC Tt, cyclin DI+ 10 . The workup step(s) include:
1G D5a CD19. CD25c5,.kMCi+, cyclin DI+ a. Cytochemical staining, including myeloperoxidase and
6. In chronic lymphocytic leukemia: nonspecific esterase, and TdT
a. The absolute lymphocyte count is usually equal to or b. Immunohistochemical study of trephine biopsy sections
exceeds 5000 X 10° cells/L. c. Immunophenotypic study by flow cytometry
b. Hemoglobin level, platelet count, and absolute number d. Cytogenetic studies
of neutrophils may be normal or elevated. e. All of the above
 5
46 Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders

. Immunophenotypic study using flow cytometry shows 12. Cytochemical stains (myeloperoxidase, nonspecific
negative T-cell markers. However, neoplastic cells esterase) and TdT performed on samples of bone mar-
express bright CD19, CD20, and weak CD5 and show row aspirate smear were negative. The most likely
monotypic kK light-chain restriction. Cyclin D1 diagnosis is:
immunohistochemical stain is negative. These cells will a. Chronic myelocytic leukemia, accelerated phase
also express: b. Typical chronic lymphocytic leukemia
a. CD103 c. Acute lymphoblastic leukemia
b. Strong FMC7 d. Chronic lymphocytic leukemia/prolymphocytic
Cleh?3 leukemia
d. Dim surface immunoglobulin io) . B-cell prolymphocytic leukemia

e. GD38

m Laboratory findings in CLL include normocytic, nor-

mochromic red cells. Lymphocytes are small or slightly
larger than normal size and have a relatively, well-
a Chronic lymphoproliferative disorders are clonal prolifer-
differentiated appearance. Autoimmune hemolytic anemia
ations of morphologically and immunophenotypically
may precede, accompany, or follow the development of
mature B or T lymphocytes.
CLL and is characterized by secondary reticulocytosis, a
w Methods for studying lymphocytes include immunofluo- positive direct antiglobulin test, and an elevated indirect
rescence, molecular probes, cytochemistry, cytogenetics, serum bilirubin level.
electron microscopy, immunoperoxidase, and _ flow
w Differential diagnosis includes acute lymphoblastic
cytometry.
leukemia (ALL), prolymphocytic leukemia (PLL), non-
mw The Rai system, the Binet system, and the International Hodgkin’s lymphomas in leukemic phase, mantle cell
Workshop on Chronic Lymphocytic Leukemia (CLL) are lymphoma (MCL), small cleaved-cell lymphoma
the three major staging systems developed for CLL. (SCCL), hairy-cell leukemia (HCL), Sézary syndrome,
w The clinical course of CLL is indolent, but as the disease T-cell large granular lymphocytic leukemia, and reactive
progresses, chronic fatigue, recurrent or persistent infec- lymphocytosis.
tions, and easy bruising resulting from anemia, neutropenia,
B-cell immunologic dysfunction, and thrombocytopenia
may Occur.

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 Chapter 4 ;

The Lymphomas
Dan M. Hyder, MD

Introduction OBJECTIVES
Hodgkin Lymphoma At the end of this chapter, the learner should be able to:
Etiology and Pathogenesis
Pathology 1. List distinguishing features for nodular lymphocyte predominant, mixed cellularity,
Clinical Features lymphocyte-rich, lymphocyte depletion, and nodular sclerosis Hodgkin lymphoma.

Diagnostic Evaluation and 2. Describe the morphological and immunophenotypic features of Reed—Sternberg cells.
Stagin
i ah and Prognosis 3. List distinguishing features for the four stages of Hodgkin lymphoma.

Non-Hodgkin Lymphoma 4. Describe the classification criteria for non-Hodgkin lymphomas by the World Health
Etiology and Pathogenesis Organization classification.
se ee 5. Describe tests that may be needed to provide a differential diagnosis for lymphomas.
aging
Treatment and Prognosis 6. Describe the prognosis and treatment options for Hodgkin lymphoma and the more
Case Study common categories of non-Hodgkin lymphomas.

Introduction Hodgkin Lymphoma

The malignant lymphomas are a heterogeneous group Hodgkin disease was the first of the lymphomas to be recog-
of diseases that arise from cells of the lymphoid tissue nized. In 1832 Thomas Hodgkin described what he believed
(lymphocytes, histiocytes, and reticulum cells). They are to be a primary yet benign disease of the lymphoid tissue.'
broadly divided into the two major categories of Hodgkin Samuel Wilks in 1865 suggested that the disorder described
disease (Hodgkin lymphoma) and the lymphocytic by Hodgkin was a malignant process and was the first to
lymphomas. Although the vast majority of lymphomas apply the term Hodgkin disease in honor of Hodgkin’s orig-
within the second category are of lymphocytic origin, occa- inal description of the process.’ It was not until 1898 that
sional cases do appear to arise from nonlymphoid cells. Sternberg,’ and later in 1902 that Reed* described the dis-
Consequently, the lymphocytic lymphomas are also tinctive histological features of Hodgkin disease, including
frequently referred to as the non-Hodgkin lymphomas the peculiar cell that is the morphological hallmark of
(NHLs). This subdivision of the malignant lymphomas into Hodgkin disease which now bears their names, the Sternberg-
two general categories has both biologic and therapeutic Reed (or Reed—Sternberg) cell (Fig. 21-1). Current under-
implications. standing is that Hodgkin lymphoma consists of two distinct
It is important for the student and clinician alike to be clinicopathological entities: nodular lymphocyte predominant
aware of the diagnostic difficulties often faced by the patholo- Hodgkin lymphoma (NLPHL) and classical Hodgkin lym-
gist in evaluating lymphoid proliferations. Perhaps no area in phoma (CHL).
pathology has produced so many subcategories of a basic dis-
ease process as the area of lymphoma diagnosis. The distinc-
Etiology and Pathogenesis
tion between benign and malignant lymphoid proliferations,
Hodgkin lymphoma versus non-Hodgkin lymphoma, and the The continued use of the term Hodgkin disease rather than
subcategorization of these two major types of lymphoma is Hodgkin lymphoma attests to the fact that the etiology and even
frequently difficult and may require additional studies beyond the very nature (i.e., inflammatory/reactive versus malignant)
light microscopy. In some circumstances, a definitive diagno- of Hodgkin disease has been in question. There has been
sis may not be possible. considerable intrigue focusing on a possible viral etiology of

466
 Chapter 21 TheLymphomas 467

however, immunoglobulin transcription is absent owing to

inactivation of the immunoglobulin promoter.®

Pathology
The cytological hallmark of HL is the presence of an unusual
giant cell, the Reed—Sternberg cell. The features of this cell
include large size (up to 45 um in diameter), abundant aci-
dophilic cytoplasm, multinucleated or polylobated nucleus, and
gigantic (more than 5 um in diameter), inclusion-like nucleoli
(see Fig. 21-1). There 1s often clearing of the chromatin around
the macronucleoli, resulting in a distinct halo effect. Formerly,
it was necessary to identify at least one of these “diagnostic”
Reed-—Sternberg cells before a primary diagnosis of HL could be
Figure 21-1 ™ Reed-Sternberg cell (arrows). Prototypic cell of made; however, with current immunophenotyping techniques,
Hodgkin lymphoma (HL)is characterized by large size, abundant this is no longer a requirement. The identification of Reed—
eosinophilic cytoplasm, multinucleation, and inclusion-like Sternberg (or Reed—Sternberg—like) cells, however, is not a
macronuclei. sufficient condition for the diagnosis of HL. Reed—Sternberg—
like cells are commonly seen in a variety of benign and
malignant conditions other than HL (Table 21-1). The diag-
Hodgkin lymphoma (HL), with particular attention to certain nosis of HL should be based on finding Reed—Sternberg
ribonucleic acid (RNA) tumor viruses and Epstein-Barr virus cells (morphologically or immunophenotypically) in the
(EBV). Conclusive experimental evidence, however, is lack- proper cellular, stromal, and clinical setting. Many nondiag-
ing. Certain epidemiologic data, such as the occasional occur- nostic variants of Reed—Sternberg cells have been described
rence of several cases of HL within a small locale and brief (Fig. 21-2A through D). Observation of these cells is help-
space of time (time-space clustering), suggest an infectious ful in suggesting the possibility of HL and in the subclassi-
etiology. Other epiderhiologic studies, however, have failed to fication of HL.
support an infectious mode of transmission. Several classification schemes for HL are in use; how-
Although an infectious mode of transmission is unlikely, ever, they all share the fact that they are primarily based on
an infectious cofactor would appear to be operational in the growth pattern and cellular composition. Clinicians com-
pathogenesis of HL. The probable infectious agent is EBV, monly utilize the Rye classification which is a simplification
although cytomegalovirus and herpesvirus 6 have also been of the Lukes and Butler classification.’”* Recent advances in
implicated. EBV nucleic acids can be demonstrated in the understanding the molecular defects in HL indicates that there
lesions of approximately 80% of cases of HL, and EBV anti- are two pathologically distinct forms of HL, namely NLUPHL
gens such as latent membrane protein can be detected in and CHL.’ The classical group may be further subdivided
approximately 50% of the cases. The mechanism by which a based on cellular composition. The recently proposed World
virus such as EBV promotes the development of HL is Health Organization (WHO) classification of HL (Table 21-2)
unclear; however, it is postulated that a preceding state of incorporates this new information.'°
immunosuppression permits an abnormal immune response
to an EBV infection. The resulting genetic errors in the NODULAR LYMPHOCYTE-PREDOMINANT HODGKIN
immunoregulatory cells (T and B lymphocytes) are hypothe- LYMPHOMA
sized to lead to the development of HL. NLPHL 1s a relatively uncommon (5% of cases) but patho-
The current view of HL is that it is truly a malignant logically and clinically distinct variety of HL. The growth
(clonal) proliferation. The malignant cells are the Reed—
Sternberg cell and its variants, which usually represent only a
small percentage of the total cells in a HL lesion. Despite the
prevailing view that the Reed—Sternberg cell is the malignant
cell of HL, the origin of this cell has been difficult to estab-
lish. Kinship to almost every cell of the mononuclear phago-
cytic and lymphoreticular system has been proposed for the
Reed-Sternberg cell. Based on recent investigations, including
single-cell molecular techniques, the vast majority of cases of ¢ Viral infection, e.g., infectious mononucleosis
¢ Anticonvulsant-induced lymphadenopathy
both NLPHL and CHL are clonal proliferations of B cells.
e Epithelial and stromal malignancies
The neoplastic cells of NLPHL appear to correspond to ger- e Melanoma
minal center B centroblasts with productive rearrangement e Various lymphomas and leukemias
(clonal) of the IgH gene.® A germinal center B-cell origin of ¢ Myeloproliferative disorders
the neoplastic cells of CHL is also suggested by recent data;
468 Chapter 21 The Lymphomas

Figure 21-2 ® A. Mononuclear Hodgkin cell (arrows). Variant RS cell with single monolobed nucleus and inclusion-like macronucleolus.
B. L&H (popcorn) cell (arrows). Characteristic cell of nodular lymphocytic-predominant Hodgkin lymphoma (NLPHL). C. Lacunar cell. Large
cell with artifactual clear space (lacuna) due to formalin fixation that surrounds the nucleus. Wisps of cytoplasm are visible in the space.
D. Pleomorphic Reed-Sternberg cell. Large cell with bizarre multinucleated/multilobated nucleus.

pattern is generally characterized by a vague nodularity numbers of a characteristic, though nondiagnostic, Reed—
although in some cases it may be well developed Sternberg variant referred to as an L&H or popcorn cell
(Fig. 21-3). It is questionable whether a purely diffuse form (see Fig. 21-2B). This cell has a variable amount of
of NLPHL exists. The cellular milieu is composed of a mix- pale staining cytoplasm, a convoluted nucleus prosaically
ture of small, normal-appearing lymphocytes, benign histi- referred to as popcorn-shaped, and an indistinct nucleolus.
ocytes, rare or absent Reed—Sternberg cells, and variable Phenotypic and genotypic studies suggest that L&H
cells are closely related to proliferating germinal center
cells (centroblasts). The immunophenotype of the neoplas-
tic L&H cells of NLPHL is distinct from the neoplastic
' Table 21-2 WHO Classification
cells of CHL (Table 21-3). Plasma cells, eosinophils, fibro-
of Hodgkin Lymphoma sis, and necrosis are usually absent. The histological
features of NLPHL must be distinguished from various
Frequency (%)
reactive conditions such as infectious mononucleosis
Nodular Lymphocyte-Predominant 5 and progressive transformation of germinal centers as
Classic Hodgkin Lymphoma well as certain subcategories of CHL and NHL. Such differ-
Nodular sclerosis, grades I and II 60-80 entiation may require special phenotypic and genotypic
Lymphocyte-rich 5
studies.
Mixed cellularity 15-30
Lymphocyte depletion =|

Source: From Harris, NL: Hodgkin’s disease: Classification and CLASSIC HODGKIN LYMPHOMA
differential diagnosis. Mod Pathol 12:159, 1999, with permission Classic HL is morphologically defined by the observation of
classic Reed—Sternberg cells in the appropriate cellular and
 Chapter 21 TheLymphomas 469

in which the artifact of formalin fixation produces a distinc-

tive appearance of this Reed—Sternberg variant. The lacunar
cell is separated from the surrounding cells by a large clear or
pale-staining space (lacuna) (see Fig. 21—-2C). Wisps of cyto-
plasm may be seen in this space. The nucleus is large and
often polylobated, and the nucleoli are small to intermediate
in size.
In some cases of HL, the characteristic cellular milieu of
NS HL may be seen, including the presence of large numbers
of lacunar cells; however, the sclerotic bands are absent.
These cases have been assigned by some authorities to a
subcategory of NS HL referred to as the cellular phase of NS
HL. Others have assigned cases with this pattern to mixed
cellularity HL.
Sclerosis may be seen in many other lymphoid and non-
lymphoid malignancies. For example, chronic idiopathic
‘Figure 21-35 ® Nodular lymphocyte predominant Hodgkin myelofibrosis (see Chap. 18) may produce a histologic pattern
lymphoma. Low-power microscopic view showing vaguely nodular of fibrosis and cellular atypia virtually indistinguishable from
pattern. ; NSHL. Sclerosis alone, even when present as orderly bands
of collagen, is not sufficient for a diagnosis of NSHL.

stromal background. It is the background that determines the MIXED CELLULARITY HODGKIN LYMPHOMA As its
subcategory of classic HL to which the lesion belongs. name implies, mixed cellularity Hodgkin lymphoma (MCHL)
is characterized by a heterogeneous mixture of cells, including
NODULAR SCLEROSIS HODGKIN LYMPHOMA Nodular lymphocytes, histiocytes, plasma cells, eosinophils, Reed—
sclerosis (NS) Hodgkin lymphoma (NSHL) is the most com- Sternberg cells, and Reed—Sternberg variants (Fig. 21-6).
mon subtype of HL, ‘representing 60% to 80% of HL cases. Classic Reed—Sternberg cells and mononuclear variants are
The cardinal histological features of NSHL are the presence usually readily apparent. MCHL represents 15% to 30% of
of birefringent collagenous sclerosis, classic Reed—Sternberg cases of HL.
cells, and a distinctive Reed—Sternberg variant called a lacunar In addition to the cellular milieu, there is usually an
cell. The sclerosis observed in NSHL is different from that increase in the background stroma in the form of a disorderly,
seen in the other subtypes of HL. It is found in the form of noncollagenous fibrosis distinct from that seen in NSHL.
well-organized bands of collagen that subdivide the tissue Small areas of necrosis are commonly present. In some cases,
into distinct nodules (Fig. 21-4). Within the nodules is a vari- clusters of epithelioid histiocytes may be numerous, making
able mixture of lymphocytes, classic Reed—Sternberg cells, distinction from Lennert’s lymphoma (a T-cell lymphoma
lacunar cells, plasma cells, eosinophils, and neutrophils. The with high content of epithelioid histiocytes) difficult. The
lacunar cells often form distinct collections within the central lymphocytes of Lennert’s lymphoma are cytologically atypi-
region of the nodule (“grouped lacunars”), and these collec- cal as opposed to the small, uniform features of the lympho-
tions may be associated with focal necrosis (Fig. 21-5). The cytes in all forms of HL, including MCHL. There is often
lacunar cell is best identified in formalin-fixed tissue sections only partial involvement of nodes involved by MCHL. This is

Antibody TCRLBL

CD30 rs

++4 +++
+/— aahe

4+ t+

+ = all cases positive; +/— = majority of cases positive; —/+ = minority of cases positive; — = all cases negative.
NLPHL = nodular lymphocyte predominant Hodgkin lymphoma; CHL = classic Hodgkin lymphoma; TCRLBL = T-cell
rich large B-cell lymphoma; DLBCL = diffuse large B-cell lymphoma; ALCL = anaplastic large cell lymphoma.
470. Chapter 21 The Lymphomas

usually an interfollicular pattern, which may be missed on

casual examination or confused with a reactive process.

LYMPHOCYTE-RICH HODGKIN LYMPHOMA Approxi-

mately 5% of HL cases may exhibit small numbers of classic
Reed—Sternberg cells in a background rich in lymphocytes and
bearing a strong resemblance to NLPHL. The Reed—Sternberg
cells however exhibit the immunophenotype of classic Reed—
Sternberg cells rather than the immunophenotype of L&H cells
seen in NLPHL. These cases have recently been assigned to a
new category of HL referred to as lymphocyte-rich classic HL
(LRHL). LRHL shares pathologic and clinical features more
akin to MC HL than to NLPHL.

LYMPHOCYTE DEPLETION HODGKIN LYMPHOMA

a a Lymphocyte depletion HL (LDHL) is the rarest form of HL,
Figure 21-4 ™ Classic Hodgkin lymphoma, nodular sclerosis sub- with fewer than 1% of cases so classified. Lymphocytes are
type. Low-power microscopic view demonstrating nodules of cells sparse in this disorder whereas Reed—Sternberg cells and
produced by orderly bands of collagen. Reed-Sternberg variants are numerous. Other types of cells
“
such as plasma cells, histiocytes, and eosinophils are infre-
hes
AN 7 quently found. In addition, irregular sclerosis is a major his-
tologic component of. LDHL. Several histologic variants of
LDHL have been described. Their recognition primarily
serves to aid the pathologist in differentiating LDHL from
other malignancies with which it is easily confused.
There has been a marked decrease in the diagnosis of
LDHL during the past three decades. This is not due to a
decreased incidence of this form of HL but to the recognition
that many cases previously diagnosed as LDHL are actually
misdiagnosed cases of NHL, particularly polymorphous
T-cell lymphomas." It may, in fact, be impossible in some
cases, even after detailed phenotypic and genotypic studies, to
resolve the differential diagnosis between a pleomorphic
8 ay ii ae ee, a 3 vet be eee "e ;
a
Peerigs. NHL and LDHL.
ee 5 Seta eGR oe hen AOE Lee
tea PS T
Ps es OSs me ao enc tte ae Bek =
UNCLASSIFIED HODGKIN LYMPHOMA A small percent-
Figure 21-5 @ Grouped lacunar cells in nodular sclerosis Hodgkin age of cases exhibit sufficient histologic features for a diagno-
lymphoma (NSHL) showing central area of necrosis. sis of HL but insufficient features to be assigned to one of
the preceding subcategories. These cases are assigned to the
unclassified subcategory.

HISTOLOGIC PROGRESSION
The concurrent evaluation of multiple sites of involvement
with HL usually demonstrates similar histology at each site.
On the other hand, with the passage of time, reevaluation
often reveals histologic progression in the sequence: LRHL to
MCHL to LDHL. Although it is often stated that NSHL does
not undergo such progression but remains NSHL, there may be
progression over time within the histologic spectrum of NSHL.
That is, cases of NSHL initially rich in lymphocytes may
undergo a progressive decrease in the number of lymphocytes,
with concomitant increase in the number of Reed—Sternberg
cells and Reed—Sternberg variants equivalent to that seen in the
etc
pe 4 S ®
Sy 2. non-NS types. This progression, however, maintains the over-
ae. 28. all nodular sclerosing pattern of NSHL. Eventually a stage of
Figure 21-6 @ Mixed cellularity Hodgkin lymphoma. Easily identi- extreme cellular depletion may be reached, referred to as oblit-
fied Reed—Sternberg cells and RS variants are seen admixed with erative total sclerosis, in which few cellular elements, includ-
small lymphocytes, plasma cells, eosinophils, and histiocytes. ing Reed—Sternberg cells, are found.
 Chapter 21 Thelymphomas 47]

Clinical Features staging would alter the therapy. The results of the staging
evaluation are used to assign the patient to staging groups.
In the United States, HL accounts for approximately 15% of The most widely utilized staging scheme is the Cotswold’s
newly diagnosed cases of lymphoma. It is estimated that revision of the Ann Arbor classification (Table 21—5).'* These
8,190 new cases of HL will be diagnosed in the United States staging schemes may be used with the data obtained from
in 2007 (SEER Cancer Statistics, NCI). The incidence of HL either clinical or pathologic staging, or both. In addition to the
exhibits a bimodal distribution, with peaks between the ages anatomical extent of the disease implicit in the staging cate-
of 15 and 35 and in the over-50 age group. gories, the disease is further classified based on the presence
Most patients with HL present with a complaint of non- or absence of specific symptoms (Table 21-6). The letter “A”
painful lymph node swelling. Each subtype of HL is associ- (asymptomatic) or “B” (symptomatic) is then appended to the
ated with rather characteristic, but not totally specific, clinical appropriate stage number.
features. NLPHL demonstrates a male predominance and is The clinical utility of the Cotswold’s staging scheme
generally a disease of younger patients. The disease is usually stems from the predictable behavior of HL. HL is a lymph
localized at presentation to one peripheral node or node group. node-based disease and rarely, if ever, starts in extranodal
Despite excellent long term survival, patients with NLPHL sites. It spreads by the lymphatic route in an orderly and pre-
experience an increased frequency of late relapses compared dictable pattern to contiguous lymph nodes. Only late in the
with CHL and progression to a large B cell lymphoma occurs disease course when hematogenous spread may occur is a
in 3% to 5% of cases. NSHL shows a female predominance and more disorderly pattern seen.
is usually associated with cervical, scalene, or supraclavicular
adenopathy. An anterior mediastinal mass is often present.
Most patients with NSHL are asymptomatic on presentation. Treatment and Prognosis
MCHL and LDHL are most often seen in symptomatic patients
The selection of therapy is directed by the results of the staging
with widely disseminated disease. Symptoms include fever,
evaluation rather than the specific histologic subtype. In general,
drenching night sweats, and weight loss. Extranodal involve-
multiagent chemotherapy with or without involved field radia-
ment is common in these two subcategories of HL.
tion is the mode of treatment. With appropriate therapy, the
10-year survival for stages I and IT HL now exceeds 80%.
Diagnostic Evaluation and Staging Ten-year survival for stages III and IV HL has been improved
with aggressive chemotherapy and now approaches 70%.'?
The diagnosis of HL requires tissue biopsy and microscopic
evaluation. Because of the complexities involved in accurate
diagnosis and subclassification, adequate tissue should be Non-Hodgkin Lymphoma
obtained at the time of initial biopsy for routine light micro-
scopic studies as well as for ancillary studies, such as Virchow (1858)'* and Billroth (1871)!° were the first to
immunophenotypic and genotypic analysis, if these are found employ the terms lymphoma and malignant lymphoma. The
to be necessary. distinction between the two major categories of malignant
Following a tissue diagnosis of HL, the patient should be lymphoma (1.e., HL and NHL) was suggested in 1893 by
appropriately staged to determine the extent of disease and Dreschfield'® and Kundrat.'’ It is estimated that 56,390 cases
permit selection of appropriate therapy. Clinical staging of NHL were diagnosed in 2005.
should be performed on all newly diagnosed patients with
HL. The elements of clinical staging are listed in Table 21-4. Etiology and Pathogenesis
Pathologic staging requires exploratory laparotomy with
multiple node samplings and splenectomy. Except at certain It seems certain that a prerequisite for the development of
institutions, routine pathologic staging is not performed and is lymphoma is damage to the regions of the genetic code that
reserved for those cases in which the results of pathologic regulate the growth and reproduction of cells of the immune

1 . Initial tissue biopsy demonstrating Hodgkin lymphoma

7). Careful history
5). Detailed physical examination
4 . Laboratory studies (CBC, sedimentation rate, chemistry panel to include liver, kidney, and bone profiles)
5). Chest x-ray exam (anterior and lateral)
6 _CT scan of thorax, abdomen, and pelvis
Th FDG-PET or gallium scan
8 . Bone marrow biopsy if “B” symptoms* or cytopenias or advanced stage disease

*See Table 21-6.

FDG-PET = Fluorodeoxyglucose positron emission tomography.
472 Chapter 21 The Lymphomas

Definition

Involvement of a single lymph node region or lymphoid structure (e.g., spleen, thymus, Waldeyer’s ring) :
Involvement of two or more lymph node regions on the same side of the diaphragm (the mediastinum is a single site:
hilar lymph nodes are lateralized); the number of anatomical sites should be indicated by suffix (e.g.. II)
Involvement of lymph node regions or structures on both sides of the diaphragm
With or without splenic, hilar, celiac, or portal nodes
With paraaortic, iliac, or mesenteric nodes
Involvement of extranodal site(s) beyond those designated E

E = Involvement of a single extranodal site, or contiguous or proximal to known nodal site of disease. 7.
Source: Jaffe, ES, et al: World Health Organization Classification of Tumours Pathology & Genetics, Tumours of Haematopoietic
and Lymphoid Tissues. IARC Press, Lyon, France, 2001.

NHL. Certain types of NHL are highly correlated with spe-

cific chromosomal abnormalities, particularly translocations
involving chromosomes 2, 8, 14, and 22 (Table 21-8).
The pathogenic significance of these chromosomal
translocations relates to the function of the genetic material
weight in the 6 months before admission
located at or near the site of these translocations. One of the
2. Unexplained fever with temperature above 38°C
3. Drenching night sweats breakpoints commonly involved in B-cell NHL 1s at the site
of the transcriptionally active immunoglobulin heavy- or
light-chain (kappa [k] and lambda [\]) genes located on chro-
mosomes 14, 2, and 22, respectively. For many T-cell lym-
system. The inciting agents for this damage remain unknown;
however, it is felt that mutagenic factors such as chemicals,
phomas, a common breakpoint is in the region of one of the
ionizing radiation, and certain viruses may play a role in T-cell receptor genes. The other breakpoint is in the vicinity of
initiating and promoting the damage. Although viruses such a gene important in the regulation of cell growth and division.
as EBV do not appear to be directly mutagenic, they may This growth-regulating gene, referred to as a proto-oncogene
function via persistent antigenic stimulation as polyclonal when it is normally located in the genome, is translocated to
mitogens that somehow favor the eventual selection of a sin- the region of one of the immunoglobulin genes (B-cell
gle clone of non-growth-regulated (malignant) cells. Support lymphomas) or T-cell receptor genes (T-cell lymphomas). In
for this hypothesis comes from the increased incidence of this new ___location, the growth-regulating gene functions
lymphomas in individuals with conditions associated with abnormally and is now referred to as an oncogene.
primary or acquired immunodeficiency (Table 21-7). One of the best-studied examples of this process occurs
The genetic damage associated with the development of in Burkitt's lymphoma, a B-cell lymphoma, in which the
a lymphoma is often associated with numerical or structural c-myc proto-oncogene located at region q24 on chromosome
alterations of chromosomes, or both.'* With high-resolution 8 is translocated to the immunoglobulin heavy-chain locus at
karyotyping techniques, nonrandom chromosomal abnormal- 14q32 (Fig. 21-7). This occurs in about 90% of Burkitt's
ities can be demonstrated in approximately 60% of cases of lymphoma cases. It also is found in about 40% of high-grade
large-cell lymphomas and, therefore, is not specific for
Burkitt's lymphoma. The c-myc proto-oncogene normally
functions in the nuclear signaling that switches cell prolifera-
tion on and off. The translocation of c-myc to the heavy-chain
locus deregulates the function of the c-myc gene, resulting in
uncontrolled cell proliferation.
Chromosomal translocations are only one mechanism for
oncogene activation. Other mechanisms include deletions, base
pair mutations, and gene amplification. Oncogene activation—
¢ SjOgren’s syndrome
¢ Sarcoidosis deregulation is believed to play a role in the development of
* Systemic lupus erythematosus most, if not all, lymphomas.
¢ Rheumatoid arthritis
* Celiac disease
¢ Dermatitis herpetiformis Pathology
¢ Acquired immunodeficiency syndrome (AIDS)
¢ Organ transplant recipients Since the original suggestion by Dreschfield'® that the
* Congenital immunodeficiency disorders lymphocytic lymphomas represent distinct histologic entities,
a number of lymphoma classification schemes have been
 Chapter 21 Thelymphomas 473

_ Abnormality Genetic Loci Involved Associated Lymphoma

crarvmsamstvraurssse .

Trisomy 12 ? B-CLL/SLL
Del 13q14 ? B-CLL/SLL
t(9;14) PAX5-IgH Lymphoplasmacytoid cell lymphoma
t(11;14) BCLI-IgH (Prad 1) Mantle cell lymphoma
t(14;18) IgH-BCL2 Follicle center lymphoma
t(11;18) API2-MALT1 Marginal zone lymphoma (extranodal)
Trisomy 3 2 Marginal zone lymphoma (extranodal)
t(8;14) c-myc—IgH Burkitt lymphoma
t(2;8) k/c-myc Burkitt lymphoma
(8322) c-myc! Burkitt lymphoma
t(3;14) BCL6-IgH Diffuse large B-cell lymphoma
Inv 14 (ql1;q32) TCR-TCL1 T-Cell Pele
t(2:5) ALK-NPM Systemic anaplastic large cell
Tso(7q) 2 Hepatosplenic T-cell lymphoma
ont er nesses
ence DASA mam EN

B-CLL = B-cell chronic lymphocytic‘leukemia; SLL = small cell lymphocytic lymphoma; IgH = immunoglobulin heavy chain locus;
BCLI = B cell lymphoma | gene; BCL2 = B cell lymphoma 2 gene; ALK = kinase gene; NPM = nucleolar protein gene; PLL = prolymphocytic
leukemia; AP/2 = apoptosis inhibitor gene; MALT] = mucosa-associated lymphoid tissue gene; c-myc = c-myelocytomatosis oncogene;
BCL6 = B cell lymphoma 6 gene; TCR = T-cell receptor gene; TCL/ = T-cell leukemia | gene; PAXS = paired homeobox-5 gene.

proposed. At least nine major classification schemes have

been extensively utilized since the mid-1960s. Most of these
schemes are based on the growth pattern, for example, nodu-
lar or diffuse (Fig. 21—8A and B), and the cytological features
of the malignant cells. At present, the most widely utilized
scheme in use by clinicians in the United States is the Work-
ing Formulation for Clinical Usage. The Working Formula-
tion was developed by a multi-institutional study group in
response to a need for a clinically useful standardized group-
ing of the lymphomas based on prognosis.'” Despite the suc-
cess of the Working formulation, it fails to recognize several
clinically distinct types of lymphomas that have been
described since publication of the Working Formulation. It is
now generally accepted that accurate lymphoma classification
cannot be based strictly on morphological features but
requires immunophenotypic and genotypic information.

c-myc

q24 region| ©8 [932 region (yg

8 14 t(8;14)(q24;q32)

Figure 21-7 @ Reciprocal translocation t(8;14) (q24;q32) is seen

in majority of cases of Burkitt’s lymphoma as well as some non-
Burkitt’s high-grade lymphomas. A reciprocal translocation of
genetic material occurs between chromosomes 8 and 14. A distal Figure 21-8 @ A. Follicular growth pattern of lymphoma.
portion of the long arm of chromosome 8, containing the c-myc Neoplastic cells are organized into closely packed nodules (follicles).
oncogene, is translocated to a site adjacent to the immunoglobulin B. Diffuse growth pattern of lymphoma. Neoplastic cells are
heavy-chain locus on chromosome 14. arranged in sheet-like fashion without follicular organization.
474 Chapter 21. The Lymphomas

In 1994 the International Lymphoma Study Group pub- A conceptual understanding of the common classifica-
lished the Revised European-American Classification of tion schemes requires knowledge of the morphological
Lymphoid Neoplasms (REAL), which utilized clinical, ontogeny of normal lymphocytes. Although it is customary to
morphological, immunophenotypic, and genotypic features to discuss the lymphomas with reference to one of the classifica-
subclassify the lymphomas.” The utility of this more “biolog- tion schemes, taking a more conceptual approach will facili-
ically correct” classification scheme has been subsequently tate understanding the relationship between the various
verified.*! The principles of REAL classification were incor- lymphomas as well as the various classification schemes.
porated and updated in the World Health Organization classi- The lymphocytic lymphomas may be conceptualized as a ma-
fication of tumors of lymphoid tissues (Table 21-9) and is lignant population of lymphocytes arrested at a particular
currently the most widely utilized classification scheme.'° stage (morphological, genotypic, phenotypic, or functional)

table 21-9 World Health Organization (WHO) Classification ieSe eu:

B-Cell Neoplasms
Precursor B-cell neoplasm
¢ Precursor B lymphoblastic leukemia/lymphoma
Mature B-cell neoplasms
¢ Chronic lymphocytic leukemia/small lymphocytic lymphoma
¢ B-cell prolymphocytic leukemia
¢ Lymphoplasmacytic lymphoma
¢ Splenic marginal zone lymphoma
¢ Hairy cell leukemia
e Plasma cell myeloma
* Solitary plasmacytoma of bone
¢ Extraosseous plasmacytoma
* Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT-lymphoma)
¢ Nodal marginal zone B-cell lymphoma
° Follicular lymphoma
¢ Mantle cell lymphoma
° Diffuse large B-cell lymphoma
¢ Mediastinal (thymic) large B-cell lymphoma
¢ Intravascular large B-cell lymphoma
¢ Primary effusion lymphoma
¢ Burkitt lymphoma/leukemia
B-cell proliferations of uncertain malignant potential
¢ Lymphomatoid granulomatosis
¢ Post-transplant lymphoproliferative disorder, polymorphic
T-Cell and NK-Cell Neoplasms
Precursor T-cell neoplasms
¢ Precursor T lymphoblastic leukemia/lymphoma
¢ Blastic NK cell lymphoma*
Mature T-cell and NK-cell neoplasms
*¢ T-cell prolymphocytic leukemia
° T-cell large granular lymphocytic leukemia
° Aggressive NK cell leukemia
¢ Adult T-cell leukemia/lymphoma
* Extranodal NK/T-cell lymphoma, nasal type
¢ Enteropathy-type T-cell lymphoma
° Hepatosplenic T-cell lymphoma
* Subcutaneous panniculitis-like T-cell lymphoma
¢ Mycosis fungoides
¢ Sézary syndrome
* Primary cutaneous anaplastic large cell lymphoma
* Peripheral T-cell lymphoma, unspecified
¢ Angioimmunoblastic T-cell lymphoma
¢ Anaplastic large cell lymphoma
T-cell proliferation of uncertain malignant potential
¢ Lymphomatoid papulosis
 Chapter 21 TheLymphomas 475

Hodgkin Lymphoma
* Nodular lymphocyte predominant Hodgkin lymphoma
* Classical Hodgkin lymphoma
* Nodular sclerosis classical Hodgkin lymphoma
* Lymphocyte-rich classical Hodgkin lymphoma
¢ Mixed cellularity classical Hodgkin lymphoma
* Lymphocyte-depleted classical Hodgkin lymphoma
Histiocytic and Dendritic-Cell Neoplasms
Macrophage/histiocytic neoplasm
* Histiocytic sarcoma
Dendritic cell neoplasms
* Langerhans cell histiocytosis
¢ Langerhans cell sarcoma
* Interdigitating dendritic cell sarcoma/tumour
* Follicular dendritic cell sarcoma/tumour
¢ Dendritic cell sarcoma, not Otherwise ee

Rouen of uncertain pete arid stage ofdifferentiation

Source: Jaffe, ES, et al: World Health Organization Classification of Tumours Pathology & Genetics, Tumours
of Haematopoietic and Lymphoid Tissues. [ARC Press, Lyon, France, 2001.

of lymphocyte maturation. Thus, we would expect to see transformed into secondary follicles containing a germinal
lymphomas that express the attributes of normal lymphoid center surrounded by a crescent of B cells referred to as the
cells found in each of the various normal lymphoid compart- mantle zone. The germinal center is populated with centro-
ments; that is, the precursor (bone marrow), intrafollicular, cytes (resting B cells), centroblasts (proliferating B cells),
mantle zone, marginal zone, and interfollicular compart- dendritic cells, and histiocytes/macrophages. The paracor-
ments. As illustrated in Figures 21-9 and 21-10, each of the tex (inner or deep cortex) is occupied mostly by T cells. The
lymph node compartments is occupied by morphologically paracortex also contains specialized vessels called high
characteristic cells. These cells are also immunophenotypi- endothelial venules which are the point of entry of circulat-
cally and genotypically distinct. ing lymphocytes into the lymph node parenchyma. The
The three major anatomical regions of a lymph node medulla contains a mixture ofT cells, B cells, macrophages,
are the cortex, paracortex and medulla. The cortex, also and plasma cells.
referred to as the outer cortex, is populated primarily by The NHLs may arise from any of these normal cellular
B lymphocytes along with macrophages, plasma cells, and counterparts. B-cell lymphomas are by far the most common
reticular cells. The B cells are mostly organized into dis- (85% to 90%). T-cell/natural killer (T/NK) cell lymphomas
tinct nodules referred to as primary and secondary follicles. make up the majority of the remainder (10% to 15%) whereas
Primary follicles are composed predominantly of small true histiocytic and dendritic cell “lymphomas” are distinctly
B cells. On antigenic stimulation the primary follicles are uncommon (<1%).

Germinal center
(follicular center)

Mantle zone

N iM uh
Paracortex a

Efferent =) Cortex

Figure 21-9 @ Anatomical compartments of the lymph nodes.

476 Chapter 21. The Lymphomas

ONTOGENY

B Lymphocytes T Lymphocytes

B precursor lymphoblast T precursor

Lymphoid stem cell lymphoblast Lymphoid stem cell
Plasma cell BONE
BONE MARROW MARROW

@ Early B-cell
Mature virgin B-cell
rtex
PERIPHERAL BLOOD Medulla
T lymphoblast
Mature virgin T cells : g, HYMUS
Primary follicle
Marginal es
zone cell Mature virgin PERIPHERAL
Monocytoid B cell Mature virgin T cells BLOOD

Centrocyte.Z— ea Manile cell

Mature virgin T cells s @
Early primary
Centroblast-t Antigen Antigen\.
immune
stimulation aehoneé ~s ‘stimulation oe
T immunoblasts LYMPH
~ NS Mantle y y NODE
Germinal Gail T effector cells (a Co
CD4+ CD8+
Marginal zone
Mantle zone ®B immunoblast Paracortex

Plasmacytoid
A lymphocyte B

Figure 21-10 @ Maturation (ontogeny) of (A) B lymphocytes and (B) T lymphocytes in relation to the pertinent anatomical compartments.

B-CELL LYMPHOMAS (Fig. 21-11A and B). Whereas the mantle zone is well devel-
B-cell lymphomas may be subdivided into precursor and mature oped in the benign secondary follicle, it is absent or attenu-
subtypes. Precursor B-cell lymphoma is distinctly uncommon ated in malignant follicles. The normal polarization of
(<1% of NHLs) whereas mature B-cell lymphomas represent the germinal center into light and dark zones is lost in malig-
approximately 90% of all lymphoid neoplasms diagnosed nant follicles. Tingible body macrophages, which are usually
worldwide. frequent in benign secondary follicles, are usually absent in
malignant follicles.
MATURE B-CELL LYMPHOMAS Mature B-cell lymphomas Follicular lymphomas are clonal proliferations of follicular
are malignant (clonal) proliferations of B cells that correspond center cells (centrocytes and centroblasts) (Table 21-10). Cen-
to stages of B-cell ontogeny from naive B cells to mature trocytes (cleaved cells) are small to medium size cells
plasma cells. Plasma cell myeloma, because of its unique clin- (6 tol5 um diameter) with scant cytoplasm, slightly to distinctly
ical presentation and treatment, is discussed in Chap. 22. irregular nuclei (angulated, clefted), and indistinct nucleoli.
Mature B-cell lymphomas are most common in developed Centroblasts (large noncleaved cells) are 20 to 40 um in diame-
countries (United States, Europe, Australia, and New Zealand) ter and have a modest amount of pyranophilic (RNA-rich)
and predominate in older adults (median age 60 years), except cytoplasm, round to oval vesicular nuclei, and small but distinct
for Burkitt lymphoma and mediastinal large B-cell lymphoma. nucleoli. In the WHO classification the number of centroblasts
Within the broad category of mature B-cell lymphoma, two per high-power microscopic field (hpf = 0.159 mm‘”) is used to
subtypes (follicular lymphoma and diffuse large B-cell lym- grade the follicular lymphomas: grade | = 0 to 5 centroblasts/
phoma) account for the majority of cases. hpf; grade 2 = 6 to 15 centroblasts/hpf; and grade 3 =
>15 centroblasts/hpf (Fig. 21-12A and B). Grading is
Follicular B-cell Lymphoma correlated with clinical outcome although the strength of the
The follicular lymphomas by definition exhibit at least a par- correlation has been subject to great debate.” Most cases show
tial follicular growth pattern and are composed of a mixture a predominance of centrocytes (grade | and 2), which are rest-
of the two principal cell types found in the germinal center, ing B cells. This may explain the indolent behavior of most
namely centrocytes (cleaved cells) and centroblasts (large follicular lymphomas.
noncleaved cells). The follicular growth pattern may be vague Although all follicular lymphomas demonstrate at least a
as observed by routine light microscopy. partial follicular growth pattern, diffuse growth areas are
The follicles of a follicular lymphoma bear a resem- often present and may predominate. The percentage of follic-
blance to the benign secondary follicles of a reactive lymph ular growth should be included in the pathologic assessment.
node; however, subtle differences aid the pathologist in rec- Lymphomas derived from follicular center cells are, by
ognizing the malignant follicle of a follicular lymphoma definition, B-cell lymphomas. The expected immunophenotype
 Chapter 21 The Lymphomas 477

Figure 21-12 @A. Follicular lymphoma, WHO grade 1. Malignant

follicles are characterized by a predominance of centrocytes.
BA ae A iene B, Follicular lymphoma, WHO grade 3. Malignant follicles are char-
acterized by a predominance of centroblasts.
Figure 21-11 @ A. Architecture of benign (reactive) follicle.
Secondary follicle consisting of well developed mantle zone (7) and
germinal center that is polarized into light (2) and dark (3) zones. of a follicular lymphoma is monoclonal surface immunoglobu-
Tingible body macrophages (arrows) are prominent in the dark lin-positive (slg+), B-cell-associated antigen-positive (e.g.,
zone. B. Malignant follicle of follicular lymphoma. Note poorly CD19+, CD20+), BCL-6 positive (nuclear), CD10 usually
defined/absent mantle zone and lack of polarization of the germinal
center into light and dark zones.
positive (more than 50%), CDS-negative, CD23 usually nega-
tive, CD43-negative, and CDl1lc-negative (Table 21-11).
Follicular lymphomas are distinguished at the genetic level
by a unique chromosomal translocation, t(14;18), involy-
ing the BCL2 proto-oncogene on chromosome 18 and the

Lymphoma Subtype Small Cells Large Cells

B-cell CLL/SLL Round (rarely cleaved) Paraimmunoblasts/prolymphocytes

Mantle cell Cleaved (rarely round) Usually absent
Follicular center cell (CB/CC) Cleaved (centrocytes) Centroblasts
Lymphoplasmacytic Round Immunoblasts
Marginal zone Round, cleaved, and/or monocytoid Centroblasts/immunoblasts

CLL = chronic lymphocytic leukemia; SLL = small cell lymphocytic lymphoma; CB/CC = centroblastic/centrocytic.
478 — Chapter 21 The Lymphomas

Lymphoma Subtype CD5 CD10 CD23 CD43 Cyclin D1 ae

B-cell CLL/SLL ate = ae 4F =

Mantle cell ae Ir = 4F as
Follicular center cell = Gh ies = oS =
Lymphoplasmacytic = = = aes =
Marginal cell Extranodal = = = Shr =
Nodal = = = ri 3

—/+ = less than 50% of cases positive; +/— = more than 50% of cases positive; CLL = chronic lymphocytic leukemia;
SLL = small lymphocytic lymphoma.

immunoglobulin heavy-chain gene on chromosome 14 (see irregular nuclei, and inconspicuous nucleoli (Fig. 21—-13A).
Table 21-8). This translocation is detectable in 70% to 95% of Most often, MCL has a diffuse or vaguely nodular pattern;
cases of follicular lymphoma. however, occasionally a distinct mantle zone pattern is
Follicular center lymphomas accounts for approximately observed. In this form the malignant cells are distributed as an
40% of adult NHLs in the United States. The disease most expanded mantle zone surrounding benign (polyclonal) ger-
commonly affects adults and is usually widespread at diagno- minal centers. This pattern of MCL must be distinguished
sis (Table 21-12). Although the disease course is usually from mantle zone hyperplasia.
indolent, it is not often curable with current therapy (median The immunophenotype of MCL aids in differentiating
survival is 6 to 8 years). Most studies indicate that grade 3 fol- this subcategory of lymphoma from other morphologically
licular lymphomas are more aggressive than grades | and 2. similar lymphomas. Characteristically, the malignant cells
Consequently grade 3 follicular lymphoma ts typically treated express monoclonal slg, B-cell-associated antigens, CD5, and
more aggressively. Progression with time from follicular lym- CD43, but are negative for CD23, CDI 1c, and usually CD10
phoma to a more aggressive, diffuse large-cell pattern occurs (see Table 21—11).'' This immunophenotype corresponds to
in approximately 40% of cases (Table 21—13); however, spon- the normal CD5-positive, CD23-negative B cell of the inner
taneous remissions of follicular lymphoma have also been follicle mantle.* Most cases of MCL demonstrate a unique
described. chromosomal translocation, t(11;14), involving the BCL/
proto-oncogene on chromosome 11 and the immunoglobulin
Mantle Cell Lymphoma heavy-chain gene on chromosome 14 (see Table 21-8). This
Lymphomas composed of lymphocytes with the morphologi- translocation results in the overexpression of the cell cycle
cal and immunophenotypic features of mantle cells have only protein cyclin D1, which may be detected in the nuclei of
recently been accepted as a distinctive subcategory of NHL.” MCL by immunocytochemical techniques (see Fig. 21—-13B).
The cytological features of mantle cell lymphoma (MCL) are MCL is a disease of older adults with high male-
somewhat variable, creating difficulty in differentiating on to-female ratio (see Table 21-12). The disease is usually wide-
histologic grounds mantle cell lymphoma from other low- spread at diagnosis, with lymph nodes and spleen being the
grade NHLs. The cells of MCL are most commonly small- most common sites of involvement. MCL has a moderately
to medium-sized lymphocytes with scant pale cytoplasm, aggressive course (3- to 5-year median survival), but behaves

Lymphoma Male: 5 years Survival Rate at

Subtype Female Extranodal % BM (%) PB (%) (%) 5 years (%)

B-cell CLL/SLL : Rare 100. >90 <10

Mantle cell : <10 >50 >50 25 0
Follicular center : <10 <50 >50 <15 SS
cell
Lymphoplasmacytic 3 Rare 100 <30
Marginal zone
Extranodal : >80 <<215) Rare
Nodal : 50? 50? Rare

*Pure nodal marginal cell lymphomas are uncommon.

BM = bone marrow involvement; PB = peripheral blood involvement; NED = no evidence of disease.
 Chapter 21 Thelymphomas 479

Table 21-15 Low-grade B-cell Lymphomas: Large-cell Transformation

Lymphoma Secondary Large Cell Type Incidence of Transformation (%)

Chronic lymphocytic Richter’s transformation (diffuse large cell) D)

leukemia/small
Lymphocytic lymphoma Paraimmunoblastic/prolymphocytoid transformation 15
Mantle cell lymphoma Blastic variant (lymphoblastoid) 2
Centrocytoid/centroblastic (centroblastoid variant) uw
Follicular center cell lymphoma Secondary centroblastic (diffuse large cell) 40
Lymphoplasmacytic Secondary immunoblastic 5
Marginal zone lymphoma Secondary high-grade lymphoma ?
? = unknown.

similarly to other low-grade lymphomas in that it is generally been described and are associated with a more aggressive
not curable with current therapies. A peculiar extranodal clinical course.”°
presentation of the disease in the intestine is referred to as
lymphomatous polyposis (Fig. 21-14). Several histologic Marginal Zone Lymphoma
variants of MCL (pleomorphic, large cell, and blastoid) have The marginal zone of lymph nodes is a poorly defined anatomi-
cal compartment.*’ However, in other lymphoid tissues (i.e.,
spleen and Peyer’s patches), the marginal zone is_ better
defined.** The typical marginal zone cell is a centrocyte-like
cell (small cleaved cell) with more abundant pale-staining
cytoplasm than a true centrocyte. Under antigenic stimulation,
these marginal zone B cells are believed, by some investigators,
capable of differentiation into monocytoid (parafollicular)
B ceils and plasma cells (see Fig. 21-10). Monocytoid B cells
are medium-sized lymphocytes morphologically resembling
monocytes that are found predominantly in the lymphoid
sinuses and paracortex of some reactive lymph nodes but not in
normal lymph nodes. They are particularly common in the
reactive lymphadenopathies associated with human immun-
odeficiency virus (HIV) infection and toxoplasmosis.

Figure 21-13 @ A. Mantle cell lymphoma. Neoplastic cells are

“intermediate” between the small uniform lymphocytes of SLL and
the centrocytes of FL. B. Majority of nuclei of MCL express BCL-1
protein (red-brown reaction product). Immunoperoxidase stain Figure 21-14 @ Endoscopic view of large polyp of the bowel in a
with monoclonal antibody to BCL-1/cyclin D1. case of lymphomatous polyposis (mantle cell lymphoma).
480 Chapter 21 The Lymphomas

Lymphomas related to marginal zone cells are a group of

recently described and controversially related entities.”Acom-
mon form of presentation of marginal zone lymphoma (MZL)
is at extranodal sites, particularly stomach, salivary gland, lung,
thyroid, and orbit, where they have been referred to as lym-
phomas of mucosal-associated lymphoid tissue (MALT
lymphomas).*° Collections of lymphoid tissue normally occur
in extranodal mucosal sites such as the small bowel (Peyer’s
patches). Mucosal-associated lymphoid tissue, however, does
not normally occur in the most common sites for MALT
lymphomas. Under conditions of chronic antigenic stimulation
(e.g., infection or autoimmune disease), such lymphoid tissue
may develop in these sites. Examples of such conditions
are Helicobacter pylori gastritis, Sjogren’s syndrome, and
Hashimoto’s thyroiditis. Lymphomas that develop in the setting
of chronic antigenic stimulation may remain dependent on the
presence of benign antigen-driven T cells, a fact that has impor-
tant therapeutic and prognostic implications. For example, in
the case of the MALT-type marginal zone lymphoma of the
stomach, eradication of the offending antigen (H. pylori) with
antimicrobial therapy has often been demonstrated to be effec-
tive in treating this specific lymphoma.*!
Thé histologic features of extranodal MZL (MALT) are
highly characteristic (Fig. 21-15A and B; 21—16A and B).
Reactive follicles or their remnants are uniformly present.
Subjacent to the mucosal lining or glandular epithelium of
involved organs and surrounding the reactive follicles are col-
lections of small- to medium-sized lymphocytes with a mod-
erate amount of pale-staining cytoplasm and irregular nuclei
(centrocyte-like cells). In addition to centrocyte-like cells,
neoplastic cells with features similar to small lymphocytes or
monocytoid B cells, or both, are present. Plasma cell differen-
tiation in a portion of the cells is also a common finding. A Figure 21-15 @ A. Extranodal marginal zone B cell (MALT)
constant feature of these lymphomas is infiltration of the lymphoma of the stomach. MALT lymphoma (1) infiltrates the gas-
tric mucosa and extends into gastric epithelium (2), Reactive folli-
associated epithelium by neoplastic lymphocytes, resulting in
cles (3) are commonly present. B. Characteristic lymphoepithelial
the formation of lymphoepithelial lesions. The neoplastic lesions (arrows) of gastric MALT lymphoma are composed of clus-
cells may also infiltrate the reactive follicles, giving the ters of small, atypical lymphocytes (CD20 positive B cells) within
impression of a follicular center cell lymphoma, a process the glandular epithelium.
referred to as follicular colonization.
A second pattern of MZL is lymph node-based and was
previously referred to as monocytoid B cell lymphoma by CD10-, CD23-, CD43-/+, and CD11c+/- (see Table 21-11).
hematopathologists in the United States.°* MZL involving The extranodal form (MALT lymphoma) is associated with
lymph nodes is frequently observed in patients with autoim- the chromosomal abnormalities trisomy 3 and t(11;18) (see
mune disorders such as SjOgren’s syndrome or coexistent Table 21-8). Patients with gastric MALT lymphoma with
extranodal (MALT) type of marginal zone lymphoma, sug- t(11;18) usually have a poor response to antibiotics for H. pylori
gesting a close relationship between extranodal (MALT) and eradication.** Rare cases of extranodal MZL are associated with
node-based (monocytoid B cell) lymphomas. t(1;14), resulting in nuclear over expression of Bcl-10 protein,
Histologically, node-based MZL is characterized by which predicts for more aggressive behavior.** These genetic
infiltration of the paracortex and sinuses with a monoclonal abnormalities are uncommon in the purely node-based MZL.
population of small lymphocytes, centrocyte-like cells, and The clinical features of MZL correspond to the two
monocytoid B cells. Monocytoid B-cells have oval, reniform, modes of presentation discussed earlier (see Table 21-12).
or sometimes quite irregular nuclei; bland chromatin pattern; The extranodal (MALT) form is a disease of adults with a
and relatively abundant pale staining cytoplasm with distinct slight female predominance and is highly associated with
cytoplasmic borders. Benign secondary follicles with hyper- autoimmune disease or infection (Helicobacter gastritis). The
plastic germinal centers are present, which serve to accent the disease is usually localized (stage I or II) at presentation
pale-staining infiltrate of monocytoid B cells. and the prognosis is usually excellent following therapy for
The characteristic immunophenotype of MZL is as fol- localized disease. Dissemination occurs, however, in approx-
lows: sIg+, cytoplasmic Ig+ (40%), B-cell antigen+, CDS5-, imately 30% of cases. Although the disease course is usually
 Chapter 21 TheLymphomas 481

cells of splenic MZL is similar to those of nodal and extranodal

MZL. The clinical course is indolent and incurable; however,
splenectomy may result in long remissions.

Lymphomas Related to Interfollicular Lymphocytes

The normal interfollicular zone of lymphoid tissue is a com-
plex mixture of cell types. Although it is often referred to
as the T-cell zone, because of the predominance of T cells,
several other cell types are found in this region, notably recir-
culating B cells, histiocytes, and reticulum cells. Despite the
prevalence of T cells in the interfollicular zone, the majority
of lymphomas whose normal counterpart is an interfollicular
ilau
~=
ut
ss
cell are B-cell lymphomas.

re: * ee! : ;
Small Lymphocytic Lymphoma/Chronic Lymphocytic
pete. base
Leukemia
Small lymphocytic lymphoma (SLL) is the tissue equivalent
of chronic lymphocytic leukemia (CLL). SLL and CLL are
considered to be different clinical expressions of a single
disease process and therefore are combined in the WHO clas-
sification. The growth pattern of SLL is diffuse, although a
pseudofollicular pattern may be observed (Fig. 21—18A).
These pseudofollicles, also called growth centers, do not have
cS
"ries the cytological or histological features of true neoplastic fol-
AS licles. In the early phase of nodal involvement, the neoplastic
—

Se:
Pee cells may be confined to the interfollicular areas with sparing
of the normal follicles. Later, there is total effacement of
lymph node architecture. The majority of the neoplastic cells
8 are small, uniform lymphocytes with scant cytoplasm, round

Se
cmeeceses:obecl
ray
Ps
ame .t nucleus, clumped chromatin, and absent to small nucleoli; that

‘s PEAT Be
Pi
is, they appear cytologically similar to normal small lympho-
"exes
Dae
Cae
== e- -& 7 “J A

cytes (see Fig. 21—18B). Admixed with these small lymphocytes

Figure 21-16 @ A. Extranodal marginal! zone B cell (MALT)
are variable numbers of intermediate to large lymphocytes
lymphoma of the parotid gland. Neoplastic cells (red arrows) sur- referred to as prolymphocytes and paraimmunoblasts. These
round and infiltrate the metaplastic epithelium (yellow arrows) of cells are most commonly found in increased numbers within
the parotid gland. B. Detailed view of lymphoepithelial lesions (red the pseudofollicles (growth centers) (see Fig. 21-18C).
arrows) involving metaplastic epithelium (yellow arrows) in a case of
MALT lymphoma of the parotid gland.

indolent, following dissemination the disease appears to be

incurable with current therapy. Transformation to a large-cell
lymphoma occasionally occurs (see Table 21-13).
Most cases of nodal involvement by MZL represent dis-
semination of extranodal (MALT) lymphoma. However, pure
nodal presentations, usually in the cervical region, have been
observed. The majority of patients present with advanced
disease (stage III or IV).
Adding to the complexity and controversy of the category
of MZL is the entity splenic MZL with or without villous lym-
phocytes.** Its relationship to nodal and extranodal MZL is not
well understood; however, most investigators consider it a dis-
tinct entity separate from MZL of the nodal and MALT types.”
Patients characteristically have massive splenomegaly with
minimal adenopathy. Bone marrow and peripheral blood
Figure 21-17 ® Splenic marginal zone B-cell lymphoma. White
involvement are common. The spleen exhibits nodular expan- pulp nodules (7) show expansion of the marginal zone (2) by a
sion of the white pulp by intermediate-sized lymphocytes population of small monoclonal B cells which express the pheno-
resembling splenic marginal zone cells surrounding a central type of splenic marginal zone cells. Neoplastic cells infiltrate the red
core of small lymphocytes (Fig. 21-17). The phenotype of the pulp (3).
482. Chapter 21. The Lymphomas

Spee
Sites3
este
e
Fy he
SLL may show evidence of plasmacytoid differentiation,
en, 3 including the presence of cytoplasmic immunoglobulin and a
small amount of circulating monoclonal immunoglobulin in
the peripheral blood. These cases are phenotypically and
clinically similar to small lymphocytic lymphoma and as such
should be distinguished from true lymphoplasmacytic lym-
See eae
phoma (see later discussion). Diagnostic difficulty may also
viae paestee ct occur when there is overgrowth of the lymphoma by the
23) atest Sa 3 Botes
br atiteen WES
larger cells of the growth centers (paraimmunoblastic and
prolymphocytoid transformation).
The normal counterpart cell to the malignant cell of SLL
is believed to be a recirculating naive B lymphocyte. In addi-
tion to the expression of B-cell-associated antigens (CD19,
CD20, and CD22), with sensitive techniques, low-density
monoclonal sIg is detectable. SLL is aiso positive for CD5,
CD23, and CD43 but lacks CD10 (see Table 21-11). A small
number of cases are weakly positive for CDI Ic.
Approximately 80% of cases of SLL will exhibit clonal
cytogenetic abnormalities including trisomy 12 (20%),
del 13q14 (50%), and del 11q22-23 (20%). The presence of
these cytogenetic abnormalities is associated with prognosis.
The 13q14 abnormality is associated with improved survival
whereas the other abnormalities predict for worse outcome.
Recently hypermutation of the immunoglobulin gene variable
region has been shown to correlate with improved survival.*’
Expression of the ZAP-70 protein has been shown to provide
the same prognostic information as the more complicated
mutation analysis.
Small lymphocytic lymphoma/chronic lymphocytic
leukemia is a disease of older adults and is usually wide-
spread at diagnosis. The disease, although indolent in
behavior, is considered incurable with standard therapy.
Improvement in disease-free survival has been demon-
strated with purine nucleoside analogues such as fludara-
bine. Transformation to a high grade lymphoma (3%) or
HL (0.5%) can occur.**

Lymphoplasmacytic Lymphoma
A phenotypically and clinically distinct form of lymphoma of
small lymphoid cells exists in which the cells exhibit plasma-
cytoid features and a phenotype distinct from typical small
lymphocytic lymphoma.*” Specifically, they express mono-
clonal surface and cytoplasmic immunoglobulin, usually of
the IgM class; are positive for B-cell-associated antigens
(CD19, CD20, CD22, and CD79a); but lack CD5 and CD10
(see Table 21-11). Fewer than 50% of cases are positive for
CD43. The normal counterpart of this lymphoma is believed
to be a recirculating CDS-negative cell, which has been stim-
ulated to differentiate into plasma cells. Approximately 50%
of cases of lymphoplasmacytic lymphoma will demonstrate
t(9;14), involving the gH and PAXS genes (see Table 21-8).
Clinically, these lymphomas usually correspond to the syn-
Figure 21-18 8A. Small lymphocytic lymphoma. Diffuse efface- drome of Waldenstrém’s macroglobulinemia. Lymphoplas-
ment of lymph node by proliferation of small lymphocytes. Pale macytic lymphoma is a disease of older adults and most
areas are growth centers (arrows). B. Cytological detail of small
commonly involves lymph nodes, spleen, and bone marrow.
lymphocytes of SLL. Cells have scant cytoplasm with uniform nuclei,
coarsely clumped chromatin and indistinct nucleoli. C. Growth A monoclonal gammopathy of the IgM type is often present
center of SLL is composed of prolymphocytes and paraimmunoblasts, and may be associated with hyperviscosity symptoms. The
cells that are larger than the typical small lymphocyte of SLL challenge to the pathologist is to differentiate this lymphoma
 Chapter 21 TheLymphomas 483

from other low-grade lymphomas that can show plasma cell A subset of DLBCLs express CD5 (10%) or CD10 (25% to
differentiation. Similar to other low-grade lymphomas, this 50%). CD5-positive DLBCL may arise de novo or from an
disease is indolent but incurable, and large-cell lymphoma underlying low-grade CD5 positive B-cell lymphoma (SLL or
transformation may occur (see Table 21-13). MCL). The absence of BCL-1 expression rules out a blastoid
variant of MCL. The expression of CD10 and BCL-6 by a
Diffuse Large-Cell Lymphoma DLBCL suggests the possibility of transformation from an
Diffuse large B-cell lymphoma (DLBCL) represents the most underlying follicular lymphoma. The t(14;18) translocation
frequently diagnosed category of NHL worldwide (30%). By characteristic of follicular lymphoma can be found in 20% to
definition, DLBCL exhibit a diffuse growth pattern and are 30% of cases of DLBCL.
composed of clonal B cells of large size (nuclear diameter equal DLBCL occurs in a broad age range including children
to or greater than normal macrophage nucleus). Large-cell lym- although the median age is in the seventies. The disease may
phomas in general exhibit diverse morphologic features; how- present as nodal or extranodal disease. The gastrointestinal
ever, attempts to subclassify these large cell lymphomas based tract is the most common extranodal site. A distinct clinical
on morphology proved to be nonreproducible.*” Consequently subtype of DLBCL is mediastinal (thymic) large B-cell lym-
all diffuse large cell lymphomas composed of B cells were phoma. This subtype usually presents as a large anterior
grouped by the WHO into a single category of DLBCL. mediastinal mass in a young adult female. DLBCL is an
On morphological grounds DLBCL consist of a mixture aggressive lymphoma if untreated; however, cure is possible
of cells with centroblastic, immunoblastic, and anaplastic/ in many patients with multiagent chemotherapy.
pleomorphic features (Fig. 21-19). One morphologic type,
most commonly centroblastic, may predominate. In rare cases Burkitt Lymphoma
of DLBCL referred to as T-cell/histiocyte rich DLBCL Burkitt lymphoma (BL), first described in 1958 by Dennis
(TCRLBL) there is a predominance of non-neoplastic T cells Burkitt,” is endemic to Africa but also accounts for approxi-
and/or histiocytes with fewer than 10% large neoplastic B mately one-third of non-African pediatric lymphomas (sporadic
cells. These cases may be easily confused with NLPHL. BL) and a high percentage of lymphomas in immunocompro-
Most cases of DLBCL arise de novo; however, transfor- mised patients, particularly patients with acquired immunodefi-
mation from a lower grade B-cell lymphoma, particularly ciency syndrome (AIDS).
SLL, MCL, MZL, follicular lymphoma, or NLPHL may also The monotonously uniform cells of Burkitt’s lymphoma
occur. Utilizing gene expression analysis two types of are medium-sized cells with uniformly round nuclei, multiple
DLBCL lymphoma appear to exist, a germinal center B-like small nucleoli, and a modest amount of intensely basophilic
(centroblastic) DLBCL and an activated B-like (immunoblas- cytoplasm (Fig. 21—20A). Lipid vacuoles are readily evident
tic) DLBCL.*! Patients with germinal center B-like gene in the cytoplasm on smears or imprints of the tissue. The
expression have a better overall survival than those with acti- proliferation rate of Burkitt’s lymphoma is very high
vated B-like expression. (see Fig. 21—20B), and the estimated volume doubling time of
By definition DLBCL is a clonal proliferation of B cells. this lymphoma is the highest of any tumor (approximately
The expected immunophenotype include expression of pan- 1 day). Programmed tumor cell death (apoptosis) is also very
B-cell antigens, e.g., CD19, CD20, CD22, CD79a; however, high, and a starry sky pattern of tingible-body macrophages is
one or more of these pan-B-cell antigens may be absent. usually evident owing to phagocytosis of the apoptotic debris.
Variant histologies are recognized and include BL with plas-
macytoid differentiation and atypical BL characterized by
cells more pleomorphic than those of classical BL.
As previously discussed, Burkitt’s lymphoma is cytoge-
netically characterized by a t(8;14) in the majority of cases or
less often by t(2;8) or t(8;22). In the endemic (African) form
of this disease, the breakpoint on chromosome 14 involves
the heavy-chain joining region, suggesting an early B-cell
origin of the Burkitt cell. However, in the nonendemic form,
the breakpoint is in the heavy-chain switch region, consistent
with origin at a latter stage (possibly follicular center cell) of
B-cell development. The endemic and immunodeficiency-
related cases are also associated with a high frequency of
tumor cell incorporated EBV genomes. The immunopheno-
type of Burkitt’s lymphoma is surface IgM-positive, B-cell
antigen-positive, CD10+, CDS5—, CD23-, Bcl-2—, and TdT-.
The male-to-female ratio for Burkitt’s lymphoma is
approximately 2.5 to 1. The facial bones, particularly the jaw,
are the most common sites of involvement for the African
Figure 21-19 ™ Diffuse large B-cell lymphoma. This common
(endemic) variety (Fig. 21-21). In the nonendemic variety, an
lymphoma is composed of a mixture of large B cells that exhibit
centroblastic, immunoblastic, and pleomorphic morphology. abdominal mass is the most common presenting pattern. The
484 Chapter 21 The Lymphomas

Figure 21-21 @ Burkitt’s lymphoma before (/eft) and after treat-

ment (right).

Response to therapy is poor and death usually occurs soon

after diagnosis.

Primary Effusion Lymphoma

Primary effusion lymphoma is defined as a large B-cell lym-
phoma in which the malignant cells are confined to body
cavity spaces with no detectable tumor masses.“ The pleural,
pericardial and peritoneal cavities are the most common sites
of involvement and usually only one cavity is involved. The
large neoplastic cells exhibit high grade features (immunoblas-
tic, plasmablastic, anaplastic) (Fig. 21-23) and are uniformly
positive for human herpes virus type 8 (HHV8). EBV may be
detectable as well. The cells express an aberrant phenotype
however at least one pan-B-cell surface antigen is detectable.
Surface and cytoplasmic immunoglobulin is usually not
detectable; however, clonal rearrangement of immunoglobulin
Figure 21-20 @ A. Burkitt lymphoma. Small uniform cells (yellow
arrows) with moderate amount of cytoplasm, round nuclei and genes is present. Most cases of PEL occur in patients with HIV
multiple small nuclei are admixed with cellular debris and tingible infection however the disease also occurs in areas of high
body macrophages (red arrow). B. High proliferation rate of Burkitt HHV8 prevalence (Mediterranean) particularly in elderly men
lymphoma detected by high percentage of nuclei showing positive without HIV infection. The disease is usually rapidly fatal.
staining for MIB-1/Ki-1 (red-brown reaction product). Immunoper-
oxidase stain with monoclonal antibody to the proliferation antigen
MIB-1/Ki-1.

distal ileum, cecum, and mesentery are the most frequent sites
of involvement. Other less common sites of involvement “ ie * >
include the kidneys, ovaries, and breasts. Burkitt’s lymphoma AY. oy ae gs
a
is a highly aggressive and rapidly fatal disease if untreated;
however, this disease is potentially curable and prognosis
correlates with tumor bulk (see staging).
¥°

Reh
y©
Intravascular Large B-Cell Lymphoma %

Intravascular large B-cell lymphoma (ILBCL) is a rare and

“ie
highly aggressive lymphoma in which the neoplastic cells are
a
-iw ‘4
is
confined to the lumina of blood vessels, particularly capillaries “a, ea

(Fig. 21—22).* The patient usually presents with symptoms

related to vascular obstruction. ILBCL usually involves the
blood vessels of multiple organs. Skin and central nervous sys-
tem involvement are particularly common at initial presenta- Figure 21-22 @ Intravascular large cell lymphoma. Small vessels
tion. Restriction of the lymphoma cells to vascular lumina is (capillaries) are occluded by atypical large cells (arrows) without
postulated to be secondary to a homing receptor defect. extension into the parenchyma of the involved organ.
 Chapter 21 The Lymphomas 485

Figure 21-25 @ Primary effusion lymphoma (PEL). Cytocentrifuge

preparation of PEL showing large atypical mononuclear cells that
usually have immunoblastic features.

PEL must be distinguished from pyothorax associated DLBCL

which has a more favorable prognosis.

PRECURSOR B LYMPHOBLASTIC LYMPHOMA Precursor

B lymphoblastic lymphoma (B-LBL) and B-cell acute lym-
phoblastic leukemia (B-ALL) are biologically the same disor-
der being arbitrarily’ distinguished by the mode of clinical
presentation.* The lymphomatous presentation is uncommon
compared to a leukemic presentation and the B-cell type of
LBL constitutes only 10% of LBLs with T-cell type represent-
ing 90%. Skin, bone, and lymph nodes are the primary sites
of involvement. Bone marrow lymphoblasts should be less
than 25% in order to label the clinical presentation as LBL.
Figure 21-24 m A. Precursor B lymphoblastic lymphoma. Cells
The pattern of lymph node involvement is diffuse with the have scant cytoplasm, finely dispersed (“blastic”) chromatin, and
lymph node architecture effaced by an infiltrate of small to brisk mitotic activity. B. The neoplastic nuclei of lymphoblastic
medium size blasts (Fig. 21—24A). Cytoplasm is scant and the lymphoma express TdT (red-brown reaction product). Immunoper-
nuclei have finely dispersed chromatin with indistinct nucleoli. oxidase stain with polyclonal antibody to TdT.
The nuclear contours vary from round to oval and are some-
times convoluted. Mitotic figures are usually numerous. they are, with limited exceptions, among the most aggressive
The cells of precursor B-LBL exhibit an immature B-cell of hematolymphoid malignancies. Mature NK lymphomas
phenotype: TdT+, HLA-—Dr+, CD19+ (see Fig. 21—24B). demonstrate a predilection to involve certain racial groups, par-
They are usually positive for CD10 whereas CD20, CD34, ticularly Asians and the genetically related Native Americans
and CD45 are variably present. The anomalous expression of of Central and South America and Mexico. Precursor T-cell
myeloid antigens (CD13 or CD33) may be present; however, lymphoma is an aggressive lymphoma that is biologically iden-
this does not preclude a diagnosis of B-LBL. The cytogentic tical to T-cell acute lymphoblastic leukemia.
findings in B-LBL are complex but prognostically important
and may be divided into favorable and adverse groups. MATURE T AND NK CELL LYMPHOMAS Mature T-cell
B-LBL is a disease primarily affecting patients younger lymphomas correspond to a post-thymic stage ofT cell differ-
than 20 years of age. The remission rate with treatment is high entiation. Most post-thymic (mature) T-cells are of the T,,
and the median survival is approximately 5 years. type which differentiate into either CD4 positive “helper”
T cells or CD8 positive “cytotoxic” T cells. A smaller popula-
T-CELL AND NATURAL KILLER (NK) CELL tion (5%) of post-thymic T cells are of the T,; type which are
LYMPHOMAS negative for both CD4 and CD8. T,, and Ty; cells are positive
Similar to the B-cell lymphomas the T-cell lymphomas for CD3. NK cells do not have a complete T-cell receptor
are divided into mature and precursor types. Because of complex (CD3 negative) but otherwise express a phenotype
phenotypic and clinical similarities, the mature T and NK cell similar to cytotoxic T cells. T/NK cell lymphomas express
lymphomas are grouped together in the WHO classification. phenotypes that correspond, at least partially, to these various
Although relatively uncommon (12% of NHLs worldwide) normal types of post-thymic T and NK cells. Because of the
486. Chapter 21. The Lymphomas

morphological phenotypic and genotypic complexity of the

T/NK cell lymphomas clinical features are an important aid
in the classification of these lymphomas. Three patterns of
clinical presentation of the mature T/NK cell lymphoma
are observed: nodal, extranodal, and leukemic/disseminated
(Table 21-14). The T-cell lymphomas of the nodal group are
by far the most common in Western countries.

Peripheral T-Cell Lymphoma, Unspecified

Most mature T-cell lymphomas do not fall within one of the
well defined clinicopathological groups. These lymphomas
are classified as peripheral T-cell lymphoma, unspecified
(PTCL) in the WHO classification and represent approxi-
mately 50% of the mature T/NK cell lymphomas seen in
Western countries. Morphologically, this is a heterogeneous
category with individual cases usually composed of a poly-
morphous mixture of atypical small-, medium-, and large- Figure 21-25 m@ Peripheral T-cell lymphoma (PTCL), unspecified.
sized cells. Occasionally, one cell size may predominate, PTCL usually consists of a mixture of atypical lymphoid cells which
giving a more monomorphic picture. Benign eosinophils, are variable in size, giving a polymorphous appearance.
plasma cells, and epithelial histiocytes are often admixed with
the neoplastic cells, and high endothelial venules may be
prominent (Fig. 21-25). Nodal presentations are most com- 25%.*° Treatment with. multiagent chemotherapy is similar to
mon; however, extranodal (skin, subcutaneous tissue, spleen, that used for the diffuse aggressive B-cell lymphomas; how-
viscera) sites are often involved. Involved lymph nodes ever, response to the therapy is usually poor.
demonstrate either a diffuse effacement of nodal architecture
or an interfollicular growth pattern of the neoplastic cells with Anaplastic Large Cell Lymphoma
preservation of benign follicles. Immunophenotypically, this Systemic anaplastic large cells lymphoma (ALCL) is a rela-
group of lymphomas usually exhibits a mature (post-thymic) tively common form of mature T-cell lymphoma that represents
T helper cell phenotype (TdT-, CD3+, CD4+, CD8-) a distinct clinical entity.*” Systemic ALCL should not be con-
although other phenotypes (CD4—, CD8+ or CD4+, CD8+) fused with primary cutaneous ALCL to be discussed later.
may occasionally be observed. PTCL is a disease of adults but The morphology of the neoplastic cells is heterogenous but all
cases in childhood do occur. The disease usually behaves in cases contain a variable number of distinctive large cells with
an aggressive fashion with an overall 5-year survival of only kidney-shaped nuclei and an eosinophilic inclusion-like region
adjacent to the nucleus (Fig. 21—26A). In the small cell variant
of this lymphoma these “hallmark” cells are few in number,
however, their identification is an aid to establishing the correct
diagnosis. The pattern of nodal involvement is usually diffuse;
however, partial nodal involvement in a sinus pattern can easily
be mistaken for metastatic carcinoma. The neoplastic cells
Nodal presentation Peripheral T-cell lymphoma, of ALCL exhibit a membranous and Golgi pattern of CD30
unspecified positivity. ALCL is characterized at the genetic level by translo-
Anaplastic large cell lymphoma cation t(2;5). This leads to expression of the ALK-NPM
Angioimmunoblastic T-cell protein which can be detected immunohistochemically (see
lymphoma
Fig. 21-26B). Most cases of ALCL express epithelial mem-
Extranodal Extranodal T/NK cell lymphoma,
presentation nasal type brane antigen (EMA) which can lead to confusion with carci-
Enteropathy-type T-cell lymphoma noma. CD3 is usually absent whereas CD45 is variably present.
Hepatosplenic T-cell lymphoma EBV is characteristically absent. Clonal rearrangement of the
Subcutaneous panniculitis-like T-cell receptor is detectable in greater than 90% of cases.
T-cell lymphoma ALCL is primarily a node based disease and most commonly
Blastic NK-cell lymphoma
Mycosis fungoides/Sézary
affects children. Compared to most other T-cell lymphomas,
syndrome ALCL is unusual in that it is generally responsive to therapy.
Primary cutaneous anaplastic Overall survival is approximately 80% at 5 years.
large cell lymphoma
Leukemic/ T-cell prolymphocytic leukemia
Angioimmunoblastic T-Cell Lymphoma
disseminated T-cell large granular lymphocytic
presentation leukemia
In 1979, Shimoyama and colleagues described a lymphoma
Aggressive NK-cell leukemias with clinical and morphological features similar to that of
Adult T-cell lymphoma/leukemia immunoblastic lymphadenopathy (IBL) and angioimmunoblas-
tic lymphadenopathy with dysproteinemia (AILD).4* These
 Chapter 21 Thelymphomas 487

ye EE EMAL Vlas 8
Ok Oo Dee Pee

Figure 21-26 @ A. Systemic anaplastic large cell lymphoma. Figure 21-27 @ A. Angioimmunoblastic T-cell lymphoma. Mixture
Pleomorphic cells that vary in size and shape. “Hallmark” cells are of lymphocytes, some of which are atypical, dendritic cells, and
readily apparent (arrows). B. Detailed view of several “hallmark” thick-walled high endothelial venules. B. Clusters of medium to
cells (arrows) showing kidney or horseshoe-shaped nuclei and large cells with clear cytoplasm (arrows) are characteristic of
juxtanuclear eosinophilic inclusion-like zone. angioimmunoblastic T-cell lymphoma.

disorders are associated with evidence of hyperimmunity (fever, involve the skin; however, this is seldom an isolated or present-
skin rash, hypergammaglobulinemia) and often develop after ing finding in B-cell lymphoma. Hodgkin’s lymphoma rarely
exposure to certain drugs and infectious agents. There is a high involves the skin in the form of direct extension from adjacent
frequency of evolution of these clinicopathological entities that involved lymph nodes. The category of cutaneous T-cell lym-
run the gamut from benign (possibly premalignant) disorders to phoma includes a broad group of dysplastic and frankly malig-
aggressive, rapidly fatal lymphomas. The affected lymph nodes nant T-cell proliferations with a predilection for infiltration of
of angioimmunoblastic T-cell lymphoma are partially effaced the skin.* Disorders within the umbrella of this category
by a mixture of atypical lymphoid cells, follicular dendritic include mycosis fungoides, Sézary syndrome, lymphomatoid
cells, and thick-walled high endothelial venules (Fig. 21—27A). papulosis, and primary cutaneous anaplastic large-cell lym-
The lymphoid cells are predominantly CD4 positive with phoma. Mycosis fungoides and Sézary syndrome, two related
admixed CD8 positive T cells.” Clusters of CD21+ follicular disorders, are characterized by infiltration of the dermis and
dendritic cells are aberrantly located and most often found sur- epidermis by malignant T cells with a peculiar cerebriform
rounding the high endothelial venules (see Fig. 21-27B). Clonal nucleus. Thin sections of well-fixed paraffin-embedded material
T-cell receptor rearrangement is detectable in approximately or plastic-embedded sections are required to appreciate this
75% of cases. Frank angioimmunoblastic T-cell lymphoma usu- nuclear detail. In 90% of cases, these cells exhibit a mature
ally follows an aggressive clinical course. “helper” phenotype (pan-T+, CD4+, CD8-) and in 10%, a
mature suppressor phenotype (pan-T+, CD4-, CD8+). In
Primary Cutaneous T-Cell Lymphoma many cases, mycosis fungoides progresses through three clini-
Primary involvement of the skin by lymphoma is usually of the cal phases. In the premycotic (erythroderma) phase, lasting
T-cell type. B-cell lymphomas may occasionally secondarily from 6 months to 50 years, the T-cell infiltrate produces a
488 Chapter 21. The Lymphomas

nonspecific eczematous dermatosis that is difficult to differen-

tiate from a benign inflammatory infiltrate. As the disease pro-
gresses, the infiltrate thickens to form distinct plaques (plaque
stage, Fig. 21-28A and B) and finally tumor nodules (tumor
stage) (Fig. 21-29). Systemic dissemination with lymph node,
peripheral blood, and visceral organ involvement is more likely
to develop in the later stages. Transformation to a large-cell
lymphoma similar to anaplastic large-cell lymphoma may
occur and is most frequently seen as a terminal event. The prog-
nosis for mycosis fungoides confined to the skin is relatively
good, with median survival of greater than 10 years. Extracuta-
neous spread, however, is associated with a median survival of
less than | year.
In the related Sézary syndrome, the early erythroderma
stage is associated with leukemia of the characteristic cerebri-
Figure 21-29 ™ Myosis fungoides, tumor stage. Tumor nodules
form T cells (Sézary cells) (Fig. 21-30). Progression to the
are produced by massive local infiltrates of the skin by the charac-
tumor stage is unusual in Sézary syndrome. teristic cerebriform cells of mycosis fungoides.
Primary cutaneous anaplastic large-cell lymphoma
(Fig. 21-31A and B) and lymphomatoid papulosis are morphologically similar disorders are both cytologically
related, cutaneous T-cell proliferations that are extremely malignant; however, lymphomatoid papulosis generally
difficult to differentiate by histologic features alone.** These pursues a clinically benign course whereas cutaneous
anaplastic large-cell lymphoma, by definition, behaves in a
malignant fashion. With the passage of time, 10% to 20% of
patients with lymphomatoid papulosis develop overt lym-
phoma with dissemination beyond the skin. It is, therefore,
probable that these two disorders represent a continuum.
In addition to their morphological similarity, they are
immunophenotypically indistinguishable, and both appear
to be clonal proliferations of T cells.
Cutaneous anaplastic large-cell lymphoma is probably a
different disease than systemic anaplastic large-cell lym-
phoma, discussed earlier. The atypical cells of the cutaneous
disease, although CD30+, similar to the systemic disorder,
are epithelial membrane antigen (EMA)-negative, and the
t(2;5) chromosomal translocation associated with the sys-
temic disease is absent in the cutaneous disease (see Table
21-8). Furthermore, cutaneous anaplastic large-cell lym-
phoma is an indolent, incurable disorder compared with the
more aggressive but potentially curable systemic lymphoma.

ce
Figure 21-28 @ A. Myosis fungoides. Plaque lesion of MF showing rs se, Me. eeu. : a
band-like infiltrate (arrows) of dermis by atypical lymphocytes that
extent into the overlying epidermis. B. Pautrier microabscess. Figure 21-30 @ Sézary cells in the peripheral blood from a patient
Cluster of atypical lymphocytes within the epidermis. with Sé€zary syndrome.
 Chapter 21 TheLymphomas 489

active blasts. Precursor T-lymphoblastic lymphoma is a high-

risk disease requiring aggressive therapy.

HISTIOCYTIC AND DENDRITIC CELL TUMORS

The vast majority of lymphomas are derived from the vari-
ous types of lymphocytes. Rare cases present clinically as
lymphomas, and the malignant cells are morphologically
indistinguishable from the lymphocytic lymphomas; how-
ever, at the immunologic and genetic level they are nonlym-
phoid. These rare cases most often exhibit features of antigen
presenting accessory cells (dendritic cells) or phagocytic
cells (histiocytes).°!

HISTIOCYTIC TUMORS Histiocytic sarcoma is a rare neo-

plasm that is histologically indistinguishable from DLBCL or
anaplastic large-cell lymphoma. The disease presents with
nodal and/or extranodal involvement, most commonly skin or
gastrointestinal tract. By immunohistochemistry the tumor
cells express a “histiocytic” phenotype. Unfortunately there
are no specific histiocytic markers and the diagnosis is one of
exclusion. The presence of histiocyte associated markers such
as CD68, lysozyme, CDI1c and CD14 associated with the
absence of specific myeloid and lymphoid markers
(myeloperoxidase, CD33, CD34, CD20, CD3) are required to
suggest this diagnosis. By definition there must be no clinical
rearrangement of immunoglobulin or T-cell receptor genes.
This disease is usually aggressive with poor response to ther-
apy and most patients die as a result of progressive disease.
Langerhan cell histiocytosis is covered in Chap. 23.

DENDRITIC CELL TUMORS Dendritic cell tumors are

neoplasms related to accessory cells, namely interdigitating
dendritic cells (IDC) and follicular dendritic cells (FDC).
Figure 21-51 @ A. Primary cutaneous anaplastic large cell
lymphoma. Large atypical cells with marked nuclear pleomorphism Interdigitating reticular cell sarcoma and follicular dendritic
infiltrate the debris. B. Detail view of cytological features of primary cell sarcoma are neoplasms composed of spindle to ovoid
cutaneous anaplastic large cell lymphoma. shaped cells that exhibit the phenotypic profiles of IDC
and FDC, respectively (Fig. 21-32). Both tumor types are
extremely rare and present a significant diagnostic challenge
Other T/NK Mature Lymphomas to the pathologist. Their clinical behavior is unpredictable,
The mature T/NK disorders that primarily present in extran- ranging from indolent localized disease to widespread lethal
odal sites or as disseminated/leukemic disease are distinct yet disease. FDC sarcoma usually behaves in an indolent fashion.
rare disorders. They are briefly described in Tables 21—15 and
21-16. The interested reader is directed to the specific refer- DIAGNOSTIC EVALUATION
ences for more detailed information.°°~’ Tissue biopsy is required for the diagnosis and subcategoriza-
tion of lymphoproliferative disorders. The mainstay of diagno-
PRECURSOR T-LYMPHOBLASTIC LYMPHOMA Precursor sis is the histologic evaluation of well-stained tissue sections
T-lymphoblastic lymphoma is biologically identical to T-acute by an experienced pathologist. Ancillary studies, however, are
lymphoblastic leukemia.” The majority of lymphoblastic lym- often required. These include immunophenotypic studies by
phomas are of the T-cell type (85% to 90%) and usually present immunohistochemistry and flow cytometry, cytogenetics by
as a mediastinal mass in an adolescent male. The cells are karyotypic and fluorescent in situ hybridization (FISH) analy-
morphologically similar to the lymphoblasts of precursor ses, Southern blotting, and nucleic acid amplification [poly-
B-lymphoblastic lymphoma. The lymphoblasts of precursor merase chain reaction (PCR)]. Careful handling of the biopsy
T-lymphoblastic lymphoma express TdT and various T-cell anti- tissue is required to ensure that adequate material is submitted
gens. The expression of CD3 is considered to be lineage and appropriately triaged for diagnostic evaluation. Guidelines
specific. Non-T-cell antigens may be expressed, such as CD79a, for the proper handling and processing of lymphoid
CD13, and CD33. The T-cell receptor (TCR) is clonally tissue are widely published.” The diagnostic difficulties fall
rearranged. Involved lymph nodes usually demonstrate com- into a relatively small number of categories; namely, differen-
plete effacement by a monotonous population of mitotically tiating (1) benign versus malignant lymphoproliferations,
490 Chapter 21. The Lymphomas

Table 21-15 T/NK with Primary Extranodal Presentation

Lymphoma Characteristics

Extranodal T/NK lymphoma, nasal type*? Angiocentric lymphoma that predominantly involves the nasal cavity,
nasopharynx, palate, skin, GI tract and testis of Asians and related racial
groups; usually NK phenotype (CD3—, CD2+, CD56+) but may be T-cell
(CD3+, CD56—). Both types are EBV positive. TCR is usually non-clonal
(CD3— type) but clonal episomal EBV; variable clinical behavior but
usually aggressive if outside nasal cavity.
Enteropathy-type™! T-cell lymphoma most commonly involving the jejuneum and ileum;
associate with celiac disease; variable morphology but usually large cells
admixed with inflammatory cells that ulcerate the mucosa and invade
bowel wall; adjacent mucosa shows enteropathy changes; phenotype
CD3+, CD5—, CD7+, CD8—/+, CD4—, CD103+; genotype clonally
rearranged TCR; prognosis is usually poor.
Hepatosplenic» Aggressive lymphoma showing infiltration of sinusoids of spleen, liver
and bone marrow by medium sized cytotoxic T-cells of the y6 type (rare
aB type). Median survival is less than 2 years. Most commonly affects
adolescent males.
Subcutaneous panniculitis-like** Cytotoxic T-cell lymphoma that preferentially infiltrates subcutaneous tissue;
usually CD3+, CD8+T,,¢ phenotype; clonally rearranged TCR; commonly
associated with hemophagocytic syndrome; aggressive disease but may
respond to therapy.
Blasic NK* Aggressive lymphoma of blastic cells with NK phenotype (CD3—, CD56+)
that has a predilection for skin; TCR is germline; poor prognosis
Mycosis fungoides* Epidermotropic T-cell lymphoma of skin slowly progressing from patches
to plaques to tumor nodules (MF); small to medium sized cerebriform
cells infiltrate the dermis and epidermis; phenotype CD3+, CD4+, CD8—
mature T-cell with clonally rearranged TCR (Figs. 21-28 and 29A and B).
Adult disease with excellent prognosis for limited disease; worse
prognosis for tumor stage/extracutaneous spread
Sézary syndrome* Generalized T-cell lymphoma with involvement of skin, lymph node and
blood; skin lesions identical to MF; phenotype similar to MF; adult
disease with aggressive behavior (10-20% 5-year survival).
Primary cutaneous ALCL” T-cell lymphoma composed of CD30+ large pleomorphic T cells confined
to skin at diagnosis (Fig. 21-30); usually solitary or localized disease
which may spontaneously regress; excellent prognosis unless
extracutaneous disease develops. A related clinical disorder is
lymphomatoid papulosis.

Lymphoma

T-cell prolymphocytic leukemia*® d,

bone marrow, lymph nodes, spleen, liver and skin; phenotype TdT-,
CD3+, usually CD4+; inv(14) in 80%; less than 1 year median survival
T-cell large granular lymphocytic leukemia*’ Defined by chronic proliferation of large granular lymphocytes with mature
T-cell phenotype (CD3+, CD4—, CD8+) and clonally rearranged TCR;
associated with neutropenia; usually an indolent disorder
Aggressive NK leukemia Aggressive disorder of large granular lymphocytes with NK phenotype
usually affecting young Asian adults (must be differentiated from
morphologically similar but indolent NK disorder). Usually fulminant
disease with short survival following diagnosis.
Adult T-cell leukemia/lymphoma® Endemic disease of Japan, Carribean, and Central Africa caused by human
T-cell leukemia virus type (HTLV-1). Usually systemic disease with
rapidly fatal course.
 Chapter 21 TheLymphomas 491

200

Negative <—+
160
&
Al
Gy
“e EE
120
pres

80 mm
SR
acer

40

10° 10! 102 103 104

A Fluorescence intensity

104
Figure 21-52 ™ Dendritic cell sarcoma. An atypical spindle cell
proliferation characterizes this tumor which expresses a dendritic
cell phenotype.
103

(2) lymphoma versus nonlymphoid malignancy, (3) T-cell ver-

©
sus B-cell lymphoma, (4) HL versus NHL, and (5) subtyping S 402
a We
of HL and NHL. One or more of the ancillary tests may be <
required to resolve the differential diagnostic dilemma, and
rarely, the dilemma may be irresolvable. 10!

BENIGN VERSUS MALIGNANT If light microscopic evalua-

tion fails to distinguish a benign from a malignant lympho- 10°
proliferation, the demonstration of immunophenotypic or 10% 110") Ap 1107 penn, 10° snet04
genotypic monoclonality would favor a malignant diagnosis. B Lambda
For B cells, immunophenotypic monoclonality is defined as
Figure 21-33 @ B-cell monoclonality detected by flow cytometry
restriction of immunoglobulin light-chain production by a A. Overlay of two single-color (one-parameter) histograms demon-
population of cells to a single light-chain class, either k or X. strating a monoclonal lambda population. B. Two-color (dual-
Operationally, light-chain monoclonality is present if the parameter) histogram with simultaneous analysis for « (y-axis)
percentage of k-positive cells to \-positive cells (K-to- and \ (x-axis) light chains. The dense cluster of events in the lower
right quadrant indicates a monoclonal \ population.
d ratio) falls outside of the expected (“normal”) range or if
“clonal excess” can be demonstrated by statistical comparison
(Kolmogoroy—Smirnoy test) of the k and 2) fluorescence
intensity distributions (see Chap. 34). An example of reaction (PCR) (see Chap. 35). Figure 21-34 illustrates the
immunophenotypic monoclonality demonstrated by two- results of Southern blot analysis in which a clonal population
color flow cytometry is illustrated in Figure 21—33. of T cells is indicated by the presence of nongermline bands
Unfortunately, at the present time, no practical method in the Southern blot analysis. PCR analysis has largely
exists to define immunophenotypic T-cell clonality. Evidence replaced the more laborious and time-consuming Southern
for T-cell malignancy, however, may be suggested by blotting technique in the evaluation of genotypic clonality.
immunophenotypically demonstrating an aberrant T-cell phe- The identification of a clonal rearrangement is presumptive
notype. Benign T-cell proliferations generally express a evidence of a malignant proliferation. Although biphenotypic
“normal” T-cell phenotype in which all pan—T-cell antigens rearrangements (both T-cell receptor and immunoglobulin
(CD2, CD3, CD5, CD7) are expressed by the individual T cells. genes rearranged) may rarely occur, the specific type of
On the other hand, T-cell malignancies that might be confused rearrangement is generally useful in assigning lineage to the
with a benign T-cell proliferation often express an aberrant lymphoma; that is, a B-cell lymphoma if immunoglobulin
T-cell phenotype in which one or more of the pan—T-cell anti- gene rearrangement is found, and a T-cell lymphoma if T-cell
gens are not expressed. Approximately 60% of peripheral T-cell receptor gene rearrangement is found.
lymphomas will express such an aberrant phenotype, with CDS Although karyotypic analysis in a search for nonrandom
and CD7 being the most frequently absent antigens. chromosome abnormalities might be useful in distinguishing
A genotypic definition of both B- and T-cell clonality benign from malignant lymphoproliferations or subcategoriz-
is possible. Clonal rearrangements of the T-cell receptor or ing the NHLs, the technical difficulties in performing this type
immunoglobulin genes are detectable by several methods, of analysis have made cytogenetic studies an uncommon ancil-
including Southern blot analysis and the polymerase chain lary test in lymphoproliferation evaluation. The development of
492 Chapter 21. The Lymphomas

LYMPHOMA VERSUS NONLYMPHOMA The differential

1 2 3
diagnoses of large-cell lymphoma and nonlymphoid malig-
M Cc Pp ec ae a ae nancies, such as amelanotic melanoma, poorly differentiated
carcinoma, germ cell neoplasms, and round cell sarcomas, is
a frequently occurring problem in surgical pathology. Differ-
ential diagnosis is facilitated by determining the antigenic
phenotype of the tumor cells. This is most commonly done by
staining of the cells with an immunoenzyme technique using
a panel of antibodies directed against normal cellular differ-
entiation antigens.®? Although no single antibody is either
100% specific or sensitive for identifying the “cell of origin”
of a tumor, with the aid of a panel of antibodies it is generally
possible to define a phenotypic profile that permits identifica-
tion of the type of tumor. From a therapeutic perspective it is
most important to resolve the differential—carcinoma versus
lymphoma versus sarcoma versus melanoma—which can
usually be accomplished by a relatively small antibody panel
(Table 21-17). Additional refinement of the diagnosis may be
possible with larger panels.

T-CELL VERSUS B-CELL LYMPHOMA Although morpho-

logical features may«suggest a B-cell or T-cell phenotype,
ancillary tests are required for specific distinction between
B-cell and T-cell lymphoma. The immunophenotypic demon-
stration of light-chain monoclonality and the expression of
Figure 21-354 @ T-cell monoclonality demonstrated by Southern
blot analysis: Abnormal bands designated by small bars indicate B-lineage antigens (CD19, CD20, CD22), or of an aberrant
clonal rearrangement of the T-cell receptor gene. M = molecular B-cell phenotype by the cells of a malignant lymphoma, are
weight markers; C = control DNA to locate position of germ-ling indicative of a B-cell lymphoma. Similarly, the expression of
(nonrearranged) bands; P = patient DNA; 1 = BamHI digest; a normal or abnormal T-cell phenotype by the malignant cells
2 = EcoRI digest; 3 = Hindlll digest.
is associated with T-cell malignancy. Gene rearrangement
studies appear to be more sensitive than immunophenotypic
DNA probes for specific chromosomal alterations may reduce studies in distinguishing T-cell versus B-cell lymphoma; how-
the need of performing karyograms in the cytogenetic evalua- ever, relatively few lymphomas require this technically more
tion of lymphoproliferations. Fluorescent in situ hybridization demanding procedure to make this distinction.
(FISH) has emerged as a powerful technique for demonstrating
diagnostically important chromosomal translocation without HODGKIN LYMPHOMA (HL) VERSUS NON-HODGKIN
the need for complete karyotype analysis (Fig. 21-35). LYMPHOMA (NHL) On morphological grounds alone, it is
sometimes difficult, if not impossible, to distinguish HL from
NHL. For this reason, as well as important therapeutic impli-
cations, there has been considerable interest in the utility of
ancillary techniques to resolve this differential diagnosis. The
literature is replete with contradictory studies concerning the
significance of various tests in distinguishing HL from NHL.
This state of affairs reflects defects in our understanding of
the basic biologic differences between these disorders as well
as the nonspecificity of the tests. At present the only specific
immunophenotypic distinction between these disorders is the
presence of light-chain monoclonality in B-cell NHLs and
its absence in HL. Clonal rearrangement of the immunoglob-
ulin heavy-chain or T-cell receptor genes has been described
in some cases of HL, and therefore limits the utility of
such tests in the differential diagnosis of Hodgkin lymphoma
from NHLs.“
The differential diagnosis of HL includes certain NHLs
Figure 21-35 @ Fluorescent in situ hybridization (FISH) analysis of
(PTCL, ALCL, TCRLBCL) and various other benign and
B-cell lymphoma using two color break-apart probe. Arrowed cells malignant conditions. Careful attention to morphological fea-
show 1 normal fusion signal and 2 abnormal break-apart signals tures in association with immunohistochemical analysis can
indicating breakage within the IgH (heavy chain) gene switch region. usually resolve the diagnosis. CHL is mostly easily confused
 Chapter 21 TheLymphomas 493

Antibo*

Cancer Type Cytokeratin* Vimentin** LCA (CD45)!

Carcinoma +
Sarcoma ~
Lymphoma +

Melanoma

*Antibody directed again st the cytokeratin group of intermediate filament proteins found in epithelial cells.
*“Antibody directed against the 58-kD intermediate filament protein found in mesenchymal cells.
‘Antibody directed against the 200-kD leukocyte common antigen found in various hematopoietic and lymphoreticular cells.
“Antibody directed against the 25-kD protein found in selected cells of the nervous system, melanocytes, interdigitating reticulum cells,
etc. The antibodies HMB-45 and Melan A are more specifically restricted to melanocytes than anti-S100.

with PTCL and ALCL whereas NLPHL is most easily con- in a diffuse background composed predominantly of small
fused with TCRLBCL or LR-CHL. T cells. In addition, conventional PCR will often detect
The neoplastic cells of all subtypes of CHL are CD45 clonal immunoglobulin rearrangement in TCRLBCL but not
and ALK protein negative, CD30 positive, and usually CD15 in NLPHL.
positive (Fig. 21-36). They are most often negative for pan-B
and T cell antigens (see Table 21-3). PTCL and ALCL are
Staging
usually CD45 positive and express one or more pan-T-cell
antigens (CD2, CD3, CD5, CD7). Systemic ALCL also The staging evaluation of the NHLs is similar to that for HL.
expresses ALK protein (Fig. 21-37). The same staging scheme, the Cotswold modification of the
The immunophenotype of the neoplastic cells of NUPHL Ann Arbor classification (see Table 21-5), which was
and TCRLBL are virtually identical. However there are dif- designed for HL, is also used for the staging of NHLs. Because
ferences in the immunoarchitecture of the involved tissue that the natural history of HL is different from that of NHL, there
may be helpful in distinguishing these two entities. The L&H are certain problems with using the Ann Arbor classification
cells of NLPHL are generally found in a vaguely nodular in staging the NHLs. Despite these drawbacks, the Ann Arbor
background composed of CD20+ small lymphocytes. The scheme remains the staging scheme of choice. One major
individual CD20+ L&H cells are often surrounded by a exception to this statement is Burkitt’s lymphoma. In this
rosette of CD3 positive small T cells (Fig. 21-38). The lymphoma the bulk of tumor, rather than sites of involvement,
CD20+ large neoplastic cells of TCRLBCL are usually found is a primary determinant of prognosis. Consequently, a

Figure 21-36 ™ Expression of CD30 antigen by Reed-Sternberg

cells and variants in CHL (red-brown reaction product). Three Figure 21-37 @ Expression of ALK-NPM protein in cytoplasm of
patterns of staining are present—cytoplasmic membrane, diffuse neoplastic cells of ALCL (red-brown reaction product). Immunoper-
cytoplasmic, and juxtanuclear dot (“Golgi”). Immunoperoxidase oxidase staining using monoclonal antibody to ALK-NPM protein
staining using monoclonal antibody to CD30 antigen. (ALK-1).
494 Chapter 21 The Lymphomas

chemotherapy. Asymptomatic patients with advanced stage

indolent lymphomas may be managed with a “watch and wait”
approach. When symptoms warrant, or the lymphoma pro-
gresses to a higher grade, therapy is instituted. When treatment
is required various options are available that may improve
failure-free survival; however, overall survival does not appear
to be improved. Recent additions to the treatment options
include monoclonal antibody (anti-CD20) therapy and purine
nucleoside analogs (Fludarabine, 2-chlorodeoxyadenosine).
Before the development of modern chemotherapeutic
modalities, the indolent lymphomas were considered
“favorable” lymphomas and the diffuse aggressive lym-
phomas were considered the “unfavorable” category. The
situation is now reversed. The indolent lymphomas are rec-
ognized as clinically indolent but incurable with current
therapies. On the other hand, the diffuse aggressive lym-
phomas are rapidly fatal if untreated but are potentially cur-
able with aggressive tréatment strategies.°’' The 10-year
Figure 21-38 m Rosette of CD3+ T cells (red-brown reaction survival for the diffuse aggressive lymphomas treated
product) surrounding L&H (popcorn) cell of NLPHD. Immunoper- aggressively with multiagent chemotherapy is now greater
oxidase stain using monoclonal antibody to CD3 antigen. than that for the indolent lymphomas. Long-term survival
depends on achieving an initial complete remission. For
- localized diffuse aggressive lymphomas standard therapy is
different staging scheme is in use for Burkitt’s lymphoma multiagent chemotherapy (CHOP) with involved field radi-
(Table 21-18). ation therapy. Multiagent chemotherapy with or without
radiation is standard therapy for advanced stage disease.
Utilizing intensive multiagent chemotherapy, complete
Treatment and Prognosis
remission rates of 60% to 85% have been achieved in the
For the purposes of prognostic assessment and therapeutic advanced stage, diffuse aggressive lymphoma category.
selection, the NHLs can be grouped into two broad cate- Relapse-free survival at 2 years is between 40% and 70%.
gories: the indolent lymphomas (see Table 21—12) and the dif- Therapy following relapse is usually ineffective. Patients
fuse aggressive lymphomas. The indolent lymphomas are so who survive for 2 years without evidence of recurrence are
designated because the median survival without therapy for considered “cured,” because relapses seldom occur after a
this group of lymphomas is relatively long (7 to 9 years). The 2-year disease-free interval. As survival is increased with
diffuse aggressive lymphomas encompass the intermediate- aggressive therapy, the risk of secondary (chemotherapy-
and high-grade lymphomas of the Working Formulation. induced) acute noniymphocytic leukemia is also increased.
Without treatment, these lymphomas are rapidly fatal (median Approximately 8% of patients develop acute nonlympho-
survival is 6 to 12 months). cytic leukemia within 10 years of chemotherapy for
Therapy for the indolent lymphomas is controversial.°* NHL, and these leukemias are usually resistant to therapy.
As a group, relatively few present with localized disease (stages The therapeutic response of lymphoblastic lymphoma to
I and II). Strictly localized indolent lymphoma (stage I/TE) can aggressive chemotherapy has not been as impressive as with
be effectively treated with radiotherapy, resulting in durable the other aggressive lymphomas. Adults older than 45 years
complete remission. Approximately 90% of these lymphomas tend to have longer survival than younger patients; however,
are stage III or IV at presentation, and although they are median survival is still short for T-cell lymphoblastic lym-
sensitive to both radiation and chemotherapy, the disease is phoma.” The prognosis for B-cell lymphoblastic is relatively
essentially incurable with standard or aggressive multiagent more favorable.

Case Study |
Stage A Single extra-abdominal site
Stage B Multiple extra-abdominal sites A 68-year-old male auto mechanic presents to his primary
Stage C Intra-abdominal tumor care physician with a chief complaint of bilateral, painless
Stage D Intra-abdominal tumor with multiple masses in the neck region of at least 6 months’ duration. He
extra-abdominal sites has a 120 pack-year smoking history, and his family history
Stage AR Stage C disease with >90% of tumor includes a father and brother with thyroid cancer. His past
surgically removed
continued
 Chapter 21 TheLymphomas 495

medical history is positive for rheumatoid arthritis. On phys- ANSWERS

ical examination, the patient is found to have bilateral supr-
aclavicular and cervical lymphadenopathy consisting of | . The causes of lymphadenopathy are myriad and include
matted groups of lymph nodes that were painless to palpa- various benign and malignant etiologies. Benign disor-
tion. The patient was afebrile and had experienced an unin- ders producing localized or generalized adenopathy are
tentional decrease in weight from 195 pounds to 180 pounds most commonly infectious or inflammatory in nature
over a 6-month interval. (e.g., viral illness, cat scratch fever), but may be
secondary to autoimmune disorders (e.g., lupus, rheuma-
toid arthritis). Metastatic malignancies (carcinoma,
QUESTIONS
melanoma, or sarcoma) produce lymphadenopathy, but
1. What pathological processes must be considered in the on physical examination are usually more localized.
evaluation of lymphadenopathy? Lymphoma must always be considered in the differential
2. What are some of the important diagnostic tests that diagnosis of lymphadenopathy, regardless of the
should be performed? All parameters of the initial patient’s age.
complete blood count (CBC) were normal, and the . The formed elements (white cells, red cells, and platelets)
peripheral smear was without morphological abnormal- of the peripheral blood often exhibit changes that are
ity. A chest x-ray film demonstrated hilar adenopathy helpful in accessing the cause of lymphadenopathy;
without any recognizable parenchymal lung lesions. therefore, a CBC with peripheral smear evaluation is
Excisional biopsy of a group of nodes was performed. indicated. A chest x-ray study should be performed in a
On microscopic examination there was diffuse efface- search for intrathoracic disease, particularly lung
ment of lymph node architecture by a population of rela- lesions. The definitive test to distinguish benign from
tively uniform cells, averaging 20 to 40 um in diameter, malignant processes 1s microscopic examination of an
with round to slightly irregular nuclei and occasional enlarged lymph node. This may be achieved by fine-
prominent nucleoli. Immunophenotypically, the cells needle aspiration or excisional biopsy. Excisional biopsy
were characterized by the expression of CD45, CD19, is preferred if lymphoma is high on the differential diag-
CD20, and k light chains. No T-cell antigens were nostic list.
detected on the abnormal cells. . Malignant lymphoma, diffuse large B-cell (WHO classi-
3. What is the most likely diagnosis based on the available fication).
data? . For staging purposes, bilateral bone marrow biopsies
4. What additional studies should be performed before and CT scanning of the abdomen and pelvis should be
therapy is instituted? Bilateral bone marrow biopsy performed.
specimens demonstrate several aggregates of abnormal Ds Stage IV.
cells, similar to those observed in the lymph nodes, 6. Multiagent chemotherapy.
randomly distributed within the marrow. A computed Te The probability of 2-year relapse-free survival is 40% to
tomographic (CT) scan of the abdomen and pelvis 70% for advanced, aggressive-grade non-Hodgkin
reveals para-aortic adenopathy and modest enlargeinent lymphomas. If relapse does not occur within 2 years
of the spleen. after achieving complete remission, the patient is con-
5. What stage is this lymphoma? sidered cured. Therefore, the probability of cure in this
O . What therapy would be appropriate? case is 40% to 70%.
7. What is the probability of “cure”?
continued

a cetions
1. What infectious agent is most commonly associated with 3. An asymptomatic mediastinal mass affecting a young
the pathogenesis of Hodgkin’s lymphoma? woman is a common form of presentation for which type
a. Echovirus of Hodgkin’s lymphoma?
b. Herpesvirus a. Nodular sclerosing
c. Helicobacter pylori b. Lymphocyte-predominant
d. Epstein—Barr virus c. Mixed cellularity
d. Lymphocyte depletion
. The cell characteristic of all types of classic Hodgkin’s
i)

lymphoma is the: 4. All follicular center lymphomas are composed of a mixture of:
a. Lacunar cell a. Small cleaved and small noncleaved cells
b. L&H cell b. Centrocytes (smail cleaved) and centroblasts (large
c. Reed—Sternberg cell noncleaved) cells
d. Sézary cell c. Large cleaved cells and immunoblasts
d. Prolymphocytes and paraimmunoblasts
496 Chapter 21 The Lymphomas

5. Small lymphocytic lymphoma is characterized by all of eh A lymphoma associated with infection by a type C retro-
the following except: virus 1S:
a. Indolent but incurable a. Endemic to Japan
b. Usually CD19+, CD5+, CD23+, CD10- b. Associated with hypercalcemia
c. Growth centers c. Usually rapidly fatal
d. t(14:18) d. All of the above
6. Translocations involving the BCL/ and /gH genes are 14. The immunophenotypic definition of B-cell monoclonality is:
commonly associated with which low-grade B-cell a. Clonal rearrangement of the IgH gene
lymphoma? b. Loss of B-cell antigens by the malignant cells
a. Small lymphocytic c. Expression of a single light-chain class
b. Lymphoplasmacytic d. Expression of a single heavy-chain class
c. Mantle cell . Alternatives to karyotypic analysis for the demonstration
d. Marginal zone of chromosomal translocations include:
7. Lymphomatous polyposis is a type of a. Southern blot analysis
a. Small lymphocytic lymphoma b. PCR
b. Large-cell lymphoma c. FISH
c. Mantle cell lymphoma d. All of the above
d. MALT lymphoma 16. Reed-Sternberg cells usually express the following
8. MALT lymphomas are characterized by all of the immunophenotype:
following except: a. CD45+, CD30+, CD15+
a. Extranodal involvement b. CD45-, CD30+, CD15+
b. Associated follicular hyperplasia c. CD45+, CD30+, CD15-
c. CD19+, CD5+, CD10+ phenotype d. CD45-—, CD30-, CD15+
d. t(11;18) Te All of the following are true regarding the low-grade
9. A lymphoma expressing a monoclonal B-cell phenotype non-Hodgkin’s lymphomas except:
with coexpression of CD10 and rearrangement of the a. Survival without treatment averages more than 5 years
BCL2 gene is most likely derived from: b. Disease is usually advanced at diagnosis
a. Follicular center cells c. Multiagent chemotherapy can prolong survival
b. Mantle cells d. Cure is possible in more than 50% of cases
c. Marginal zone cells 18. The diffuse aggressive lymphomas are:
d. Interfollicular cells a. Seldom curable
10. Burkitt’s lymphoma is histologically characterized by b. Rapidly fatal if untreated
which of the following? c. Best treated with a combination of surgery and
a. Uniform nuclei radiotherapy
b. High mitotic rate d. Single-agent chemotherapy is the treatment of choice
c. “Starry sky” pattern
. The staging system for Hodgkin’s lymphoma and the
d. All of the above
non-Hodgkin’s lymphomas is:
11. Lymphoblastic lymphomas are associated with all the a. Working Formulation
following except: b. Duke’s staging system
a. High frequency of developing acute lymphoblastic c. Rye staging system
leukemia d. Ann Arbor staging system
b. Mediastinal mass
20. A patient with Hodgkin’s lymphoma that is localized to
c. Usually express B-cell phenotype
lymph nodes above and below the diaphragm and is asso-
d. Potentially curable in young patients
ciated with drenching night sweats would be classified as:
12. All of the following are T-cell disorders except: a. Stage INE
a. Mycosis fungoides b. Stage ITB
b. Sézary syndrome c. Stage IIIB
c. Hepatosplenic gamma/delta lymphoma d. Stage INA
d. Burkitt's lymphoma
awe 7 eres at the back of this book. —
case
. =
 Chapter 21 Thelymphomas 497

g The usual histological progression in Hodgkin’s disease is

lymphocyte-rich to mixed cellularity to lymphocyte
depletion lymphoma.
m The malignant lymphomas are a heterogeneous group of
diseases that arise from cells of the lymphoid tissue (lym- m@Most patients with Hodgkin’s lymphoma present with
nonpainful lymph node swelling.
phocytes, histiocytes, reticulum cells).
mwThe most widely used staging scheme in Hodgkin’s
m The Reed—Sternberg cell is the hallmark of Hodgkin’s dis-
disease is the Ann Arbor classification.
ease; the morphological features of the Reed—Sternberg cell
include large size (up to 45 tm in diameter), multinucleated, @ The World Health Organization (WHO) utilizes clinical,
and inclusion-like nucleoli. The typical immunophenotype morphological, immunophenotypic, and genotypic fea-
of Reed—Sternberg cells are CD30+, CD15+, and CD45-. tures to classify the lymphomas.
a The probable infectious agent of Hodgkin’s lymphoma is @ The follicular center lymphomas (non-Hodgkin’s lym-
Epstein—Barr virus. phoma) are composed of various mixtures of centrocytes
(small cleaved) and centroblasts (large noncleaved) cells
= Lymphocyte-predominant Hodgkin’s lymphoma consists
and represent the B-cell phenotype (CD19+, CD20+,
of a mixture of small, normal-appearing lymphocytes;
CDS5-—); t(14;18) is associated with this disorder.
benign histiocytes; rare, classic Reed—Sternberg cells; and
many variant Reed—Sternberg cells (L&H cells). m Mantle cell lymphoma is characterized by small-to
medium-sized lymphocytes with scant pale-staining cyto-
w The most common type of Hodgkin’s lymphoma is nodular
plasm, irregular nucleus, and inconspicuous nucleoli;
sclerosing Hodgkin’s lymphoma, which is characterized by
cells express surface immunoglobulin, CD5, and CD43;
birefringent collagenous sclerosis, classic Reed—Sternberg
t(11;14) is also found.
cells, and lacunar cells.
@ The MALT lymphomas are related to marginal zone cells
gw Mixed cellularity Hodgkin’s lymphoma is characterized by
and usually present in extranodal sites (stomach, salivary
a heterogeneous mixture of cells. including lymphocytes,
gland, lung, thyroid, and orbit); hence, the name mucosal-
histiocytes, plasma cells, eosinophils, Reed—Sternberg
associated lymphoid tissue (MALT).
cells, and Reed—Sternberg variants.
m The immunophenotype of Burkitt’s lymphoma is surface
mw Lymphocyte-rich Hodgkin’s lymphoma is characterized
IgM+, CD10+, CDS5-, CD23-, Bcl-2-, and TdT-.
by smal] numbers of Reed—Sternberg cells in a back-
ground of lymphocytes. @ Characteristics of the systemic form of anaplastic large-
cell lymphoma include overexpression of the ALK-NPM
a Lymphocyte depletion Hodgkin’s lymphoma is the rarest
protein, t(2;5), CD30+, and infiltration of the lymph node
form of Hodgkin’s disease. It is characterized by numer-
sinuses by large atypical malignant cells.
ous Reed-Sternberg cells and variants, and rare lympho-
cytes, plasma cells, histiocytes, and eosinophils; irregular
sclerosis is also present.

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—
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 Chapter / jj

Multiple Myeloma
and Related Plasma
Cell Disorders
Ashraf Z. Badros, MD

Overview OBJECTIVES
Plasma Cell Development At the end of this chapter, the learner should be able to:
Immunoglobulin
Le Describe the normal structure and function of immunoglobulin and the significance of
Structure and Function
the M-spike.
Laboratory Recognition and
Méasurements . Describe the workup for patients suspected of plasma cell dyscrasia.
Clinical Consequences of
. List the diagnostic criteria for multiple myeloma and its variants.
Increased Monoclonal
Immunoglobulin . Compare multiple myeloma and monoclonal gammopathy of undetermined
WwW
R

Monoclonal Gammopathy of significance.

Undetermined Significance . Describe other variants of monoclonal gammopathy such as Waldenstrém’s
Multiple Myeloma macroglobulinemia and list the characteristics of the disorders.
Epidemiology . List the laboratory tests used to diagnose plasma cell disorders.
Etiology
Pathophysiology . Define amyloidosis.
Clinical Features . Define heavy chain disease.
Diagnostic Workup
Laboratory Studies
Diagnostic Criteria
Staging
Treatment
Atypical Variants of Plasma
Cell Syndromes
Smoldering Myeloma
Solitary Plasmacytoma of
the Bone
Extramedullary
Plasmacytoma
Plasma Cell Leukemia
Nonsecretory Myeloma
POEMS Syndrome
Supportive Care in Myeloma
Patients
Waldenstrom’s
Macroglobulinemia
Amyloidosis
Light Chain Deposition and
Heavy Chain Diseases

500
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders SQ]

Case Study 1
Case Study 2
Case Study 3

Overview of the immunoglobulin heavy chain genes (IgH) leading to

pre-B cells. Subsequent rearrangement of the light chain
This chapter summarizes various stages of plasma cell develop- enables the cells to express surface immunoglobulin; conse-
ment and the difference between the normal and monoclonal quently immature B lymphocytes leave the bone marrow,
immunoglobulins, followed by a brief review of selected rele- migrate to the peripheral blood, and rest in the G, (dormancy)
vant monoclonal disorders. These include monoclonal gam- phase of the cell cycle. On exposure to antigens that occurs in
mopathy of undetermined significance (MGUS), multiple the lymph nodes, the cells differentiate into short-lived “low
myeloma, Waldenstrém’s macroglobulinemia, and amyloidosis affinity” plasma cells. A few become long-lived “memory
(Table 22-1). The discussion focuses on the pathophysiology B cells” and reside in the primary follicle of the lymph nodes.
of each disorder and the use of the clinical and radiological Once rechallenged with the same antigen, the primary folli-
procedures in the evaluation of each disease. The roles of cyto- cles are transformed into secondary follicles with germinal
genetics, gene array, and molecular biology are briefly intro- centers. The memory B cells differentiate into centroblasts
duced. The relevance of clinical data in staging various after immunoglobulin isotype switching and somatic muta-
diseases and in providing a prognostic model is briefly dis- tions in the variable region of the immunoglobulin generating
cussed in the context of the currently available therapy. Details “high-affinity” antibodies. These cells differentiate into plas-
of the treatment of various disorders are beyond the scope of mablasts, leaving the lymph node and entering the peripheral
this chapter. Several references and suggested readings are pro- blood, where the cells acquire several adhesion molecules
vided for additional information. such as: CD56, syndecan-1, collagen, ICAM-1, leukocyte
function-associated antigen 1, as well as vascular cellular
adhesion molecule or fibronectin. These terminally differenti-
Plasma Cell Development ated plasma cells “home” back to the bone marrow and
Plasma cells are terminally differentiated, nondividing cells adhere to the stromal cells. These bone marrow plasma cells
representing the final stage in the development of B lympho- produce most of serum immunoglobulin and have a life span
cytes.' These cells are responsible for the production of anti- of approximately | month (Fig. 22-1). ?
bodies called immunoglobulins that respond to microbial
pathogens with a high degree of specificity. Stem cell com- Immunoglobulin
mitment to B-cell lineage starts in the bone marrow and is
dependent on many transcription factors after rearrangements Structure and Function

The intact immunoglobulin is composed of two identical

heavy chains (50 kD) and two identical light chains (25 kD).
These chains are held together by varying numbers of disul-
fide bonds. The antibody produced by each plasma cell
has only one type of light chain and one type of heavy chain
(Fig. 22-2). There are five types of heavy chains: gamma (jy),
Malignant monoclonal gammopathies alpha (a), mu (w), delta (5), and epsilon (€). These heavy
Multiple myeloma (IgG, IgA, IgD, IgE, and free light chain) chains are designated as IgG, IgA, IgM, IgD, and IgE, respec-
Smoldering multiple myeloma tively. The heavy polypeptide chains are further subdivided;
Plasma cell leukemia
IgG has four subclasses: IgG1, IgG2, [gG3, and IgG4, and
Nonsecretory myeloma
Osteosclerotic myeloma (POEMS syndrome) IgA has two: IgA1 and IgA2. There are two light chain types
Plasmacytoma either kappa (k) or lambda (A); only one is found in any
Solitary plasmacytoma of bone/extramedullary plasmacytoma isotype.° IgM is a large molecule composed of five such units;
Monoclonal gammopathies of undetermined significance it does not cross the placenta. IgA and IgG aggregate in pairs,
Waldenstrém’s macroglobulinemia
or dimers. Each antibody has a specific role in the immune
Heavy-chain diseases
y-HCD, a-HCD, -HCD
system. IgM is the initial “transient” antibody produced in
Primary amyloidosis response to infection; it becomes unmeasurable within weeks
after it first appears. Later, IgG levels increase and are
502 Chapter 22. Multiple Myeloma and Related Plasma Cell Disorders

ari) Natural
c @ antibodies
wy NZL and IgA
B1 cell ae

“ae |
o
Marginal-
zone
ee
B cell _Y ake
Plasma @) >

ind
cell Se loM

Apoptotic |
cell
Germinal center

Proliferation
and mutation
—

Follicular
¥ B cell

Selection
and CSR

Week 1 Week 2 Week 3

Figure 22-1 ™ Development of plasma cells; on encounter with an antigen (indicated by week 1), naive marginal-zone B cells differentiate
into plasma cells. CSR = class-switch recombination.

Amino terminal Light chain

hypervariable Light chain
regions

Heavy chain
Antigen
binding ab Heavy chain
hypervariable
regions

Interchain Hinge region

disulfide bonds Complement binding region
Carbohydrate
Biological ;
activity F, Intrachain
mediation disulfide bonds

Carboxy terminal

Figure 22-2 ™ Structure of the basic immunoglobulin unit. V4, = variable region heavy chain; V, = variable region light chain;
C, = constant region heavy chain; C, = constant region light chain.
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 503

maintained in variable amounts after the antigenic stimulus is QUANTITATIVE IMMUNOGLOBULINS

removed. High levels can be readily available on re-exposure Immunoglobulin classes are quantitated by rate nephelome-
through memory cells. IgA is an antibody that provides pro- try, which is based on measuring the degree of turbidity by
tection of epithelial surfaces in the gastrointestinal tract and light scatter caused by antigen—antibody complexes by mix-
the airways. The IgE antibodies are involved in allergic/ ing a patient’s serum sample with an appropriate specific
hypersensitivity reactions. The role of IgD is unclear. antibody reagent. Estimation of quantitative immunoglobulin
Immunoglobulin overproduction is the hallmark of by nephelometry does not allow an assessment of monoclon-
plasma cell disorders. The overproduction of the same ality. Increased levels can be due to polyclonal or monoclonal
immunoglobulin is called monoclonal, and is usually associ- elevations; clonality needs to be established using elec-
ated with low levels of other uninvolved immunoglobulins. In trophoresis and immunofixation, as discussed in the preced-
addition, the normal equal production of light chains and ing text. Quantitation of immunoglobulin using nephelometry
heavy chains is unbalanced, usually with overproduction of is a useful adjunct to electrophoresis in following multiple
free light chains that are spilled in the urine as Bence-Jones myeloma patients. It is important to remember that when
proteins. assessing response, electrophoresis values are more reflective
of the actual change in the M-spike.°

Laboratory Recognition and Measurement FREE SERUM LIGHT CHAINS

In 20% of multiple myeloma patients, only free light chain
ELECTROPHORESIS
(FLC) is often produced in concentrations too low to be detected
Protein in the serum consists mostly of albumin, immunoglobu- by routine measures mentioned earlier.’ Normal serum values of
lin, and smaller amounts of other proteins such as «,-antitrypsin, BLC 1s kK G3 to, 19-4 mg/L), ’ (5.7 to 26.3 me/L); and
a,-macroglobulin, transferrin, and B-lipoprotein. Total serum k/X ratio (0.26 to 1.65).° Recently, a highly sensitive, automated
protein is measured on routine automated chemistry panels. immunoassay for measurement of FLC concentrations in serum
Measurement of albumin allows approximate quantitation of the has been used for identification and monitoring of patients with
immunoglobulin-containing fraction by subtraction (i.e., Total monoclonal gammopathy (Fig. 22-4). Serum FLC assays were
protein minus albumin = immunoglobulin fraction). Thus, one sensitive in detecting monoclonal FLC that were not detected by
can detect a substantialincrease in immunoglobulin by an auto- routine electrophoresis and immunofixation. These tests are
mated chemistry panel alone. It is important to differentiate used for diagnosis and monitoring of light-chain-only myeloma,
between polyclonal and monoclonal increase in immunoglobu- nonsecretory myeloma, and AL amyloidosis (amyloid with
lin levels that is seen in infections and plasma cell disorders, presence of light chain section).’? The widespread use of these
respectively. This is performed by protein electrophoresis.* tests have not been fully integrated in the staging system for
Electrophoresis separates the serum proteins into albumin, a, B, multiple myeloma nor in evaluating response or detection of
and y globulins (Fig. 22-3). The speed of different proteins relapse.
depends on their size and electrical charge. The spike on protein
electrophoresis caused by monoclonal immunoglobulin is called BENCE-JONES PROTEINURIA
a monoclonal spike or M-spike.” The primary reason for per- Imbalanced immunoglobulin production most frequently
forming a protein electrophoresis is to determine whether an yields an excess of free light chains (K/A).'° These light chains
M-spike is present or not. The same procedure is used for are rapidly metabolized in the blood; with increased produc-
analyzing a 24-hour urine collection. Further identification of tion of FLC being filtered into the urine as Bence-Jones pro-
the proteins causing the M-spike is carried out by a technique teinuria. The protein is named after the physician who first
called immunofixation, which is critical in the diagnostic evalu- noted their unique properties to precipitate at 40° to 60°C, dis-
ation of plasma cell disorders. solve at 100°C, and reprecipitate on recooling. Today, 24-hour
urine collection is necessary for determination of the total
IMMUNOFIXATION amount of protein excreted in the urine per day and the
Immunofixation is used when investigating an abnormal Bence-Jones proteins. FLC can be deposited in the kidneys
M-spike on serum protein electrophoresis. After separation of and can lead to leakage of protein into the urine (proteinuria)
the various proteins by electrophoresis into at least five sepa- and kidney failure.
rate lanes, mono-specific antibody, usually three for the heavy
chain component (y, «, 1) and two for the light chain compo-
nent (k and )) are added. Precipitation of proteins (i.e., the Clinical Consequences of Increased
antigen-antibody complex) is allowed to occur, followed by Monoclonal Immunoglobulin
washing (nonprecipitated proteins wash out) and staining of
the remaining immunoprecipitates. An M-protein is charac- HYPERVISCOSITY SYNDROME
terized on immunofixation by the combined presence of a The presence of excess immunoglobulin can lead to viscous
sharp, well-defined band associated with a single heavy-chain blood resulting in higher resistance to flow in the blood
class and a sharp and well-defined band with similar mobility vessels. The result is decreased blood flow in the small
characteristics which reacts with either kappa or lambda light vessels of various vital organs, thus increasing the workload
chain antisera (see Fig. 22-3). on the heart. Patients may become confused and may suffer
504 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

CELLULOSE ACETATE PATTERN DENSITOMETER TRACING

Normal serum

O2
IgG Myeloma with y spike “M” or monoclonal spike
and reduced albumin

Polyclonal
hypergammaglobulinemia

Oy, 2 B Y
Waldenstrom’s
macroglobulinemia
with IgM spike

ALB 01 02 B
Hypogammaglobulinemia

Figure 22-3 ™ Patterns of serum protein electrophoresis showing characteristic patterns of normal serum, monoclonal M-spike, polyclonal
antibody production, IgM M-spike, and the absence of antibody production seen in hypogammaglobulinemia.

from headache, dementia, disturbances of consciousness, to the abnormal immunoglobulin.'* The low immunoglobulin
stroke, and/or coma. Failure of the heart to compensate for levels lead to increased susceptibility to infections such as
widespread hypoxemia, increased intravascular volume, and sinusitis, bronchitis, and pneumonia, which is a main cause of
high pressure may lead to congestive heart failure. Although the death of multiple myeloma patients. Intravenous infusions of
correlation between serum viscosity and clinical manifestations immunoglobulin can help decrease the frequency of these
is not precise, clinical manifestations are rarely attributable to infections.'?
hyperviscosity if serum viscosity is less than 4 centipoises (CP),
CRYOGLOBULINS
(normal value =1.8 CP). Most patients are symptomatic when
Cryoglobulins are serum immunoglobulins that precipitate
serum viscosity is greater than 6 CP. Most cases of hyperviscos-
reversibly on exposure to cold. This is a rare complication
ity syndrome are seen with IgM, IgA, and IgG3 because of their
of plasma cell disorders. In cryoglobulinemic syndrome, a
molecular weight and physical properties. Plasma exchange
multitude of organs can be involved, but usually patients
(plasmapheresis) and removal of the immunoglobulin is an
suffer from painful extremities on exposure to cold, palpable
emergency therapy for symptomatic patients."
purpura, arthralgia, and fatigue. In severe cases it may result
in vasculitis and kidney damage.'* Cryoglobulins are classi-
DECREASED PRODUCTION OF NORMAL
fied into:
IMMUNOGLOBULINS
Patients with the plasma cell disorders of multiple myeloma Type I: The presence of isolated monoclonal
and Waldenstr6m’s macroglobulinemia have decreased levels immunoglobulin (IgG or IgM). This is common in
of the normal “uninvolved” immunoglobulin. The low levels Waldenstr6m’s macroglobulinemia and/or multiple
result from suppression of normal plasma cells as a response myeloma.
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 505

+ Normal (n = 282)
® x light-chain multiple myeloma (n = 120)
65
O Renal impairment from non-light-chain diseases (n = 31)
50
4 i light-chain multiple myeloma (n = 104)
© Nonsecretory myeloma (n = 28) Relative risk of
full progression 25
100,000 at 20 years

=) 13.6
Ss
= 10,000
2 3.6
=
i 0.5 1.0 Wes 2.0 2.5 3.0
—
7 Serum M-spike value
ee FOOO
>
@ Figure 22-5 @ Actuarial risk of full progression by serum mono-
2 clonal protein (M-protein) value at diagnosis of monoclonal
S 100 gammopathy of undetermined significance (MGUS) in persons from
i=
2 southeastern Minnesota.
s
. 10
)
(es marrow (BM) aspirate, and no end-organ damage or other
e)
o
en
o
findings of multiple myeloma such as lytic bone disease.
After completing the initial screening tests for the presence of
n
D
monoclonal protein, computed tomographic (CT) scans of the
fo} chest and abdomen, with biopsies of suspicious enlarged
ot
0.1
lymph nodes, may also be required. Reassurance for the
aN \ SS Ors patient and full explanation of the condition is crucial.
x SS Patients will have to understand that no immediate therapy is
needed. However, close monitoring initially every 3 months,
Log serum concentration of free-light-chain « (mg/L)
and if the M-spike remains stable, every 6-month intervals is
Figure 22-4 ™ Concentrations of « and } free-light-chains in sera needed for life.'’
from healthy individuals and those with myeloma. (From Bradwell,
AR: Serum test for assessment of patients with Bence-Jones myeloma.
Lancet 361:489, 2003. Reprinted with permission from Elsevier.) Multiple Myeloma
Multiple myeloma is the most common plasma cell dyscrasia,
Type II: A mixture of polyclonal immunoglobulin in affecting terminally differentiated B cells. The biology of
association with a monoclonal immunoglobulin, multiple myeloma suggests a multistep process as illustrated
typically IgM or IgA, with rheumatoid factor by the clinical progression from MGUS, to a symptomatic
activity. Type II is seen in patients with persistent multiple myeloma (Fig. 22-6). A B-cell precursor, after
viral infections, particularly hepatitis C and human immunoglobulin gene rearrangement is presumed to be the
immunodeficiency virus infections. origin of the malignant clone in multiple myeloma. The
events that determine the susceptibility of B cells to undergo
Type III: Mixed cryoglobulins consisting of polyclonal
such malignant transformation are at best speculative. Plasma
immunoglobulin that is secondary to connective
cells are generally localized in the bone marrow until late in the
tissue diseases."°
disease when the cells grow independent of the bone marrow
microenvironment, usually causing a more aggressive presen-
Monoclonal Gammopathy tation of the disease. Multiple myeloma is currently an incur-
able hematological malignancy; although complete responses
of Undetermined Significance
can be obtained with various therapies as outlined below.
Although monoclonal gammopathy of undetermined signifi- Patients’ survival varies widely— ranging from a few months
cance (MGUS) was previously named benign monoclonal to more than 15 years. The disease is characterized by the
gammopathy, 20% of these patients will develop overt malig- triad of an abnormal proliferation of plasma cells in the bone
nancy such as multiple myeloma, non-Hodgkin’s lymphoma marrow, over production of monoclonal immunoglobulin, and
(NHL), and chronic lymphocytic leukemia (CLL).'° The lytic/destructive bone disease. This clinical triad causes vari-
incidence of MGUS varies by age, sex, and race. MGUS is ous disease manifestations which include: life threatening
diagnosed in approximately 1% of individuals older than end-organ damage with renal insufficiency, anemia, immuno-
the age of 50 and up to 3% of those older than the age of 70 suppression and infections. Probably the most devastating
(Fig. 22-5). MGUS is defined by the finding of a small feature of myeloma is the osteolytic lesions, resulting in bone
M-spike in patients with normal levels of uninvolved pain, pathological fractures, spinal cord compression, and
immunoglobulin, fewer than 10% plasma cells on bone hypercalcemia.'*
506 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

8 (KSHV/HHV8) and hepatitis C have been considered etio-

logic pathogens in multiple myeloma. Biologically, it is plau-
pe
MGUS ——————> _SMM ———— MM
sible that the antigen-driven response to the viral infection and
the associated increased levels of cytokines (e.g., interleukin-6)
<3 g M spike 23 g M spike 210% PC and many angiogenic factors (e.g., vascular endothelial growth
<10% PC 210% PC + M spike initiate and
factor and basic fibroblastic growth factor) can
Plus sustain the malignant clone in multiple myeloma.
Calcium Nuclear atomic and industrial high-grade radiation expo-
Renal effect
sures were associated with the risk of leukemia and possibly
Anemia
Bone lesions non-Hodgkin’s lymphoma but not multiple myeloma. Pro-
longed low-grade exposure, as seen in factory workers and in
those living near nuclear facilities, seems to increase the risk
Figure 22-6 @™ Progression from monoclonal gammopathy of of multiple myeloma. Other occupations with a high risk of
undetermined significance to smoldering myeloma to malignant include workers in metals, rubber, wood,
multiple myeloma
myeloma.
leather, paint, and the petroleum industries; although the
strongest association for multiple myeloma is agricultural
work.” Industrial exposure to benzene is not a risk for devel-
Epidemiology oping multiple myeloma.”* It is worth noting that there are
clusters of various monoclonal disorders (such as multiple
Multiple myeloma accounts for 10% of all hematological malig- myeloma and Waldenstrém’s macroglobulinemia) in families,
nancies and one percent of all cancers. In the United States, although neither specific genetic link nor environmental
there was an estimated 15,770 new multiple myeloma cases in exposures have been.clearly identified.
2005 and over 11,070 yearly deaths.'? There was an overall
increased incidence of multiple myeloma between 1970 and
1990. This increase was attributed to under-reporting in the Pathophysiology
earlier years and to improved diagnosis and more intensive sur-
There are three primary processes that lead to the complex
veys, especially in the elderly in the later years (diagnostic
disruption of various organ systems in multiple myeloma.
phenomenon), rather than an actual increase in cases. Median
First is the expanding plasma cell mass in the bone marrow,
age at presentation is 67 years. Recent data indicate that the
second is the overproduction of monoclonal immunoglobulin,
disease is occurring more frequently in individuals younger than
and third is the overproduction of various cytokines that
55 years. Multiple myeloma incidence like all hematological
affect bone structure and function (Fig. 22-8). In the follow-
malignancies is higher in men. This is despite the fact that
ing section, each process is discussed further.
multiple myeloma is a disease of the elderly, and that women in
general live longer than men. PLASMA CELL EXPANSION
Multiple myeloma is the only hematological malignancy As the plasma cell establishes a malignant clone; normal BM
with a higher incidence among blacks. Asians have the lowest is gradually replaced by the slowly expanding malignant
rate of multiple myeloma with fewer than | per 100.000 inci- plasma cell colonies. As the replacement progresses, normal
dence of the disease.*? Multiple myeloma incidence in US blood cells decrease in number, a condition referred to as
white and black males and females categorized by age at pancytopenia. These cellular deficiencies commonly appear
diagnosis is described in Figure 22—7. There are no definitive in sequence. First, a decrease in the number of red blood cells
data to suggest that the higher incidence of multiple myeloma (anemia) is observed. Later, decreases in the number of
in blacks is associated with a biologically different disease. platelets occur (thrombocytopenia), and in advanced stages,
However, in some studies, black multiple myeloma patients neutrophils also decrease, although this is usually related to
were on average 10 years younger than white patients and had the effect of chemotherapy rather than an actual disease pro-
a higher incidence of fractures, paraplegia and infections. gression. Anemia results in fatigue, shortness of breath, and
Lower socioeconomic status and life style between blacks and rapid heart rate. Thrombocytopenia results in delayed hemo-
white have been suggested as a possible explanation for such stasis, with resultant prolonged bleeding and easy bruising.
differences, though no definitive data are available.
Neutropenia causes an increased susceptibility to infections.”
The expanding plasmacytoma (plasma cell tumors) orig-
Etiology inating in the bone marrow frequently cause destruction of the
bone surface “cortex.” Stretching of the overlying nerve-rich
The cause of multiple myeloma is unknown; several environ- periosteum leads to pain. Pain is the most common presenta-
mental, occupational and genetic factors have been associated tion at diagnosis in more than two thirds of multiple myeloma
with the increased risk of developing multiple myeloma.*! patients. Plasmacytoma may extend beyond the boundaries of
Many medical conditions associated with chronic stimulation the bone to compress adjacent neurological structures. This
of the immune system, such as repeated infections, allergic occurs most frequently in the vertebrae, where the nerve roots
conditions, or autoimmune disease, have been reported to exiting from the spine, and even the spinal cord itself, may be
increase the risk of multiple myeloma. Human herpes virus affected. This causes pain and may lead to paralysis. Less
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 507

White male Black male White female Black female

Rate per 100,000 Rate per 100,000 Rate per 100,000 Rate per 100,000

® Incidence
® Mortality

15

0
1975 1980 1985 1990 1995 2000 1975 1980 1985 1990 1995 2000 1975 1980 1985 1990 1995 2000 1975 1980 1985 1990 1995 2000

Figure 22-7 @ SEER incidence and U.S. death rates from myeloma, by race and sex. (From SEER 9 areas and NCHS public use data file.)
Rates are age-adjusted to the 2000 U.S. standard million population by 5-year age groups. Regression lines are calculated using the Join Point
Regression program.

commonly, plasmacytomas may affect soft tissues away from the formation of new bone by osteoblasts. Bone lesions in
the bone and bone marrow. multiple myeloma present as lytic lesions as well as osteo-
porosis that manifest as pathological factures and/or spinal
BONE MARROW STROMA cord compression. Late in the disease course, many patients
The bone marrow microenvironment plays an important role in report loss of height because of vertebral collapse. Multiple
supporting the malignant plasma cells in multiple myeloma by myeloma bone disease results from dissociation of bone
promoting their growth and preventing apoptosis (Fig. 22-9). absorption and formation secondary to stimulation of osteo-
Adhesion of myeloma cells to the stromal cells induces the clasts and inhibition of osteoblasts, respectively.*° Myeloma
secretion of several cytokines (proteins that promote or inhibit cells adhere and interact with bone marrow stromal cells
cell functions) from the stromal cells, endothelial cells, and/or resulting in the production of osteoclast-activating factors,
osteoclasts as well as from the plasma cells themselves. These including interleukin (IL)-18, IL-6, and IL-3 and tumor
molecules play an important role in multiple myeloma patho- necrosis factor; these cytokines stimulate the tumor necrosis
genesis and mediate many of the disease signs such as bone factor-related induced cytokine (TRANCE). TRANCE is
destruction, tumor cell proliferation, drug resistance, and responsible for the activation and maturation of osteoclasts.
genetic instability of plasma cells. The details of each cytokine TRANCE activity is blocked by osteoprotegerin. Osteoprote-
and the various pathways involved are beyond the scope of this gerin is a cytokine and a member of the tumor necrosis
review. In brief, they include osteoclast activating factors, such factor (TNF) receptor superfamily. It is also known as
as interleukin (IL)-18, IL-6, and the tumor necrosis factor osteoclastogenesis inhibitory factor (OCIF). The balance of
family proteins, as well as vascular endothelial growth factor, TRANCE/osteoprotegerin is totally disrupted in multiple
transforming growth factor-B, and insulin-like growth factors. * myeloma patients leading to overproduction of TRANCE and
inactivation of osteoprotegerin by binding to syndecan- | that
BONE DISEASE IN MULTIPLE MYELOMA is secreted by plasma cell surface (Fig. 22-10).?’
Normal bone is a dynamic structure undergoing continuous Recently, inhibition of osteoblasts is established as a
renewal through the resorption of old bone by osteoclasts and major contributor to bone disease in myeloma. Dickkopf |
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

MULTIPLE MYELOMA

Skeletal destruction Marrow infiltration

MALIGNANT PROLIFERATION
OF PLASMA CELLS

Figure 22-8 ™ Mechanisms of disease in multiple myeloma. Skeletal destruction, abnormal immunoglobulin production, marrow failure,
and decreased production of normal immunoglobulin all play a role.

(DKK1), an inhibitor of the Wnt signaling pathway, which is to hypercalcemia, renal insufficiency, or amyloidosis. Bone pain
crucial for osteoblast differentiation, leads to reduced bone is the most common presentation in multiple myeloma. Spinal
formation. In the Wnt signaling pathway, signaling mole- cord compression from extramedullary plasmacytoma is an
cules (Wnt proteins) regulate morphology, proliferation, acute emergency that should be diagnosed and treated promptly
motility, and cell fate. to prevent long-term disability. Magnetic resonance imaging
(MRI) or computed tomographic myelography of the entire
HYPERCALCEMIA spine must be done immediately, with appropriate follow-up
Calcium balance plays a critical role in regulation of cellular treatment by chemotherapy, radiotherapy, or neurosurgery to
function. With increased bone turn- over, calcium is released avoid permanent damage.
in the blood causing several symptoms. One of the earliest is The serum creatinine concentration is elevated in 20% of
constipation and cramping from altered motility of the intes- patients at diagnosis (>2 mg/dL).** The most common cause
tine followed by increased urination, dehydration and muscle of renal insufficiency in multiple myeloma is the precipitation
weakness. Increased calcium in the urine can lead to forma- of monoclonal light chains in the renal tubules called cast
tion of kidney stones and kidney failure. Later on, change in nephropathy. Other causes of renal dysfunction in multiple
mental status may develop, mostly confusion. The severity of myeloma include deposition of light chains in the kidney
the symptoms is dependent on both the level of calcium and parenchyma (light-chain deposition disease) and amyloid
the rate of rise. Hydration and forced diuresis and bisphos- fibrils in AL amyloidosis. Acute renal failure can occur in
phonate therapy are the main therapies for hypercalcemia (see multiple myeloma after administration of radiocontrast media
supportive care section below). during CT scans, or the use of none steroidal pain medica-
tions. In a few cases, acute renal failure may occur secondary
to hypercalcemia.
Clinical Features

The various manifestations and possible causes of multiple Diagnostic Workup

myeloma are outlined in Table 22-2. Anemia, which is a
major manifestation in multiple myeloma, can lead to weakness Once the diagnosis of multiple myeloma is suspected based
and loss of energy.” Infection and fever are quite common in on the presence of symptoms such as anemia, renal insuffi-
myeloma patients. Patients with more than two episodes of ciency, bone pain, neuropathy, and by the presence of high
pneumonia or multiple sinus infections should undergo screen- protein levels, confirmatory testing is needed to establish the
ing for multiple myeloma. Patients may have symptoms related tumor load “stage” and prognosis (Table 22-3).
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 509

Myeloma Cell Growth

Osteoclasts Increase

BMECs Angiogenesis

Bone Reabsorption

©D. Harmening

Figure 22-9 @ Bone marrow microenvironment. Effects of cytokines and transforming growth factors on the bone marrow (BM) microenvi-
ronment in patients with multiple myeloma (MM). Bone marrow stromal cells (BMSC) secrete: IL-6, VEGF, SDF-1a, and RANKL. . This IL-6
secreted in the BM microenvironment promotes the binding of MM cells to the BMSCs augmenting more secretion of IL-6 and other
cytokines from the BMSCs and MM cells. For example, TGF8, TNFa, and VEGF from malignant myeloma cells enhance IL-6 secretion from
BMSCs. The MM cells secrete IL-6, VEGF, bFGF, TNFa, TGFB, and MIP-1a. The IL-6, VEGF, bFGF, secreted by the MM cells and the VEGF from
BMSCs initiate angiogenesis in bone marrow endothelial cells (BMECs). The cytokines MIP-1a@ secreted by the MM cells and RANKL secreted
by BMSCs initiate or activate osteoclast formation, leading to bone reabsorption and bone destruction in MM patients. Osteoclasts also
secrete IL-6, inducing the growth of the malignant clone of MM cells. IL-6 = Interleukin 6; VEGF = vascular endothelial growth factor;
SDF-1a = stromal cell-derived factor alpha; RANKL = receptor activator of nuclear factor a—B ligand; bFGF = basic fibroblast growth factor;
TNF = tumor necrosis factor; TGFa = transforming growth factor beta; MIP-la = macrophage inflammatory protein alpha.

Myeloma cells
(2) IL-6 ®
TNF-B
Stroma © @ C@® va
—__ EDI SS... (> Osteoblast
TRANCE \ Fait
PTHrP U0

@)
1.
2.
MMcells adhere to stroma.
Stromal cells secrete OAFs.
Bo
a Syndecan
3. OAFs induce stroma and osteoblasts to secrete TRANCE.
4a. TRANCE is blocked by OPG; Syndecan from MM cells U
traps OPG and reduces OPG concentrations.
6) © 4b. Excess TRANCE is available to stimulate osteoclast. U0
5. Increased osteoclastic activity increases cytokine release
'e from bone matrix. U 4b)
6. These cytokines stimulate MM cell growth.
7. These cytokines also cause release of PTHrP from MM cells,
which activates stromal cells to secrete additional TRANCE.
TGF-B
FGF-1&2 e
IGF-1&ll Osteoclasts nn
PDGF Pe Osteoclast
progenitors

Figure 22-10 w Bone destruction in multiple myeloma. (From Tricot, G: New insights into role of microenvironment in multiple myeloma.
Lancet 355:248, 2000. Reprinted with permission from Elsevier.)
510. Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

d Sympt
om s immunoglobulin in the blood, causing red blood cells to
‘Sig ns an
Table 22-2 adhere to each other. The same phenomenon results in the
increased erythrocyte sedimentation rate. Circulating plasma
cells can be occasionally seen in multiple myeloma patients
Possible Cause and it carries a poor prognosis. Later in the disease, course
replacement of the bone marrow with plasma cells may also
Bone pain (68%) Fracture, lytic lesions
Easy fatigue (62%) Anemia (73%) cause teardrop-shaped red cells and the appearance of earlier
Polyuria (30%) Hypercalcemia (13%) forms of white blood cells in the peripheral blood.
Nausea and vomiting Renal failure, hypercalcemia
Recurrent infections Low normal Ig levels CHEMISTRY STUDIES
Paraplegia Cord compression
Routine chemistry panels are essential in evaluating myeloma
Confusion (15%) Hyperviscosity, hypercalcemia
Bleeding Thrombocytopenia patients. Serum blood urea nitrogen (BUN) and creatinine mea-
Arrhythmia (7%) Amyloidosis (4%) sures the kidney function that is often affected in myeloma.
Peripheral neuropathy Amyloidosis Serum lactate dehydrogenase (LDH) is a nonspecific marker of
Fever Infection tissue breakdown and elevated levels are associated with shorter
Renal insufficiency (19%) Cast nephropathy, light chain
survival. Calcium levels are often elevated in myeloma patients,
deposition
indicating bone destruction. Calcium is primarily bound to
Source: Modified from Kyle, RA, et al: Review of 1027 patients with albumin; the unbound or free calcium is the major cause of
newly diagnosed multiple myeloma. Mayo Clin Proc 78:21, 2003.
symptoms. Calcium levels must always be interpreted with the
albumin level in mind. For each mg/dL of albumin below
normal, the serum calcium should be increased by 0.8 mg/dL.
Laboratory Studies Albumin levels can be low in multiple myeloma due to inhibi-
tion by IL-6 as well as loss in the nephrotic kidney. In either
COMPLETE BLOOD COUNT AND PERIPHERAL BLOOD
case low albumin is a major prognostic criterion in multiple
SMEAR
myeloma (see international staging system below).
The automated complete blood count (CBC) is an easy avail-
8.-Microglobulin is the light chain of the histocompati-
able assessment of bone marrow function. The most common
bility locus antigen (HLA). It is the most useful predictor of
finding in multiple myeloma is a decrease in the number of
tumor load and disease activity, and in predicting the progno-
red blood cells with no change in size (normocytic nor-
sis of multiple myeloma patients. C-reactive protein is another
mochromic anemia). On peripheral smear examination, the
nonspecific marker of disease activity, mostly reflecting the
most characteristic finding in multiple myeloma is rouleaux
activity of IL-6, an important growth factor for myeloma
formation of the red cells (stacking of red cells together like
cells. Its prognostic value is limited by the non-specificity of
coins) (Fig. 22-11). It is caused by increased amounts of
the test.

BONE MARROW EXAMINATION

A bone marrow aspirate and biopsy is essential in the evalua-
tion of multiple myeloma patients. Marrow biopsy findings in
myeloma patients include increased numbers of plasma cells,
often forming “sheets,” with immature, binucleate, and large
M-Protein in Serum
Electrophoresis and immunofixation cells (Figs. 22-12 and 22-13). Sometimes certain features
Quantitation of immunoglobulins
Serum viscosity if symptomatic
Free serum light chain
24-Hour Urine for Urinary Protein Electrophoresis
(UPER)AFE (Immunofixation) and Creatinine
Clearance
Bone Marrow Aspirate and Biopsy
Cytogenetics
Flow cytometry (plasma cell labeling index)
CBC with Reticulocyte and Differential
Chemistry Panel (renal, Ca, albumin, uric acid)
6,-Microglobulin, C-Reactive Protein, and LDH
Radiological Evaluation
Skeletal survey
MRI (spine, pelvis, and skull)
PET/CT scan
Bone densitometry
Organ Function
Echocardiogram, EKG, pulmonary function tests and viral
testing Figure 22-11 @ Peripheral blood showing marked rouleaux
formation. Note the “stacked-coin” appearance of the red cells.
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 511

lial }

, 4

Mili,
Figure 22-12 ® Bone marrow aspirate showing atypical and Figure 22-14 m Flame cell, sometimes associated with IgA
binucleated plasma cells and Russell bodies (arrow). myeloma.

such as flame cells (large, intensely staining plasma cells) can CYTOGENETICS IN MULTIPLE MYELOMA
identify IgA subtype (Fig. 22-14). A differential count of Chromosomal studies (karyotyping) on bone marrow plasma
marrow cells (usually 500 cells are counted) is performed to cells in multiple myeloma have been slow to be recognized
establish percentages of various cell types. Patients with and implemented in clinical practice, mostly due to the low
myeloma have a variable level of plasmacytosis ranging from proliferative state of the plasma cells.
10% to 100%. This variability is partially related to the Chromosomal abnormalities can be found in 30% to 40%
pattern of bone marrow involvement in multiple myeloma, of patients. Frequently, cytogenetic abnormalities in multiple
which is often focal. Special stains are usually performed to myeloma are characterized by complex karyotypes with fre-
confirm clonality with k or ) restriction. quent iumerical and structural aberrations. *? Those with mono-
Bone marrow plasma cell labeling index (% of dividing somy 13, 13ql4 deletion and hypodiploidy have the worst
plasma cells) can differentiate between benign and malignant prognosis. Interphase fluorescence in situ hybridization (FISH)
plasma cells and is a major prognostic factor. Plasma cells detected a higher trequency of abnormalities even in patients
have a characteristic immunophenotype. In addition to stain- with normal karyotype (Fig. 22-15). Chromosomal transloca-
ing positively for cytoplasmic immunoglobulin, malignant tions involving 14q32, the site of the IgH (heavy chain) locus,
plasma cells stain positive for CD38, CD56 (neural celi adhe- is the most frequent abnormality in multiple myeloma. The
sion molecule), and CD138. They are usually negative for most common of these are translocations involving t(11;14)
surface immunoglobulin and the pan-B-cell antigen CD19; (q13;q32), t(4:14)(p16.3;q32.3), t(6;14)(p25;q32), and t(14;16)
15% to 20% will stain positively for CD20 and CD52. These (q32.3;q23) (Table 22-4). The t(4; 14) and t(14; 16) are associ-
latter markers led to the use of monoclonal antibodies (such ated with a poor prognosis (Fig. 22—16).°° In contrast, t(11;14)
as rituximab and Campath, respectively) in several clinical (q13;q32) patients have a better prognosis with up-regulation of
trials for the treatment of multiple myeloma. cyclin DI (a gene located on 11q13, which can function as an
oncogene), and lower levels of serum monoclonal proteins and
lymphoplasmacytic or small mature plasma cell morphology,
along with CD20 expression (see Table 22-4).*!
In recent years, gene expression profiling has emerged as
a useful tool to identify subgroups that follow a similar pro-
filing. These subgroups confirm the previously recognized
clinical observation of the diversity of multiple myeloma
patients. Recently, two major pathogenetic pathways for the
development of plasma cell tumors were defined: one that is
associated with hyperdiploidy and one that is characterized by
the presence of chromosome translocations involving the
immunoglobulin heavy chain locus (IgH).** While these studies
are in their infancy, this technology, as well as comparative
genomic hybridization, and studies of tumor-associated
antigens will ultimately define the critical genetic lesions in
multiple myeloma, providing new diagnostic and prognostic
indicators as well as a therapeutic paradigm for multiple
Figure 22-13 m Bone marrow biopsy sample showing replace-
ment of marrow by plasma cells. myeloma patients.
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

Table 22-4 Common Chromosomal

Translocations |
in Multiple Myeloma |
Translocations Affected Gene

t(11;14) Cyclin DI
t(4;14) MGFR-3 and MMSET
t(6;14) Cyclin D3
t(14;16) C-MAF
CEPI7 LSI p53§CEP17 t(14;20) MAF-B

RADIOLOGICAL INVESTIGATIONS
The role of imaging in the management of multiple myeloma
includes defining the extent of bone disease, and, in the uncom-
mon cases, defines soft tissue involvement. This 1s a critical step
in the diagnosis; as well as establishing adequate interventions
to prevent fractures and cord compression, etc. Full discussion
of the various modalities is available in an excellent recent
16q23§14q32
review listed in the references.**** The following is a brief
Figure 22-15 ™@ PCs with both the normal and abnormal pattern outline.
of hybridization. The depicted PCs show (A) a cell with the normal
configuration of 2 pairs of signals for the probes localizing to the
centromere 17 (CEP17; aqua) and the 17p13.1 (LSI p53) (red) PLAIN RADIOGRAPH—X-RAY EXAMINATION Skeletal
probe. (B) A cell with deletion of 17p13.1. There are two green survey remains the standard method for radiological screen-
signals arising from the centromeric probe but only one red signal ing of suspected multiple myeloma cases. Multiple lytic or
from the p53 locus probe. (C) A normal configuration of probes punched out lesions are very suggestive of multiple myeloma
used to detect the t(14;16)(q32;q23). The locus-specific 14q32
and are present in 76% of patients at diagnosis (Figs. 22—17
probes are labeled in green, and the 16q23 probes are labeled in
red. (D) A cell with fusion of probes for 14q32 (green) and 16q23 and 22-18). X-ray exams provide a prognostic parameter for
(red). (From Fonseca, R, et al: Clinical and biologic implications of assessment of the tumor load at diagnosis in the classic
recurrent genomic aberrations in myeloma. Blood 2003;101:4570. Durie/Salmon staging system. X-ray films of the long bones
Reprinted with permission from the American help identify critical cortical thinning that may lead to patho-
Society for Hematology.)
logical fracture, allowing for preventive interventions. How-
ever, plain radiography, demonstrating lytic disease only
when at least 30% of trabecular bone substance has been lost,
p<0.001 provides an inadequate assessment of generalized osteopenia
that affect more than 25% of the patients with no lytic disease.
2 oo All others including t(11;14)

es
oOiN
(=) rep)
Survival
probability
t(4;14) or
S Ne)
t(14;16) or
—17p13
2 ro)
0 10 20 30 40 50 60 70 80 90 100110120130140150
Months

Figure 22-16 @ Overall survival of patients stratified by the

hierarchic classification model. The survival curves show clear sepa-
ration of patients into the good, intermediate, and poor prognosis
category. The poor prognosis group includes patients with
—17p13.1, t(4;14)(p13;q32), and/or t(14;16)(q32;q23), the inter-
mediate prognosis group includes those patients with A13; and the
good prognosis group includes remaining patients, including those
with the t(11;14)(q13;q32) and those with normal karyotype. (From
Fonseca, R, et al: Clinical and biologic implications of recurrent
genomic aberrations in myeloma. Blood 2003;101:4571. Reprinted Figure 22-17 @ Extensive lytic skull lesions in a patient with
with permission from the American Society for Hematology.) multiple myeloma.
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 513

powerful magnetic field. It surpasses other modalities in

imaging neurological tissues and the spine because of its abil-
ity to detect differences between normal and abnormal
soft tissues and its ability to image the spine longitudinally
(Fig. 22-19). MRI is useful in assessment of soft tissue plas-
macytoma and in patients with neurological presentations
suggestive of cord compression. MRI provides an accurate
evaluation of the level and extent of cord or nerve root com-
pression and the size of the tumor. MRI yields information
about the pattern of bone marrow involvement such as focal
and diffuse involvement. Some preliminary data suggest that
normalization of MRI after therapy is predictive of longer
remission (Radiological complete remission). MRI should be
performed in all patients suspected of having solitary plasma-
cytoma, and possibly in many MGUS patients to avoid down
staging of their disease.

OSITRON
RJ LN EMISSION TOMOGRAPHY Positron emission
tomography (PET) imaging with 18-fluorine-fluoro-
deoxyglucose (FDG) is a promising new scanning tech-
nique under evaluation in multiple myeloma. PET scan
appears to be very sensitive in detecting occult sites of
disease and appears to be most useful in diagnosing and
follow up of nonsecretory multiple myeloma (only 1% to
2% of all myeloma patients) and extramedullary plasma-
cytoma (Fig. 22-20).

8) 7 Vala:
} 4 & 4 Y A
UNE OWL

Dual energy X-ray absorptiometry scanning (DEXA) is the

Figure 22-18 @ Radiographic film of the left humerus of a patient standard procedure for diagnosing osteoporosis. In multiple
with multiple myeloma. Areas with severe cortical bone destruction
may be fractured by everyday activities such as lifting or walking
(pathologic fractures).

These x-ray films do not detect soft tissue plasmacytomas and

have low sensitivity and specificity.

CT scans are more sensitive than radiographs in detecting

lytic bone disease as well as soft tissue plasmacytoma. It fur-
ther defines suspicious areas on plain films and parts of the
skeleton that cannot be evaluated fully by plain films, e.g.,
scapulae, ribs, and sternum. CT is routinely done before biop-
sies and radiotherapy.

NUCLEAR MEDICINE—SCINTIGRAPHY Nuclear medicine

studies, which are extremely important in malignancies involv-
ing bone, such as breast and prostate cancers, have a limited
role in multiple myeloma because the technetium radioisotope
used in bone scans is actively taken up by osteoblasts, and as
mentioned earlier, osteoblastic activity in multiple myeloma is
inhibited so most multiple myeloma lesions are not detected
except in limited areas of active bone remodeling.

MAG aX g |ES(
Ne) CE IMAG Magnetic resonance
imaging (MRI) produces computer-generated images based Figure 22-19 @ MRI showing compression fracture at L-2 (red
on tissue absorption of radiofrequency energy while in a arrow) and focal plasmacytoma (white arrows and circles).
514 — Chapter 22. Multiple Myeloma and Related Plasma Cell Disorders

CT Transaxial

CT Scout View

eT Coronal PET Coronal — Fused Coronal

PET Transaxial

=
ve
&i
@to.
*
*

oa
rt
oO
ae
os
%

on
==
ta]
CY
vy
rye @°9
“©
Fused Transaxial

s+ n900
Figure 22-20 mPET-scan showing metabolic activity in multiple plasmacytomas throughout the skeleton.

myeloma patients, low-lumbar spine bone mineral density at

diagnosis is correlated with an increased risk of vertebral col-
lapses and fractures. It is an important test to consider, espe- The currently agreed-on diagnostic criteria are listed in
cially if there is no lytic bone disease, and in the presence Table 22—5.*° Multiple myeloma is diagnosed if the following
of lytic bone lesions and vertebral body collapse, DEXA three criteria are present: (1) a monoclonal protein in serum
scanning is difficult to interpret. Follow-up imaging is not and/or urine, (2) bone marrow clonal plasma cells or plasma-
routinely indicated in multiple myeloma. cytoma, and (3) related organ or tissue impairment (e.g.,
Lytic bone lesions never heal in multiple myeloma even increased calcium, renal insufficiency, anemia, lytic bone
in patients achieving a complete remission. The use of radio- lesions). In general, the type and amount of immunoglobulin
logical evaluations such as plain x-ray films for follow- up is does not predict outcome or response to therapy. IgG is
therefore of no value in assessing disease response. However, the immunoglobulin produced in more than 50% of cases,
developing new bone lesions can indicate progressive dis- followed by IgA (20%); 20% produce only light chains and,
ease, and any multiple myeloma patients with new pain or rarely, IgD (fewer than 1%). Rarely (in fewer than 1% of
neurological symptoms should have repeat appropriate radio- cases), multiple myeloma may produce no immunoglobulin
logical evolutions. “nonsecretory.” Two immunoglobulins classes can be
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 515

on Table 22-6Durie Salmon Clinical

: iy Myeloma piping.
I. Biopsy-proven plasmacytoma
II. Bone marrow plasmacytosis
Ill. Monoclonal protein (M-spike); Serum or urine iter Median: Survival
PLUS Evidence of End-Organ Damage (CRAB) Stage I (ioe.Hivos Ser $60 Beene
Calcium; Ca >11 mg/dL (all criteria must be met)
Renal; creatinine >2 mg/dL 1. Hemoglobin >10 g/dL
Anemia; Hgb <10 g/dL 2. Corrected serum calcium <12 mg/dL
Bone; lytic lesions, osteoporosis 3. Fewer than two lytic bone lesions
Others: recurrent bacterial infections, hyperviscosity, 4. Small M-spike
amyloidosis, etc. IgG <5 g/dL
IgA <3 g/dL
Urine light chains < 4 9/24 hr
Stage II (intermediate 41 months
produced in 3% “biclonal myeloma” of cases that reflect myeloma mass)
the coexistence of two different malignant clones. The differ-
1. Does not meet all stage I criteria
ential diagnosis of myeloma includes related plasma cell
2. Does not meet any stage III criteria
disorders such as monoclonal gammopathy of undetermined
Stage III (high myeloma mass) 23 months
significance, smoldering multiple myeloma, primary amy-
(any of the following criteria)
loidosis, and other lymphoproliferative disorders such as
Waldenstrém’s macroglobulinemia and other non-Hodgkin’s 1. Hemoglobin < 8.5 g/dL
2. Corrected serum calcium > 12 mg/dL
lymphomas.
3. Two or more lytic bone lesions
4. Large M-spike
IgG >7 g/dL
Staging IgA >5 g/dL
{ , eis ; Urine light chains >12 g/24h
Once a diagnosis of multiple myeloma has been established, Substaging
an estimation of the extent of the disease “staging” is needed. ihe Sepuanter nine 22.0 mera
Staging 1s important for reporting treatment outcomes and pro- B.Serum creatinine >2, 0 mg/dL
vides a common means of comparing clinical trials. Various naan ng RRRRER RReReenneRemenReNRanepeERmenaEnEnREEEET

aging systems have been proposed and are cently in|

use. The most widely used staging system was described by
Se Atal onDu OM at Solon,SE Sen
P, et al (eds): Neoplastic Diseases of the Blood. Churchill
Durie and Salmon in 1975 (Table 22-6). This system uses Livingstone, New York, 1985, with permission.
hemoglobin, M-spike magnitude, serum calcium level, bone
radiographs, and serum creatinine level (kidney function) to
estimate the mass of myeloma cells in the body. It associates
low myeloma mass (stage I) with a median survival of greater
than 5 years. An intermediate myeloma mass (stage I) is
associated with a survival of approximately 3.5 years and
a high myeloma mass (stage III) with survival of less than
PESTAGS ee
A new International Staging System (ISS), based on
10,750 previously untreated multiple myeloma patients from
17 institutions worldwide, has been developed (Table 22-7).
It is based on the levels of serum beta 2 microglobulin ({;,,)
Median Survival
and serum albumin. It relates normal levels of B;,, less than Stage Critertia (months)
3.5 mg/L and serum albumin greater than or equal to 3.5 g/dL ern anne tenements ar enetnannininanintnntanscennsna
(stage I) with a median survival of 62 months, and high B>., I Serum siteocean 62
>5.5 mg/L (stage III) with a median survival of 29 months. . ey5 mg/L
Z i ; : 3 A erum albumin 23.5 g/dL
Stage II is associated with a median survival of 44 months.’ ll Not stage I or IIT’ 44
Ul Serum B,-microglobulin 29
2. > mele

There are two categories for stage II: serum 8.microsiobaln

For more than 40 years, the standard therapy for multiple <3.5 mg/L but serum albumin <3. 5 g/dL; or serum 8.-microglobulin
2 | Iphalan and prednisone SY) (Oy SBS) mg/L irrespective of the serum albumin level.
myeloma patients has been oral melpha 2 P : Source: From Grippe, PR, et al: International staging system for
This regimen had a 50% overall response rate, although com- multiple myeloma. J Clin Oncol 23:3412, 2005.
plete remissions were rare, and the median duration of
516 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

response was approximately one and a half years. Overall sur- 27% was achieved in heavily treated relapsed and refractory
vival for patients was less than 3 years and was mostly depen- multiple myeloma patients with a median duration of
dant on the stage at diagnosis rather than therapy.*” More response of 13 months and a median overall survival of
intensive combinations with various chemotherapy drugs did 17 months. Responses to bortezomib have been rapid and
not show improvement of overall survival in comparison to are seen after one or two cycles, in most patients. A Phase
melphalan/prednisone. The introduction of dexamethasone in III trial confirmed the superiority of single-agent borte-
the VAD regimen (vincristine, doxorubicin, and dexametha- zomib over dexamethasone in terms of response rate, time to
sone), initially for relapsed patients and later for newly diag- progression, and overall survival. The additive effect of
nosed patients has been associated with increased complete dexamethasone was seen in nonresponders and in those
remission (CR) rates (5% to 10%) with no significant impact who progressed on single-agent bortezomib. The main side
on overall survival.*° effects of the velcade include peripheral neuropathy and
In the mid-1990s, high-dose chemotherapy with autolo- thrombocytopenia; both appear to be reversible on stopping
gous stem cell transplant (auto-SCT) support changed the therapy. Currently bortezomib is being evaluated in the
way we treat myeloma patients, especially those younger first-line setting, alone and in various combinations with
than age 65.*' Auto-SCT has improved the outcome in newly dexamethasone, thalidomide, doxorubicin, and with mel-
diagnosed multiple myeloma patients, with higher complete phalan with and without stem-cell transplantation, with very
remission rates (40%), longer event-free survival (4 to 5 years), encouraging results.°?°?
and improved overall survival (greater than 7 years). Many of Lenalidomide (Revlimid) is another novel therapy that
these patients had durable remission, especially the subset of has shown significant activity in relapsed multiple myeloma.
patients with no chromosomal abnormalities and normal f,,, In March 2005, two Phase III trials in relapsed and refractory
at diagnosis.*” Several studies have shown that tandem trans- myeloma patients were stopped prematurely when interim
plants can further increase the duration of remission and over- efficacy endpoint “time to disease progression” was found to
all survival.**4 be statistically better in patients receiving Revlimid plus
Allogeneic stem cell transplants have been advocated dexamethasone compared with patients receiving dexametha-
as the only curative therapy for multiple myeloma patients.* sone alone. The trials were un-blinded and Revlimid was
The role of allo-SCT has been controversial; standard con- added to patients receiving dexamethasone alone. Revlimid
ditioning allo-SCT was associated with 50% treatment has now been approved by the Food and Drug Administration
related mortality in the first 100 days. However, 30% to in June 2006 for use in relapsed multiple myeloma patients
50% of patients who survive the first year remain disease- that have received at least one prior therapy. Lenalidomide is
free at 3 to 6 years, with few well documented cases of sus- being explored in combinations with other drugs, and in the
tained molecular remissions. These durable remissions have newly diagnosed multiple myeloma patients.
been attributed to immunological effects of T cells known as Additional therapy after achievement of a maximal
graft versus myeloma effect.*° Several reports have shown response following chemotherapy or transplant (called mainte-
that a nonmyeloablative conditioning followed by allo- nance) produced conflicting results.°° Continuing conventional
geneic graft (T cells) had lower mortality and can lead to chemotherapy therapy (often referred to as consolidation) is not
prompt engraftment.*’ Despite initial success, long-term sur- beneficial following the achievement of the plateau phase, espe-
vival remains low, especially in high-risk relapsed myeloma cially in high-risk patients, as most relapse while on therapy.
patients.** Continuing conventional chemotherapy is associated with high
Radiation therapy has limited efficacy in treating sys- morbidity and adversely affects the patient’s quality of life.
temic manifestation of multiple myeloma. However, it is Interferon (IFN) maintenance results in prolonged relapse-free
highly effective in treating the localized disease process such survival by 4 to 9 months with a small gain in overall survival.*°
as painful lesions resulting from plasmacytoma, and lytic However, the drug has many side effects, and its use in
bone lesions especially in spinal cord compression or impend- myeloma is decreasing in favor of novel therapeutics mentioned
ing fractures. above.”’
In 1999, single-agent thalidomide (Thalomid) showed sig- One study investigated alternate day, oral prednisone at
nificant activity in relapsed refractory multiple myeloma with an two different dose levels (10 mg versus 50 mg) in multiple
overall response rate of 30%; few patients remain in continuous myeloma patients after they responded to induction. The
remission at 6 years. In combination with dexamethasone, higher dose of prednisone significantly improved progression-
response rates increase to approximately 50%. The combination free survival (14 months versus 5 months), and overall sur-
of thalidomide plus dexamethasone is now the main induction vival (37 months versus 26 months).** The study did not
therapy for newly diagnosed multiple myeloma patients with an include a formal evaluation of side effects. Several newer
overall response rate of 65% to 70%.*° Thalidomide/dexametha- agents are now being studied as maintenance therapies in
sone has the advantage of being an oral regimen; however it car- the plateau phase following initial chemotherapy or high-
ries a risk of neuropathy and deep venous thrombosis (15%).°°>! dose chemotherapy, including thalidomide, lenalidomide, and
The proteasome inhibitor bortezomib (Velcade) was bortezomib. Their role in this setting remains experimental.
approved in 2003 in the United States for relapsing multiple Preliminary data suggest that remission and overall survival
myeloma patients. Initial approval was based on the results may be prolonged with thalidomide/pamidronate mainte-
of the Phase II trial “SUMMIT” in which a response rate of nance after high-dose chemotherapy.°*?
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 517

Atypical Variants of Plasma Cell patients remaining free of disease for at least 10 years. New
Syndromes modalities for diagnosis (PET, free light chain assays, etc.)
should help further define these cases.°?
Smoldering Myeloma
These are patients who meet the criteria for diagnosis of mul- Plasma Cell Leukemia
tiple myeloma but have a low tumor mass (Durie-Salmon
stage I) with an M-protein greater than 3 g/dL and/or greater Plasma cell leukemia (PCL) is a rare variant of multiple
than or equal to 10% bone marrow plasma cells and no end- myeloma (2% to 3%), with an aggressive presentation and
organ damage from the disease (normal kidney function, short survival. It is defined as circulating peripheral blood
normal serum calcium, and no lytic bone lesions).°° These plasma cells exceeding 2000/uL, and 20% of peripheral blood
patients should not be treated as long as they are asympto- white cells (Fig. 22-21). Half of these cases are found in
matic, but they need careful follow-up as their incidence of advanced multiple myeloma, usually late in the course of the
progression to multiple myeloma is higher than that of MGUS disease, and usually herald a terminal event. The other half,
patients, and most will progress to advanced stages requiring often referred to as primary plasma cell leukemia, affect
therapy within 3 years.°' The administration of bisphospho- newly diagnosed patients. Patients present with high calcium
levels, kidney failure, and much more anemia, thrombocy-
nates to patients with smoldering disease, to delay the
progression to overt multiple myeloma, is undergoing clinical topenia, and involvement of various organs, such as the
trials. It is thought that bisphosphonates change the bone mar- liver, spleen, and lymph nodes. Treatment is the same as for
row “soil” to become an ineffective microenvironment to newly diagnosed multiple myeloma. Prognosis is poor, with
expected survival of only several months, even after autolo-
plasma cell “seeding.” The benefit of this treatment has to be
balanced against the long term risks of therapy.” gous and/or allogeneic transplantation.

Solitary Plasmacytoma of the Bone Nonsecretory Myeloma

Fewer than 5% of patients with a plasma cell dyscrasia pre- Fewer than 1% of multiple myeloma patients present with no
sent with a single bone lesion without evidence of systemic abnormal immunoglobulin in the blood or urine, even with
disease (normocalcemia, absence of anemia, normal renal sensitive assays for free light chains. These patients present
function). The tumor may secrete immunoglobulin, resulting with bone marrow plasmacytosis (high number of plasma
in a small serum M-spike which usually disappears after cells), lytic bone disease with elevated serum calcium, and
therapy. Solitary plasmacytoma of the bone (SPB) occurs plasmacytomas. Treatment is the same as for multiple
more often in men, at an average age of 60, 7 years younger myeloma. Because of normal levels of immunoglobulin,
than the age of multiple myeloma patients. Diagnosis requires infectious complications are fewer. The absence of light-chain
a biopsy to confirm the presence of a monoclonal plasma cell excretion, via the kidneys, preserves renal function, and sur-
infiltrate. The treatment of choice is radiotherapy given with vival is often better than associated with other multiple
curative intent (greater than 4000 cGy) resulting in long-term myeloma subtypes. Evaluation of response to therapy is often
disease-free survival in approximately 40% of patients at difficult, but has been markedly improved by the use of PET
10 years. Although the majority of these appear to be cured, scans.
rare late recurrences have been reported. Sixty percent of SPB
patients develop multiple myeloma after a median of 2 to
3 years. The disease course tends to be more benign than that
of multiple myeloma, with a median overall survival of 7 to
12 years. Treatment decisions on relapse are the same as for
multiple myeloma.’

Extramedullary Plasmacytoma
Extramedullary plasmacytoma (EMP) is a soft tissue plasma
cell tumor. Similar to SPB, there should be no evidence of
bone destruction or evidence of marrow plasmacytosis. EMP
must be distinguished from reactive plasmacytoma, plasma
cell granuloma, and lymphoma (MALT, marginal zone, and
immunoblastic) (see Chap. 21). EMP occurs mostly in
mucosal sinuses in the head/neck; less frequently it may
affect the gastrointestinal tract, skin, lungs, bladder, thyroid, Figure 22-21 @ Peripheral blood in plasma cell leukemia showing
testis, ovaries, and tonsils. Treatment is radiotherapy just like presence of circulating plasma cells. (From Dutcher, T: Hematology.
Solitary Plasmacytoma of the bone (SPB). The risk of conver- In: Listen, Look, and Learn. Health and Education Resources, Inc,
sion to multiple myeloma is lower, with 50% to 65% of Bethesda, MD, with permission.)
518 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

POEMS Syndrome vacuoles containing IgM monoclonal protein (Dutcher bod-

ies) within the malignant cells of Waldenstrém’s macroglob-
POEMS syndrome is an unusual syndrome of complex clinical ulinemia are common (Fig. 22—22).
features that are closely related to the presence of a monoclonal In the United States, Waldenstrém’s macroglobulinemia
plasma cell disorder. POEMS is classically defined by the affects 1400 individuals each year. The median age at diagno-
presence of a peripheral neuropathy (P), organomegaly (O), sis is 64 years, and again, like multiple myeloma, has a male
endocrinopathy (E), a monoclonal plasma cell disorder (M), and predominance. There is a clear familial clustering in families
skin changes (S). Other features include sclerotic bone lesion(s), that can been seen in as many as 20% of the cases.
thrombocytosis, edema, and pulmonary hypertension. Not all Patients with Waldenstrém’s macroglobulinemia complain
features of the disease need to be present to make the diagno- of fatigue, weight loss, and generalized weakness.’' Progressive
sis. The mainstay of therapy for patients with POEMS includes sensory motor peripheral neuropathy is another presenting
irradiation to isolated bony lesions. In systemic cases, myeloma- feature of this disease. Severa! patients may have a cryoglobu-
like therapy, with the goal of eliminating the plasma cell clone, lin (type I), proteins (often IgM) that precipitate on exposure to
is often used with various degrees of success.°° cold, many patients with Waldenstrém’s macroglobulinemia
develop symptoms of cryoglobulinemia, such as pain and color
changes in cold-exposed areas (Raynaud’s phenomenon),
Supportive Care in Myeloma Patients clotting or thrombosis of small blood vessels, and kidney
Once a decision is made to treat the patient, attention should be damage. Patients with Waldenstr6m’s macroglobulinemia often
directed to supporting the patient, minimizing the side effects have extensive bruising called cryoglobulinemic purpura and
of therapy. Hypercalcemia (high calcium levels) is managed by may have bleeding from the gums and nose (Fig. 22—23). More
intravenous saline and medications that inhibit osteoclasts, severe bleeding may be seen, which is caused by interference
such as glucocorticoids, bisphosphonates, and calcitonin.°’ with platelets and blood clotting factor proteins by the abnormal
Vatcines for the encapsulated bacteria (Streptococcus IgM. Bleeding time is often prolonged, as are the clotting factor
pneumoniae, Haemophilus influenzae, and Neisseria meningi- assays, prothrombin time (PT) and activated partial thrombo-
tis) should be given early in the disease course to prevent plastin time (APTT). Lymphadenopathy and organomegaly
infections. In patients with recurrent bronchitis or sinusitis, are seen in more than half the cases at presentation, mostly
intravenous pooled immunoglobulin given at monthly through infiltration with malignant cells or deposition of IgM or
intervals may be of significant benefit, especially if the IgG amyloid fibrils. Anemia is the main feature in most symptomatic
level is less than 500 mg/dL. In addition, patients are given Waldenstr6m’s macroglobulinemia patients (80%). Anemia
erythropoietin to maintain adequate hemoglobin levels. Pain results from impaired hematopoesis as well as hemolysis.
is areal problem in multiple myeloma patients and is treated Recurrent infection is common and is related to reduction of
with chemotherapy, or radiation therapy if the lesion is local- uninvolved immunoglobulin (IgG and IgA). Such suppression
ized to one bone. Pain medications should be given liberally persists even after therapy in responding patients.”
to multiple myeloma patients, and anti-inflammatory nons- Chemotherapy is the primary treatment, using alkyla-
teroidal drugs avoided, as they can precipitate renal failure. tors such as cyclophosphamide and nucleosides analogues
Bisphosphonates—pamidronate (Aredia) and zoledronate such as fludarabine. Recently, Rituximab, a monoclonal anti-
(Zometa)—act at sites of active bone remodeling by binding to body against CD-20, has shown promise in Waldenstrém’s
hydroxyapatite, inhibiting osteoclast development and migra-
tory activity, inducing cell death, and thereby decreasing bone
resorption without affecting bone mineralization. Monthly
infusions of bisphosphonates have been shown to reduce skele-
tal events and modify the natural history of bone disease in
multiple myeloma.”

Waldenstrém’s Macroglobulinemia
Waldenstr6m’s macroglobulinemia is a malignant lymphopro-
liferative disorder involving small lymphocytes that exhibit
plasma cell differentiation (plasmacytoid lymphocytes); these
cells produce IgM monoclonal protein. Waldenstrém’s
macroglobulinemia cells express pan-B lymphocyte surface
antigens (CD19, CD20, and CD24), with light chain restric-
tion (mostly k). Chromosomal abnormalities are common in
Waldenstr6m’s macroglobulinemia; the most common is Figure 22-22 @ Plasmacytoid lymphocytes in marrow aspirate
from a patient with Waldenstrom’s macroglobulinemia. (From
deletion of 6q21, which is detected in 42% of Waldenstrém’s Hyun, BH: Morphology of Blood and Bone Marrow. American
macroglobulinemia patients. The bone marrow is extensively Society of Clinical Pathologists. Workshop 5121, Philadelphia,
infiltrated with lymphoid and plasmacytoid cells. Intranuclear September 7, 1983, with permission.)
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 519

Pigure 22-25 m@ Arm of patient with cryoglobulinemic purpura.

Note the skin manifestations. Figure 22-24 ™ Amorphous amyloid deposits replacing normal
liver architecture.

macroglobulinemia therapy. If severe symptoms are present at Light Chain Deposition and Heavy
diagnosis, IgM may be quickly removed by plasmapheresis,
Chain Diseases
resulting in the rapid improvement in symptoms.”
Light chain deposition disease (LCDD) is similar to primary
amyloidosis; both are clonal plasma cell proliferative disor-
Amyloidosis ders, but the light chain fragments do not form amyloid fibrils
Amyloid is a protein conformation disorder that renders the and while most cases of AL-amyloidosis are related to
protein insoluble and fesults in its deposition in the extra cel- secretion, LCDD is usually k. As with AL-amyloidosis,
lular matrix of various organs. There are many precursor pro- LCDD may be associated with multiple myeloma or other
teins that may result in amyloidosis.” The most common is conditions, such as lymphoma or Waldenstr6m’s macroglob-
AL amyloid (amyloid with presence of light chain secretion); ulinemia. In the kidney, LCDD is often associated with renal
the precursor protein is derived from immunoglobulin light failure, in contrast to AL-amyloidosis, which results in excess
chain fragments. Less common is AA amyloidosis (secondary protein excretion. Treatment is directed at eliminating the
amyloidosis associated with inflammation). This usually plasma cell clone, and if the patient is young and had end-
complicates chronic diseases in which there is ongoing or stage renal disease, renal transplants may be considered.
recurring inflammation, such as rheumatoid arthritis or Heavy chain diseases (HCDs) are very heterogeneous
spondyloarthropathy; chronic infections; or periodic fever group of disorders and do not represent true plasma cell disor-
syndromes. The fibrils are composed of fragments of the ders. They are mentioned here owing to the presence of
acute phase reactant serum amyloid A. Discussion of other abnormal serum immunoglobulin component. These are
familial amyloidosis syndromes is beyond the scope of this usually related to lymphoma, chronic lymphocytic leukemia,
chapter. and marginal zone lymphoma. The B cells in these disorders
Amyloidosis is a rare and potentially fatal disease that exclusively produce monoclonal heavy chains and no light
occurs when insoluble protein fibers are deposited in tissues chains. The heavy chains are divided into three main types:
and organs, impairing their function (e.g., heart, kidneys, y, pw, and a. Patients have systemic symptoms and
liver, spleen, nervous system, and gastrointestinal tract) organomegaly. The details of these disorders are beyond the
(Fig. 22-24). Amyloidosis causes progressive loss of function scope of this book and can be found in reference 78.
of the involved organ. Amyloid patients have a wide range
of presentations depending on the organ involved. Renal
amyloid may present with asymptomatic proteinuria. Cardiac
involvement results in systolic/diastolic dysfunction leading Case Study 1
to heart failure. Mixed sensory and motor peripheral neuropa- Multiple Myeloma
thy and/or autonomic dysfunction is a prominent feature in
many patients with amyloidosis. Skin may provide clues of A 71-year-old man presented with a 6-month history of back
amyloid involvement when thickening, easy bruising, and pain. His wife noted that he was frequently confused, slept
periorbital purpura are seen. Treatment is aimed at eliminating much of the day, and made occasional nonsensical state-
the source of the abnormal precursor protein. In AL-amyloid, ments. On evaluation, he was anemic with a hemoglobin
treatment involves chemotherapy or stem cell transplantation (Hgb) level of 10 g/dL and a mean corpuscular volume
to eliminate the plasma cells, the source of the abnormal light (MCV) of 91 fL. He had a calcium level of 10.6 mg/dL
continued
Chainsy es!
520 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

Case Study I: Case Study 2:

Multiple Myeloma -cona Monoclonal Gammopathy
(normal range is 8.8 to 10.5) with a low albumin level of
of Undetermined |
2.6 g/dL and a corrected calcium of 11.7 g/dL. The creatinine
level was 1.1 g/dL. The patient was hydrated with normal
Significance (MGUS)
saline and received bisphosphonate infusion (pamidronate) A 55-year-old man presented with back pain after lifting some
that rapidly lowered the calcium levels, and the patient’s heavy boxes. Examination of the patient’s back revealed some
mental status quickly improved over the following 3 days. tenderness in the muscles with no bone tenderness. X-rays
Serum protein electrophoresis revealed an M-spike measur- of the back were normal. Serum protein electrophoresis
ing 4.7 g/dL. Immunoelectrophoresis revealed that the revealed an M-spike of 1.1 g/dL. Immunofixation identified
M-spike was composed of an IgG-« antibody. His serum the M-spike as IgG-lambda. A full skeletal survey was normal
IgM level was 63 mg/dL, and the IgA level was 120 mg/dL. and 24-hour urine collection for protein evaluation showed no
The 8, microglobulin was 3.4 mg/dL. A 24-hour urine col- monoclonal protein. Serum calcium and serum quantitative
lection demonstrated no protein. The bone marrow was immunoglobulins were normal. The bone marrow revealed
hypercellular with 70% plasma cells. Cytogenetics showed 7% normal-appearing plasma cells. CT scans of the chest and
normal male karyotype. A radiological skeletal survey abdomen revealed no enlarged lymph nodes suspicious for
revealed classic lytic lesions in the skull, pelvis, both arms, lymphoma, and confirmed a normal size spleen.
and both femurs, with diffuse osteoporosis.

QUESTIONS
QUESTIONS
1. What is the most likely diagnosis for this patient?
1. What major and minor criteria for multiple myeloma are
a. Multiple myeloma
met in this patient?
b. Waldenstr6m’s macroglobulinemia
2. What stage myeloma does he have?
c. Monoclonal gammopathy of unknown significance
3. What is the prognosis of this patient?
d. Non-Hodgkin’s lymphoma
4. What is the best therapy for this patient? What are the
2. Which explanation should be given to the patient?
main toxicities of such therapy?
a. You have an incurable disease, probably a cancer that
has not yet been found. I want to see you back every
ANSWERS 3 months for tests.
1. Major criteria met were (a) more than 30% plasma cells
b. Nothing of concern was found. There is nothing to
in the bone marrow; (b) IgG-k M-spike greater than worry about. Have a nice day.
3.5 g/dL. Minor criteria met were (a) lytic bone lesions; c. Your body is producing some abnormal antibodies. It
(b) low-normal immunoglobulins (see Table 22-6).
is acommon disorder that will probably never cause
2. The patient has clinical Durie Salmon stage HI and you any problems. A small percentage of patients may
international stage II. develop a more serious problem later, so I need to see
3. He will be intermediate risk with a median surival of you back in 3 months, and then periodically to see if
44 months (see Table 22-7). there has been any change.
4, Dexamethasone and thalidomide and/or lemalidomite is 3. What malignancy is likely to occur in this patient?
currently the most widely used regimen in therapy of a. Multiple myeloma
myeloma patients. The main toxicities are dexametha- b. Chronic lymphocytic leukemia
sone related such as hyperglycemia, hypertension and c. Amyloidosis
fluid retention. Thalidomide combinations have been d. Non-Hodgkin’s lymphoma
associated with increased risk of deep venous thrombo- e. All of the above
sis and neuropathy. The patients should receive monthly 4. What follow-up schedule would be appropriate?
infusions of bisphosphonates to prevent skeletal compli- a. Monthly serum protein electrophoresis.
cations such as fractures b. Bone marrow biopsy repeated every 6 months for
2 years. If there is no evidence of myeloma at that
time, the patient does not have to return anymore.
QO . Repeat the complete evaluation every 6 months.

d. Repeat the serum protein electrophoresis in 3 months,

then every 6 months thereafter if there has been no
change.
continued
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 5 i)

ANSWERS QUESTIONS
1. c. Although a few patients develop multiple myeloma, 1. Which of the following is a possible cause of the
non-Hodgkin’s lymphoma, or chronic lymphocytic patient’s problems in concentrating and reading?
leukemia, most patients with MGUS are not adversely a. The calcium level is actually high when the low albu-
affected by this disorder. min level is considered.
2a€ b. The increased viscosity of the blood is causing poor
3. All of the above. MGUS patients can develop any lym- circulation through the blood vessels in the brain,
phoid malignancy. resulting in these symptoms.
4. d. Once the initial evaluation is complete, no further test- c. On exposure to cold, cryoglobulins in the patient’s
ing needs to be done unless there is a change in the pro- head precipitate and block blood vessels.
duction of the M-spike immunoglobulin. Three months is d. Osteolytic lesions in the spine.
considered a reasonable interval because of the known 2. Which statement is most correct about the patient’s
growth rate of these disorders. If the patient’s condition diagnosis?
is stable after that time interval, the period of time may a. This is a terminal disease that may end her life within
safely be increased to 6-month follow-ups for life. a year.
b. This is a chronic disorder that often requires treatment,
but she will probably live a relatively normal life.
c. This disease is rapidly fatal and does not respond to
treatment.
‘Case Study 3: d. This is a terminal disease, but the numbers of abnor-
mal cells in the bone marrow suggest that, with
Waldenstrom’s treatment, she may live another 4 or 5 years.
3. If the patient developed more severe neurologic prob-
Macroglobulinemia lems, what could be done to rapidly lower the IgM in
the blood?
A 59-year-old woman presented with a 35-pound uninten-
tional weight loss oyer the past 18 months. She had been ANSWERS
having trouble concentrating at work, had decreased energy,
|. b. Hyperviscosity syndrome is common with Waldenstrém’s
and had two frightening episodes during which she could
macroglobulinemia, and these are classic symptoms. The
not make sense of the words she was reading in a novel.
plasmacytoid lymphocytes may infiltrate nerves, meninges,
On physical examination, she was noted to be a tall, thin
and the brain and may be a less common cause of mental
woman with no enlarged lymph nodes. Her spleen was
status changes and neurologic changes. Hypercalcemia is
enlarged, and easily felt approximately 7 cm below her left
uncommon with Waldenstr6m’s macroglobulinemia, and
ribs. Her neurologic examination was normal. Blood tests
correcting the albumin to 4 g/dL (1.3 g/dL increase) would
revealed a Hgb of 9.7 g/dL, hematocrit (Hct) of 29.2%, and
result in a correction of serum calcium level of 0.8 <
MCV of 89 fL. Platelet count, white cell count, and white
1.3 = 1.04 mg/dL, totaling 9.34 mg/dL. This is within the
cell differential were normal. A chemistry panel was normal
normal range. Chilling of the scalp would not result in pre-
with the exception of a total protein level of 9.7 g/dL and a
cipitation of cryoglobulins in the brain, but might affect
low albumin of 2.7 g/dL. The calcium level was 8.3 mg/dL.
the tips of the ears and nose.
A serum protein electrophoresis was ordered and revealed a
2. d. The prognosis of patients with Waldenstrém’s
5.7 g/dL M-spike.
macroglobulinemia is associated with extent of plasma-
Immunoelectrophoresis identified the protein as an IgM-k.
cytoid lymphocytes in the bone marrow. This patient
The bone marrow was hypercellular with 23% plasmacytoid
may live for 3 to 4 years after diagnosis.
lymphocytes consistent with Waldenstr6m’s macroglobu-
3. Plasmapheresis is useful in removing IgM-rich plasma
linemia. Blood viscosity was measured and found to be
from the patient within | to 2 hours and may result in
moderately increased.
dramatic resolution of symptoms.
continued
= P19) Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

f). esncone
r Which laboratory test(s) provide the most important prog- a. IgM M-spike and hyperviscosity
nostic information in multiple myeloma? b. Lytic:bone lesions and rouleaux
a. Hemoglobin c. Renal failure and more than 30% plasma cells
b. Albumin d. M-spike of IgM, IgG, or IgA; low-normal levels of
c. B.-microglobulin other immunoglobulins; inability to make light chains
d. All of the above 4. What does staging refer to?
bo. Which list would contain diagnostic criteria for multiple a. Bone marrow compatibility
myeloma? b. Estimate of disease severity
a. Biopsy-proven plasmacytoma and 10% to 30% plasma c. Chemotherapy regimen
cells d. Determination of specific immunoglobulin in heavy-
b. More than 30% plasma cells in bone marrow chain disease
c. Biopsy-proven plasmacytoma and M-spike present
d. Monoclonal protein and M-spike present See answers at the back of this book.
. Which of the following would be diagnostic criteria for
Waldenstr6m’s macroglobulinemia?

”) SUMMARY CHAR biopsy examination, chemistry panel, beta, microglobulin

(B.m), and C-reactive protein (CRP). Diagnostic proce-
dures include radiography and magnetic resonance imag-
a Multiple myeloma is a disorder characterized by:
ing (MRI).
¢ Proliferation of abnormal plasma cells in the bone mar-
row. w Staging in multiple myeloma provides an estimated extent
¢ Plasmacytoma, a tumor of plasma cells. of disease to help determine the appropriate treatment
¢ Over production of identical antibodies in the serum is plan and patient prognosis.
referred to as monoclonal gammopathy m Chemotherapy is the primary treatment in multiple
* Bence-Jones proteins are light chains secreted in the myeloma.
urine which precipitates at 56°C.
# Monoclonal gammopathy of undetermined significance
¢ Patients present with bone destruction, hypercalcemia,
(MGUS) is evident when a patient has a small M spike,
kidney failure and hyperviscosity as well as pancytope-
urine light chains of less than | g per 24 hours, no lytic
nia, causing anemia, bleeding diathesis, and increased
bone lesions, and less than 10% plasma cells in the bone
susceptibility to infection.
marrow and no end organ damage from the monoclonal
mAmyloidosis occurs in multiple myeloma when protein overproduction.
immunoglobulin kappa («) or lambda (A) light chains set-
m Waldenstr6m’s macroglobulinemia is the overproduction
tle or deposit in organs such as the heart, kidneys, nerves,
of monoclonal IgM antibodies by cells called plasmacy-
liver, spleen, and gastrointestinal tract.
toid lymphocytes or prolymphs.
w IL-6 and osteoclast-activating factor (OAF) play major
m Manifestations associated with Waldenstrém’s macroglob-
roles in the production of the lytic bone lesions associated
ulinemia include hyperviscosity syndrome, increased
with multiple myeloma by stimulating the development of
levels of IgM, fatigue, cryoglobulinemic purpura, and
osteoclasts and subsequent release of calcium from bone,
bleeding diathesis.
thereby weakening the bone structure.
@ Standard treatment in Waldenstrém’s macroglobulinemia
g Laboratory evaluation in multiple myeloma includes elec-
includes chemotherapy and plasmapheresis to remove
trophoresis, immunofixation, quantitative immunoglobu-
increased levels of IgM.
lin assay, complete blood count (CBC), peripheral blood
smear, erythrocyte sedimentation rate (ESR), bone marrow
 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders 523

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524 Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders

7
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—

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 Siatelelcag :

Lipid (Lysosomal)
Storage Diseases and
Histiocytosis
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
Catherine M. Spier, MD

Lipid (Lysosomal) Storage OBJECTIVES

Diseases
Gaucher's Disease At the end of this chapter, the learner should be able to:

Niemann-Pick Disease 1. Name the enzyme deficiency seen in Gaucher’s disease.

Tay-Sachs Disease
Mucopolysaccharidoses . List characteristics for type I, type H, and type HI Gaucher’s disease.

Histiocytosis . Describe the appearance of Gaucher’s cells.

Sea-Blue Histiocyte . Name the enzyme deficiency seen in Niemann—Pick disease.
Syndrome :
Other Histiocytic Disorders . List clinical features of Niemann—Pick disease.
(Eosinophilic Granuloma, . Name the enzyme deficiency seen in Tay—Sachs disease.
Hand-Schuller—Christian
Disease, Letterer—Siwe . List clinical features of Tay-Sachs disease.
Disease) . Describe clinical features of Hurler’s syndrome, Hunter’s syndrome, and other
ahs
Go
yi
JO
TION
=I
OOF

Case Study mucopolysaccharidoses.

9. List laboratory findings in mucopolysaccharidosis disorders.

10. Describe the characteristic cell of sea-blue histiocyte syndrome.

Lipid (Lysosomal) Storage Diseases Lipid storage diseases have a wide clinical expression,
ranging from essentially asymptomatic to severe and inca-
The lipid storage diseases are rare, autosomally inherited disor- pacitating with early death. The aim of control in these
ders. They are known as lysosomal storage diseases because disorders has been directed at prenatal detection. The only
there is subcellular accumulation of unmetabolized material in currently effective practical therapy is enzyme replacement,
the lysosomes of various cells. Lipid or lysosomal storage dis- which has initiated a new age of treatment for genetic dis-
eases are caused by various enzyme defects (inborn errors) in orders and has improved the lives of many patients. The
lipid metabolism linked to an enzyme deficiency (Fig. 23-1). greatest controversy regarding enzyme replacement ther-
Although many different types of lipid storage disorders have apy, however, is the optimum amount and frequency of
been documented, the most widely known and well established treatment.' In addition, allogeneic bone marrow transplan-
include Gaucher’s, Niemann—Pick, and Tay—Sachs diseases tation has been used to treat some patients and can be con-
and mucopolysaccharidoses (Table 23-1). Although all ethnic sidered curative.* However, bone marrow transplantation is
groups are known to be affected by lipid storage diseases, an extremely aggressive, expensive, and high-risk therapy
there is an increased incidence of selected disorders such as considering that these conditions now have an effective and
Gaucher’s and Tay-Sachs diseases in certain ethnic groups, practical treatment in enzyme replacement therapy. The
most notably Ashkenazi Jews (Jews who trace their origin to general characteristics of lipid storage diseases are summa-
the Baltic Sea region). rized in Table 23-2.
Bos)
526 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

GLOBOSIDE
Ceramide

Fatty acid-Sphingosine ——- Glucose —— Galactose —— Galactose —— N-Acetylgalactosamine

Hexosaminidase A & B
(Deficiency — Sandhoff’s Disease)
a-Galactosidase
(Deficiency — Fabry’s Disease)
B-Galactosidase
(Deficiency — Lactosy! ceramidosis)
B-Glucosidase
(Deficiency — Gaucher’s Disease)

GANGLIOSIDE
Ceramide N-Acetylneuraminic acid

Fatty acid-Sphingosine —- Glucose —— Galactose —— N-Acetylgalactosamine —— Galactose

TB-Galactosidase
(Deficiency >
Generalized gangliosidosis)

Hexosaminidase A
(Deficiency — Tay-Sachs)
B-Galactosidase
(Deficiency > Lactosy! ceramidosis)
B-Glucosidase
(Deficiency — Gaucher's Disease)

Figure 25-1 ™ Schematic structure of globoside and ganglioside to show site of action of the several catabolic enzymes, which, when defec-
tive, result in one of the storage diseases. (From Wintrobe, MM, et al: Clinical Hematology, ed 8. Lea & Febiger, Philadelphia, 1981, p 1341,
with permission.)

Gaucher’s Disease
Table 23-1 Lipid Storage Diseases
HISTORICAL PERSPECTIVES
Gaucher’s disease This disorder was first described in 1882 by Philippe C.
Niemann—Pick disease
Gaucher in a 32-year-old woman with an enlarged spleen.
Tay-Sachs disease
Mucopolysaccharidoses Gaucher believed that the abnormal cells found in her spleen
at autopsy were part of a primary splenic tumor. This abnormal
cell, later named Gaucher's cell, is the result of the deficiency
of the enzyme beta ()-glucocerebrosidase, which leads to an
accumulation of unmetabolized substrate glucocerebroside in
cells, predominantly the monocyte-macrophage system (the
“4 Table 23-2 General Characteristics | reticuloendothelial system; Fig. 23-2). Gaucher’s observa-
. of Lipid Storage | tions were studied further, and the entity known as Gaucher’s
Diseases disease was defined and characterized as a familial disorder at
the turn of the last century.* In 1920, another variation or type
¢ Rare, inherited autosomal-recessive disorders } of Gaucher’s disease that is characterized by neurologic
¢ Also known as lysosomal storage diseases because of involvement was first described. It was after this date that
accumulation of unmetabolized material in lysosomes
Gaucher’s disease was classified as a lysosomal storage disor-
* Caused by enzyme deficiencies in lipid metabolism
* Increased incidence of some lipid storage diseases in der resulting from an enzyme deficiency with an autosomal-
certain ethnic groups (i.e., Gaucher’s disease in Ashkenazi recessive inheritance pattern. Although Gaucher’s disease is
Jews) the most frequent lysosomal storage disease, it was not until
* Great variation in clinical expression (i.e., asymptomatic to | 1965 that the actual enzyme deficiency was identified as glu-
severe with early death) cocerebrosidase. In 1984 the gene for glucocerebrosidase was
¢ Effective therapy: enzyme replacement
cloned, and in 1991 an important breakthrough occurred with
* Most well-known and characterized: Gaucher’s disease,
Niemann-—Pick disease, Tay—Sachs disease, and the initiation of clinical trials for enzyme replacement therapy
mucopolysaccharidoses at the National Institutes of Health (NIH).? Gaucher’s disease
became the first enzyme-deficiency disorder to be successfully
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis BPA

Hepatosplenomegaly
Gaucher’s cells in the bone marrow
Increase in serum acid phosphatase

Gaucher’s cells can be deposited in any bone in the body, which

leads to a predisposition for fractures, loss of bone, and avascu-
lar necrosis (particularly in the femoral head).
Bone involvement in Gaucher’s disease may present as
chronic bone pain or severe crises similar to those described in
Figure 25-2 = Gaucher's cell, bone marrow aspirate.
patients with sickle cell anemia.’ The severity of such episodes
of bone involvement are unpredictable. Skeletal involvement in
Gaucher’s disease includes a spectrum of findings radiograph-
treated with infusion of replacement enzyme.*” The chronol-
ically ranging from minimal bone loss and osteopenia to severe
ogy of Gaucher’s disease is summarized in Table 23-3.
evidence of bone destruction, including osteolytic and sclerotic
lesions.” The radiological classification of bone pathology is
CLASSIFICATION AND CLINICAL FEATURES
summarized in Table 23-6.° The osteolytic lesions seen on the
Gaucher’s disease has three clinically recognizable types: the
x-ray study of the knee of a patient with Gaucher’s disease is
adult or non-neuronopathic form (type I); the infantile, acute,
demonstrated in Figure 23-3.
or malignant neuronopathic form (type II); and the juvenile or
The underlying mechanisms of the bone complications
subacute neuronopathic form (type III). Gaucher’s types I, II,
of Gaucher’s disease are not well defined. It is assumed that
and III all have in common the triad of hepatosplenomegaly,
Gaucher’s cells infiltrate the medullary space and eventually
Gaucher’s cells in the bone marrow, and an increase in serum
replace trabecular bone and initiate a series of events that
acid phosphatase (Table 23-4).* The severity of the disease
lead to osteopenia, osteolytic lesions, and osteonecrosis.
and the patient’s age when the disease is first manifested are
Gaucher’s disease is clearly a multisystemic disorder charac-
related to the magnitude of the enzyme deficiency. The char-
terized not only by skeletal disease but also by organomegaly,
acteristics of each type of Gaucher’s disease are briefly sum-
hematologic complications, and occasionally pulmonary
marized in Table 23-5.
involvement.*
The accumulation of glucocerebrosides, the result of the
enzyme deficiency of B-glucocerebrosidase, produces the dis-
TYPE I: ADULT GAUCHER’S DISEASE (CHRONIC NON-
tinctive Gaucher’s cells (see Fig. 23-2). These cells are histio- NEURONOPATHIC) Type I non-neuronopathic Gaucher’s
cytes that are 20 to 100 um in diameter with a displaced disease is the most common type of this disorder and the most
nucleus. One or more round to oval nuclei are present in each
common of the lipidoses. It is the most frequently inherited
cell. The cytoplasm is faintly blue with white stain and has a
disorder in the Ashkenazi Jewish population. There is a
“crumpled tissue paper” or finely folded appearance, possibly a remarkable degree of variability in the clinical signs and
result of glycolipid deposition. The presence of Gaucher’s cells symptoms. Some patients with type I Gaucher’s disease may
alone is not pathognomonic for Gaucher’s disease, because display anemia, thrombocytopenia, massively enlarged livers
these cells are also found in other lymphoproliferative disorders.
and spleens, and extensive skeletal disease. In contrast, other
type I Gaucher’s disease patients have no symptoms at all,
and the disorder is identified in their adult years only during
the screening or evaluation for other diseases. The average
age of onset is between 30 and 40 years. In most cases of a
severe disorder, the diagnosis is made in childhood or early
¢ 1882: First described by Philippe Gaucher adulthood. Approximately two-thirds of the patients with type
¢ Abnormal cell named Gaucher’s cell I Gaucher’s disease are of Ashkenazi Jewish descent.* The
¢ 1900: Gaucher’s disease defined as a familial disorder remaining one-third of patients with type I disease have a
¢ 1920: Another type of Gaucher’s disease described, charac- panethnic distribution.
terized by neurologic involvement
Three clinical presentations occur in the type I non-
¢ Gaucher’s disease classified as a lysosomal storage disor-
der inherited as autosomal-recessive neuronopathic form of Gaucher’s disease:
¢ 1965: Enzyme deficiency identified as glucocerebrosidase
1. The mild presentation, which represents from 10% to
¢ 1984: Gene for glucocerebrosidase cloned
¢ 1991: Enzyme replacement therapy in clinical trials 25% of patients who are essentially asymptomatic and live
without the need for any treatment or intervention
528 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

Table 23-5 Gaucher's Disease: Clinical Subtypes 2

Type I: Acute Type ILI: Subacute

Type I: Non-neuronopathic Neuronopathic Neuronopathic (Juvenile
Clinical Features _ duit coms (Infant Form) Form)

Clinical onset Childhood/adulthod™ infancy Childhood/juvenile

Estimated frequency 1/450-1/1,000 1/100,000 1/100,000
Hepatosplenomegaly + + =
Hematologic complications ar + +
secondary to hypersplenism
Skeletal deterioration ar ae

(bone crises/fractures)
Neurodegenerative course os +++ ++
Life expectancy 6-80+ years 2 years 20-40 years
Ethnic predilection Ashkenazi Jew Panethnic Swedish (Norrbottnian)

2. The moderate clinical presentation, in which the patient has than 40 genetic mutations have been described.' The muta-
hepatosplenomegaly, near-normal blood counts, and a nor- tions include both single insertional and point mutations as
mal physical appearance well as crossover mutations. All of the mutations that cause
3. The most severe form, in which patients present with mas- Gaucher’s disease have complex effects on the properties of
sive hepatosplenomegaly, significant thrombocytopenia, this enzyme.'' The normal enzyme, glucocerebrosidase (acid
anemia, and skeletal complications.'° 8-glucosidase), is a lysosomal enzyme responsible for the
degradation of the AMES SOS EMS molecule, preventing its
A few patients with the severe forms of the disease may
buildup in tissue cells.'* Protein synthesis of the normal
develop central nervous system damage, delayed sexual mat-
enzyme occurs in the endoplasmic reticulum, with transport
uration, severe wasting, and eventually die. It should be noted
to the Golgi apparatus for glycosylation and delivery to the
that clinical presentations can be misleading because some of
lysosomes of the cell. Mutations at the genetic level that code
these patients may still develop underlying skeletal complica-
for the production of this enzyme have direct effects on the
tions (Table 23-7). It is important, therefore, to perform a
catalytic activity, with decreases from 5- to 100-fold.'* In
baseline skeletal evaluation even in patients with a mild case
addition, enzyme stability and half-life activity (normal is
of the disease. The femoral head is the most common initial
60 hours) are also decreased for acid B-glucosidase as a result
bone site to be affected by the disease, and this area should be
of these mutations.'*
evaluated by magnetic resonance imaging (MRI) to assess for
avascular necrosis.”
The definitive diagnosis of Gaucher’s disease is made TYPE If: INFANTILE GAUCHER’S DISEASE (ACUTE OR
with an assay for the enzyme acid f-glucocerebrosidase MALIGNANT NEURONOPATHIC) Type If Gaucher’s disease
activity of the leukocytes. Bone marrow examination alone is is a much rarer form that occurs in infancy, and patients rarely
insufficient for confirming the diagnosis because Gaucher’s survive past the age of 2 years. Type II acute neuronopathic
cells may be present in other disorders. Gaucher’s disease is seen in all ethnic groups, although it is
The gene for the enzyme glucocerebrosidase is located uncommon in the Jewish population.” The frequency of type II
on chromosome 1q21—31. Since the characterization, cloning, disease is estimated at approximately | in 100,000.? The hall-
and sequencing of the glucocerebrosidase gene in 1984, more mark of type Il Gaucher’s disease is neurologic involvement,

Stage Bescon ®

il.Cie Coarse RabecnirAner of decreased bone density, Shieh may be localized

or diffuse
2. Medullary expansion Loss of normal concavity above femoral condyles
3. Localized destruction (osteolysis) Small erosions, well-defined or moth-eaten, cortex rarefied and endosteally
notched, ground-glass veiling
4. Ischemic necrosis of long bone, sclerosis, Patchy densities and erosions serpiginous sclerotic streaks, layered periostitis >
osteitis sequestra
5. Diffuse destruction, epiphyseal collapse, Flattening or irregular destruction of femoral heads with mixed lytic and
osteoarthrosis _sclerotic foci, larger “soap bubble” ty
Source: Wenserus: RJ ex een es
boneReus in ‘Guiche eee Gaicien Clin Persp 6: 12, 1998.
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis 529

TYPE Ul: JUVENILE GAUCHER’S DISEASE (SUBACUTE

NEURONOPATHIC) Type II Gaucher’s disease may be pre-
sent from early childhood to the teenage years and is charac-
terized by clinical and physical findings and survivals ranging
between those of type I and type II.* Type III has been noted,
especially in a group of children from northern Sweden, the
off-spring of several related intermarriages. The frequency of
type III Gaucher’s disease among some Swedes is approxi-
mately | in 100,000.* Neurologic involvement is also charac-
teristic of type II] Gaucher’s disease; however, the clinical
manifestations are much more heterogeneous than those
observed in type II.
Two distinct subtypes of type III Gaucher’s disease have
been described.'® The classic form, type Illa, usually presents
itself clinically between early childhood and midadult life.
Type IIIb Gaucher’s disease is characterized by a clinically
aggressive systemic disease with neurologic involvement
of isolated horizontal supranuclear gaze palsy as the major
sign.'’ Ocular motor disorder is the distinctive neurologic fea-
ture of type IIb Gaucher’s disease. Generally, in type II
Gaucher’s disease regardless of the subtype, the more severe
the neurologic disease, the shorter the survival. The clinical
characteristics of type IIIa and type IIIb Gaucher’s disease are
compared in Table 23-8.

LABORATORY DIAGNOSIS
Figure 25-3 ™ Anteroposterior radiograph of the knee shows
Peripheral blood, bone marrow, and spleen are the sites most
diffuse mottled increased density of the distal femur and proximal
tibia, characteristic of widespread bone infarction in Gaucher's frequently examined in patients with Gaucher’s disease. The
disease. The metaphyseal regions are broader than normal (arrow), peripheral blood nearly always demonstrates a moderate
resembling an Erlenmeyer flask deformity. (Courtesy of Charles S. normocytic, normochromic anemia with thrombocytopenia
Resnik, MD, Department of Diagnostic Radiology, University of because of the replacement of normal hematopoietic cells
Maryland Medical Center, Baltimore, MD.)
with Gaucher’s cells in the bone marrow. There is pooling of
blood in the enlarged spleen and some degree of ineffective
erythropoiesis, with decreased incorporation of iron in
including multiple signs such as difficulty swallowing, erythroid precursors in the bone marrow. As a result, active
opisthotonos (extreme arching of the spine), and other mani- signs of a compensated anemia such as polychromasia and
festations of brain stem involvement that are noted early in nucleated red blood cells are usually absent on the peripheral
infancy.'> The infant has difficulty in feeding and fails to grow. smear.'* Leukocytes are commonly decreased in number.
Death usually occurs before the age of 2 years. Familial inter- Platelets are also usually decreased in number as a result
marriage is frequently found in the infant’s family history. of splenic sequestration. These patients may have a bleeding
The clinical presentation of this type II disease is much tendency, with nosebleeds especially common. Gaucher’s
more uniform than that observed in type I Gaucher’s dis- cells are noted only rarely in the peripheral blood. Bone mar-
ease and is very severe. The disease exhibits a progressive row aspirates are often the first tissue in which Gaucher’s
pattern of clinical symptoms, with hepatosplenomegaly cells are detected; these cells are required for diagnosis (see
evident within the first 6 months of life, and is often discov- Fig. 23-2). They are histiocytes, 20 to 100 um in diameter,
ered by 3 months of age. The principle cause of death in found in moderate numbers and as clumps of cells in the
infants with type II Gaucher’s disease is brain stem thickest areas of the smear. One or more round to oval nuclei
damage. are present in each cell. The cytoplasm is faintly blue with

‘table 23-7 Clinical Presentations of Type | Gaucher's Disease

Mild Moderate Severe

10%-25% of patients Hepatosplenomegaly Massive hepatosplenomegaly

Asymptomatic Near-normal blood counts Significant thrombocytopenia
No need for treatment Normal physical appearance Anemia, skeletal complications
 Type Ila

Clinical onset Early childhood to mid-adult Infancy to early childhood

Clinical course Mild to moderately severe Aggressive systemic disease
Neurologic involvement At onset; multifocal rapid jerky movements, Ocular motor disorder, horizontal
ataxia, spasticity, dementia, and seizures supranuclear gaze palsy; mild
cognitive impairment

Wright’s stain and has a “crumbled tissue paper” or finely The excess is therefore stored in histiocytes, with their end
folded appearance, possibly as a result of glycolipid deposi- morphological expression identical to that of true Gaucher’s
tion. Electron microscopy has demonstrated that this appear- cells. This phenomenon is also seen in a variety of other
ance is the result of lamellar bodies stacked inside secondary disorders, including acute myelocytic leukemia, chronic lym-
phagolysosomes. These cells stain positive with periodic phocytic leukemia, plasma cell myeloma, aplastic anemia,
acid-Schiff (PAS), acid phosphatase, Giemsa, iron, Sudan idiopathic thrombocytopenic purpura, thalassemia major, and
black B, and oil red O stains because of the accumulation of rheumatoid arthritis'? (Table 23-10). The presence of Gaucher-
the unmetabolized glucocerebroside. It is important to note like cells in patients with these diseases has no known prognos-
that the presence of Gaucher’s cells is not pathognomonic for tic significance. It should be emphasized that in each of these
Gaucher’s disease, because these cells are also found in other diseases there is no deficiency of the B-glucocerebrosidase, as
lymphoproliferative disorders. there is in Gaucher’s disease, but rather an overtaxation of a
The spleen is variably enlarged, owing to the accumula- normal system.
tion of masses of Gaucher’s cells. The enlargement is com-
monly up to 10 times normal splenic weight and can cause PROGNOSIS
considerable discomfort to the patient. Other organs and sys- As previously stated, the length of survival in patients with
tems commonly affected include the liver and, in type II, the Gaucher’s disease is variable and depends on the type. The
nervous system, pituitary gland, kidneys, lung, and ovaries. adult form (type I) has the longest survival, with patients sur-
These organs contain massive deposits of Gaucher’s cells. viving commonly into adulthood. In the infantile form (type ID),
The serum acid phosphatase level is increased, and survival beyond 2 years of age is rare. Like the clinical fea-
isozyme measurement of this enzyme has shown that the tures, survival in the juvenile form (type II) is intermediate
tartrate-resistant fraction is what is increased in patients with between the first two, and patients usually live into adoles-
Gaucher’s disease. The laboratory findings in Gaucher’s dis- cence. A relatively increased risk of cancer in patients with
ease are summarized in Table 23-9. Gaucher’s disease has been reported,*? primarily because of
Although the Gaucher’s cell is associated with the an increased incidence of hematologic malignancy. Patients
disease, so-called pseudo-Gaucher’s cells have also been with Gaucher’s disease are 15 times more likely to develop a
described. They are seen in disease states with increased cel- hematologic malignancy than a healthy individual. The most
lular turnover, especially chronic myelogenous leukemia, in frequently reported hematologic malignancies are listed in
which the phenomenon was first described. In theory, the Table 23-11. This increased risk of malignancy may be asso-
increased cell turnover presents so much glycosylceramide to ciated with immunologic abnormalities found in patients
the reticuloendothelial system that its enzyme system is over- with Gaucher’s disease. These abnormalities include increased
whelmed and cannot adequately metabolize all of the material. helper-to-suppressor (T,:T;) cell ratio, decreased natural killer

Normocytic, normochromic or normocytic, hypochromic

anemia
Leukopenia
Thrombocytopenia
Gaucher’s cells in bone marrow aspirate Acute myelocytic leukemia (AML)
Increased serum acid phosphatase Chronic lymphocytic leukemia (CLL)
Positive staining of Gaucher’s cells in the bone marrow Plasma cell myeloma
with PAS, acid phosphatase, Giemsa, iron, Sudan black B, Aplastic anemia
and oil red O stains Idiopathic thrombocytopenic purpura (ITP)
Thalassemia major
PAS = periodic acid—Schiff. Rheumatoid arthritis
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis 531

Niemann-Pick Disease
This inherited form of lipid storage disease was first described
in 1914 by Niemann and subsequently by Pick in 1933.?’
Niemann-Pick disease is caused by a deficiency of the
enzyme sphingomyelinase, with a secondary accumulation of
the unmetabolized lipid sphingomyelin as well as cholesterol.
Chronic lymphocytic leukemia (CLL) Sphingomyelin is a sphingophospholipid that is a common
Hodgkin’s disease and non-Hodgkin’s lymphoma
constituent of cell membranes as well as cellular organelles.
Acute leukaemia
As a result, a deficiency of sphingomyelinase is a serious dis-
order. The general characteristics of Niemann—Pick disease
are summarized in Table 23-13.
(null) cells, polyclonal B-cell lymphocytosis, and plasmacyto- A wide variety of clinical manifestations of variable sever-
sis.°° The immunologic abnormalities described in Gaucher’s ity have been reported in patients with Niemann-Pick disease.
disease are listed in Table 23-12. These include growth retardation, hepatosplenomegaly, lym-
phadenopathy, pigmentation, and impaired neurologic functions
TREATMENT (Table 23—14).** A large number of lipid-laden giant foam cells
Before the advent of the newer treatment modality of known as Niemann—Pick cells can be found in affected tissues
enzyme replacement therapy, Gaucher’s disease was tradi- and organs (Fig. 23-4). The detection of Niemann—Pick cells in
tionally managed by supportive therapy. Total or partial patients with this disorder is essential for the diagnosis of this
splenectomy was frequently performed. In addition, trans- disease. There is an increased incidence of Niemann—Pick dis-
fusions, orthopedic procedures, and occasionally bone mar- ease in the Jewish population, especially in consanguineous
row transplantation were used in some patients. Although groups. Because of the very different clinical manifestations of
potentially curative, allogeneic bone marrow transplanta- the disease, five types, A through E, have been described.” Only
tion is an extremely aggressive and high-risk therapy. In types A, B, and C are discussed here. Type E, which is very
1991, a major advancement occurred in the treatment of rare, has been found only in adults and is characterized by a
Gaucher’s disease type I. The U.S. Food and Drug Admin- mild chronic course and a lack of neurologic manifestations.
istration approved the use of enzyme replacement therapy Types A, B, and C of Niemann—Pick disease are compared in
for this disorder. Gaucher’s disease is the first lysosomal Table 23-15.
storage disorder for which enzyme replacement therapy is avail-
able. Enzyme replacement therapy has successfully reversed CLASSIFICATION AND CLINICAL FEATURES
many of the clinical complications of this disorder, including
correcting blood counts and reducing the organomegaly that TYPE A This form is also known as infantile or classic
occurs in these patients.?!*° Niemann—Pick disease. \t is the most common form, account-
The first enzyme replacement therapy utilized was a puri- ing for up to 85% of all cases of Niemann—Pick disease. The
fied enzyme from human placenta. This enzyme, which is an onset is early in infancy and is associated with failure to
alglucerase injection, is manufactured by Genzyme Corporation thrive, difficulty feeding, and retarded physical and mental
as Ceredase and has demonstrated effectiveness by the reversal development. The skin has a waxy consistency. There 1s often
of signs and symptoms of Gaucher type I, non-neuronopathic jaundice at birth and, usually, hepatosplenomegaly with a dis-
disease.* Another form of the enzyme, the recombinant form, tended abdomen. The lymph nodes are enlarged as well.
which is also produced by Genzyme Corporation as Cerezyme, A cherry-red spot in the macula of the eye 1s found in approx-
is genetically engineered and has the advantage of being unlim- imately 50% of the affected infants. The neurologic symp-
ited in supply. In addition, the recombinant Cerezyme has the toms are more pronounced in this type of Niemann—Pick
advantage of a very low risk of transmitting any infectious agent disease than in any of the other types. Deterioration is rapid,
and also has a lower rate of patients developing IgG antibodies and survival past the age of | or 2 years is rare.
to the glucocerebrosidase enzyme.”

Inherited lipid storage disease

Caused by a deficiency of the enzyme sphingomyelinase
Niemann—Pick cells (lipid-laden giant foam cells) found in
Increased helper-to-suppressor (T,:T,) cell ratio bone marrow aspirate, tissues, and organs
Decreased natural killer (null) cells Increased incidence in the Jewish population
Polyclonal B-cell lymphocytosis Five types: A to E have been described
Plasmacytosis Wide variety of clinical manifestations
532. Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

type is characterized by a milder defect in sphingomyelinase

activity and an abnormality in cholesterol transport reflected
in an alteration in cholesterol esterification from exogenous
cholesterol.
Type C Niemann-Pick disease is an autosomal-recessive
disease characterized by a gradual and ill-defined onset of
neurologic abnormalities, which include unsteady gait, poor
motor coordination, slurred speech, dysphagia, and ophthal-
moplegia (paralysis of the eye muscles).” When neurologic
symptoms appear, psychotic manifestations may also be
predominant. Neuro-ophthalmologic findings, in which the
oculomotor system is often affected, are also characteristic.
In addition, seizures often appear with neurologic involve-
ment, with grand mal seizures most frequently observed.
Frequent seizures may contribute to the mental deterioration
Figure 25-4 ® Niemann-Pick cell, bone marrow aspirate. observed in patients with type C Niemann—Pick disease.
Hepatosplenomegaly is a constant finding in the infantile or
juvenile form, whereas hepatomegaly is often absent in the
TYPE B Also called the chronic or adult form, type B adult form. Splenomegaly, however, is present in the adult
Niemann—Pick disease is much more rare than type A, with form, with thrombocytopenia being a sign of hypersplenism.
approximately 20 reported cases in the literature. Clinical onset The characteristic foam cell, Niemann—Pick cell, or sea-blue
consisting of hepatosplenomegaly usually occurs in infancy, histiocytes, or a combination, are a consistent finding in the
but the central nervous system is not involved. Individuals with bone marrow of patients with type C Niemann-—Pick disease.
this type of disease may live longer than those with type A, but Diagnosis of type C Niemann—Pick disease is made with the
they do not survive beyond childhood or early adolescence. finding of the characteristic abnormality in cholesterol trans-
port and esterification from exogenous cholesterol.
TYPE C Type C Niemann-Pick disease has been described in
two forms, an infantile or juvenile form with a prolonged life LABORATORY DIAGNOSIS
span, and an adolescent or adulthood form, which is generally There is a distinct pattern to the histiocytes in Niemann—Pick
slower in evolution and progression.*” The primary defect in disease. These cells are most commonly seen in bone marrow
type C Niemann-—Pick disease is still unknown. However, this and spleen, although they accumulate throughout the body and
in the nervous system (patients with type A disease). They are
large cells, 20 to 90 um in diameter, with an inconspicuous
‘linical Manifestations nucleus. The cytoplasm is filled with and distended by round,
Pick Mise ae
uniformly sized droplets of accumulated lipid, turning the cell
a very pale or light blue when Wright-stained (see Fig. 23-4).
Stains producing a positive reaction with Niemann—Pick cells
Growth retardation are the lipid stains oil red O, Sudan black, and Luxol fast blue;
Hepatosplenomegaly
Lymphadenopathy and acid phosphatase and nonspecific esterase. The PAS stain-
Pigmentation ing is weak, and the myeloperoxidase stain is negative.
Neurologic impairment The bone marrow of some adult patients with certain
varieties of Niemann—Pick disease contains a mixture of

se 25-15Comparison ofTyTypespes A,B, and C Niel

Type C, Two Forms: Infantile
Type A, Infantile Type B, Chronic or Juvenile Form and
Classification or Classic or Adult Adolescent or Adult Form

Incidence Accounts for 85% of all cases Rare Rare

Clinical manifestations Jaundice at birth, Hepatosplenomegaly Hepatosplenomegaly in
hepatosplenomegaly, enlarged infantile form, splenomegaly
lymph nodes, cherry-red spot in the adult form, neurologic
in macula of the eye, neurologic abnormalities, neuro-
symptoms, retarded physical and ophthalmologic findings,
mental development seizures
Survival 1-2 years Childhood or early Juvenile or adulthood
adolescence
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis NnWwWwW

Niemann-Pick cells and sea-blue histiocytes (histiocytes dis- deficiency of the enzyme hexosaminidase A, with an increase
tended with blue-staining ceroid on Wright’s stain). It is of the other isoenzyme, hexosaminidase B. The gene for
believed that the sphingomyelin is gradually metabolized to hexosaminidase A is located on chromosome 15.** Inheritance
ceroid, thus generating the sea-blue histiocytes. A marrow of two abnormal alleles (one from each parent) accounts for
specimen with these findings would then need to be distin- almost all infantile Tay-Sachs cases in the Ashkenazi-Jewish
guished from the entity of sea-blue histiocytosis (see the sec- population. The severity of the disease correlates with the
tion at the end of this chapter). level of residual enzyme activity. The unmetabolized GM,
Other disorders that may cause Niemann—Pick-like cells ganglioside accumulates in almost all tissues and has its most
in the bone marrow are GM, gangliosidosis, lactosyl cerami- devastating effects within the central nervous system and
dosis, and Fabry’s disease. eye. The general characteristics of Tay-Sachs disease are
The peripheral blood is most remarkable for the vacuoles summarized in Table 23—16.*°
that may be found in lymphocytes and monocytes of a routine
peripheral blood smear (Fig. 23-5). These vacuoles are round, CLINICAL FEATURES
and from 2 to 20 may be found within one cell. Anemia and Although affected infants appear normal at birth, by 6 months
leukopenia may be present but do not usually present any of age both physical and mental deterioration are notable.
threat to the patient. Serum lipids are not usually increased. They have an exaggerated physical response to noise (the
An assay of the enzyme sphingomyelinase activity in leuko- startle reflex) beginning at age 3 to 5 months. There is a pro-
cytes and fibroblasts can also be performed. gressive loss of motor function with weakness, decreased
attentiveness to surroundings, hypotonia (diminished tone of
PROGNOSIS AND TREATMENT skeletal muscles), and poor head control between the ages
There may be a slightly longer survival in patients with the other of 6 and 10 months.” In addition, a cherry-red spot in the
types of Niemann-Pick disease, but those with type A have a macula of each eye is found; this is the most characteristic
very short life expectancy. Survival past the age of 2 years is feature of Tay-Sachs disease. The central nervous system
uncommon. Currently there is no treatment for Niemann—Pick steadily degenerates after 1 year of age. Along with the con-
disease. However, successful allogeneic bone marrow trans- tinual deterioration, there is enlargement of the head (macro-
plants have been reported for type B.'* In addition, research cephaly), seizures, and paralysis. Spasticity with hyperactive
studies have focused on finding a source of enzyme replacement reflexes, deafness, and blindness follow. The neurons are
for sphingomyelinase*' and gene therapy using retroviruses.” greatly enlarged by accumulation of the unmetabolized
ganglioside in vacuoles in the cytoplasm. In contrast to many
other lipid storage diseases, the spleen, liver, and lymph
Tay-Sachs Disease nodes are not enlarged. Feeding is poor, and death occurs by
4 years of age. It should be noted that cherry-red spots are not
Also known as GM, gangliosidosis, Tay—Sachs disease was first
pathognomonic for Tay—Sachs disease; however, this clinical
described in 1881 by the British ophthalmologist Warren Tay.
finding in a Jewish infant with the absence of organomegaly
In 1886, the New York neurologist Bernard Sachs used the
is strongly suggestive of Tay—Sachs disease.*°
term familial amaurotic infantile idiocy to describe this disor-
der. Its incidence in the Ashkenazi Jewish population is
LABORATORY DIAGNOSIS
100 times greater than that in the non-Jewish population.” It
A deficiency of hexosaminidase A is the basic cause of this
is estimated that this high-risk group has a | in 30 carrier rate.
disease. Hexosaminidase A is the enzyme responsible for
This autosomal-recessive sphingolipidosis is the result of a
hydrolyzing GM, ganglioside, the glycolipid that accumulates
in neurons. This deficiency can be demonstrated in the serum,
plasma, leukocytes, and cultured fibroblasts of infants with
Tay-Sachs disease.

Known as GM, gangliosidosis

Autosomal-recessive inheritance
Caused by deficiency of hexosaminidase A
Higher incidence in Ashkenazi Jewish population
Central nervous system degeneration
Physical and mental deterioration
Cherry-red spot in the macula of each eye
Macrocephaly (enlargement of head)
Seizures and paralysis
Figure 23-5 ™ Tay-Sachs disease, vacuolated lymphocytes (also Death by 4 years old
characteristic of Niemann-Pick disease).
534 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

The major site of pathology is in the central nervous

system, and examination of other tissues is less informative.
The peripheral blood contains vacuolated lymphocytes (see
Fig. 23-5). The number and size of the vacuoles are related
to the duration of the disease. It is postulated, but not defi-
nitely proven, that they contain the unmetabolized lipid
GM, ganglioside. Vacuolated lymphocytes, however, are not
pathognomonic for Tay—Sachs disease, because they are also
seen in Niemann—Pick disease and in certain types of
leukemias. Foam cells, or vacuolated histiocytes, are found in
the bone marrow. The presence of these cells is helpful but
not diagnostic for the disease.
Because of the high frequency of this disease in certain
populations, prenatal detection has taken on greater impor-
tance. Culture of fetal fibroblasts from the amniotic fluid can be
undertaken to detect hexosaminidase A levels in the fetus. Mass Figure 23-6 ® Hurler’s anomaly.
screening programs of adults at possible risk for transmitting
the disease have been undertaken, with variable success.
constellation of abnormalities now recognized as Hunter’s syn-
drome. Table 23-17 summarizes the general characteristics
PROGNOSIS AND TREATMENT
of MPSs.
The infantile form of Tay—Sachs disease is uniformly fatal
before age 4. Enzyme replacement is now being attempted, CLASSIFICATION
and theefinal results of the potential therapy are not yet The MPSs have been arranged into seven categories, but there
known. Patients with the juvenile and adult forms of are only four possible unmetabolized products that build up in
Tay-Sachs disease have longer survival than those with the tissues: keratin sulfate dermatan sulfate, heparin sulfate, and
infantile form, although it is quite variable. Supportive treat- chondroitin sulfate. Table 23-18 gives an abbreviated classi-
ment remains the mainstay of this disorder, which includes fication scheme for MPSs.** With the exception of Hunter’s
management of hydration, recurrent infections, and seizures syndrome, which is X-linked recessive, these disorders have
with conventional fluids, antibiotics, and drugs, respec- an autosomal-recessive mode of inheritance. There does not
tively. Prenatal diagnosis can be performed by measuring appear to be a significant increase of affected individuals
hexosaminidase A in amniotic fluid, cultured amniocytes, or within any one ethnic group.
chorionic villus samples. In terms of the biochemical classification of MPS, very
different clinical phenotypes can result from different muta-
tions at the same locus. Characterization of these mutations
Mucopolysaccharidoses showed deletions for MPS I, II, and III, and a point mutation
for MPS IV.*’
The mucopolysaccharidoses (MPSs) are rare disorders that
For example, in MPS I H (Hurler’s disease) and in
constitute a group of lysosomal storage diseases caused by a
MPS IS (Scheie’s syndrome), mutations at the same locus of
deficiency in one of the enzymes involved in the breakdown
the enzyme alpha («)-iduronidase occur for both disorders, pro-
of mucopolysaccharides.**
ducing quite different clinical phenotypes.** In Hurler’s dis-
Similar to the previously described disorders, the MPSs
ease, there is a progressive mental and physical deterioration,
show accumulations of unmetabolized material within lyso-
with death occurring usually in the first decade of life. This is
somes (Fig. 23-6); however, it is mucopolysaccharides, not
in contrast to the course in Scheie’s disease, in which the con-
sphingolipids, that accumulate. Products are found in the
dition is milder, intellect is normal into adult life, and life
reticuloendothelial system (spleen, bone marrow, liver),
expectancy is normal. The presence of many different muta-
lymph nodes, blood vessels, brain, heart, connective tissue,
tions at the a-iduronidase enzyme locus that may be inherited
and urine. The clinical severity of these disorders varies
in either the homozygous or heterozygous state accounts for the
widely, with mild, intermediate, and severe forms. Multiple
wide variation in clinical phenotypes. The use of molecular
clinical presentations exist, including skeletal abnormalities,
probes to characterize these mutations will eventually allow
organomegaly, facial dysmorphism, and corneal opacities.
correlations to be made between mutations and phenotypes.
The original description of children affected with different
forms of the MPSs was published within a relatively short CLINICAL FEATURES
time span at the turn of the last century. In London in 1900, Many clinical abnormalities are found within each type of
Dr. John Thompson first described three young brothers with the MPS, which are outlined in Table 23-18. The findings in
characteristics of MPS. Gertrud Hurler elaborated on his Hurler’s syndrome are given in the most detail, because it is
description, describing two unrelated boys in Munich in 1919 considered the prototype of the MPSs.
with very similar characteristics, now known as Hurler’s In patients with Hurler’s syndrome (MPS 1), there may
syndrome. In 1917, Hunter described two brothers with a be a short period of apparently normal development, but this
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

Rare lysosomal storage disease

Autosomal-recessive inheritance
Deficiency of one of the enzymes involved in the breakdown of mucopolysaccharides
Panethnicity
Different clinical phenotypes resulting from different mutations at the same locus

Mode of Accumulated Enzyme Clinical Life

Category Inheritance Product Deficiency Features Expectancy
9 ne eoRR RSE RRersemsesorvmmfcemined

PS IH Autosomal- Heparan sulfate a-L-iduronidase Onset 6—8 months, 6-10 years

(Hurler’s) recessive Dermatan severe mental
sulfate retardation, dwarfism,
large long head, flat
broad nose with
upturned nostrils
(coarse facies), corneal
clouding, hepato-
splenomegaly, valvular
lesions, coronary artery
lesions, skeletal]
deformities, joint
stiffness
MPSIS Autosomal- Heparan sulfate a-L-iduronidase Onset after 5 years, Normal
(Scheie’s) recessive Dermatan normal intelligence,
sulfate stiff joints (especially
of the hands), near-
normal height, corneal
clouding, valvular
lesions, coronary artery
lesions
MPS I H-S Autosomal- Heparan sulfate a-L-iduronidase Onset infancy, mild 3rd decade
(Hurler- recessive Dermatan retardation (may be
Scheie) sulfate normal), dwarfism,
facial and bony
lesions of Hurler’s
syndrome, cardiac
lesions
MPS II X-linked Heparan sulfate Iduronate a-sulfatase Mild retardation to 2nd decade
(Hunter’s) recessive Dermatan normal intelligence, to normal
(wide range sulfate similar to Hurler’s
of severity) syndrome, but not
corneal clouding,
retinal degeneration,
deafness, nodular
skin infiltrates
MPS III Autosomal- Heparan sulfate Heparan N-sulfatase, Onset after 3 years, 2nd—3rd decade
recessive a-N-acetyl- mild to severe mental
glucosaminidase, retardation, normal
a-glucosaminide growth, Hurler’s
transferase facies, no corneal
N-acetylgluco- clouding, no heart
samine-6-sulfatase disease, no
hepatosplenomegaly,
mild skeletal changes
continued
536 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

table 25-18 Mucopolysaccharidoses (MPSs)—cont'd

Mode of Accumulated Enzyme Clinical Life
Inheritance Product Deficiency Features Expectancy. .
Category

(Sanfilippo’s A)
(Sanfilippo’s B)
(Sanfilippo’s C)
(Sanfilippo’s D)
(wide range of
severity)
MPS IVA Autosomal- Keratan sulfate N-Acetylgal- Normal intelligence, 3rd—6th decade
(Morquio’s) recessive Chondroitin actosamine, severe skeletal
(wide range sulfate 6-sulfate deformities,
of severity) sulfate dwarfism,
thoracolumbar
gibbus
8-Galactosidase Kyphoscoliosis, facies
similar to Hurler’s
syndrome, corneal
clouding, valvular
and coronary artery
lesions, joint
hypermobility,
genu yalgum
MPS IV B
(Morquio’s)
MPS VI Autosomal- Dermatan N-Acetylgalactosamine Similar to Hurler’s 2nd decade
(Maroteaux— recessive sulfate 4-sulfatase, syndrome, but
Lamy) (arylsulfatase B) normal intelligence,
longer survival
MPS VII Autosomal- Dermatan 8-Glucuronidase Variable from severe Variable,
(Glucuronidase recessive sulfate mental retardation 1-40 years
deficiency with dysostosis
disease) multiplex and
hepatosplenomegaly
to a milder form;
also severe neonatal
form with hydrops
fetalis

Source: Modified from Fensom, AH, and Benson PF: Recent advances in the prenatal diagnosis of the mucopolysaccharidoses.
Prenatal Diagn 14:1, 1994.

is only temporary. These individuals are abnormally short and common. Patients affected with Sanfilippo’s syndrome
have coarse facial features, with a broad, flat nose, widely (MPS III) have a more normal stature but unfortunately many
spaced eyes, and thickened tongue and lips.*® Some authors more severe neurologic problems and decreased survival.
have described their appearance as similar to that of a gar- Compared with patients with Hurler’s syndrome, those with
goyle (the carved heads sometimes found on older European Scheie’s syndrome (MPS I S) have more prominent corneal
churches). The amount of body hair is increased, dark, and clouding but less abnormality in stature, facial appearance,
especially prominent on the forehead. The skin is thickened. and mental development. Patients with Maroteaux—Lamy
Patients are mentally retarded. Clouding of the corneas of the syndrome (MPS VI) have growth and skeletal abnormalities
eyes 1s present. These individuals may have hearing loss or be but no mental retardation. In Morquio’s syndrome (MPS IV),
completely deaf. The heart is damaged, owing to the accumu- patients have numerous skeletal changes, giving a markedly
lation of mucopolysaccharides in the valves and blood abnormal physical appearance; however, there is no mental
vessels. There is a hump on the back and a prominent retardation.**
abdomen, with enlarged liver and spleen. The arms and legs
are abnormal, with contractures of many joints. In addition, LABORATORY DIAGNOSIS
the hands are very wide and the fingers shortened. An accurate enzymatic diagnosis should be established for
In Hunter’s syndrome (MPS II), the changes are similar, all suspected cases of MPS, as clinical diagnosis alone is
although not as severe. Corneal clouding is much less often impossible because of overlapping phenotypes. The
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis 5 oS)—

diagnosis of MPS can be made by performing simple striking blue color of the histiocytes after staining with
enzyme assays using leukocytes, serum, or fibroblasts.1° Wright’s or May-Griinwald-Giemsa stain gives the syndrome
The identification of heterozygotes, however, is still a dif- its name.
ficult process because of the overlays of normal and The mode of transmission has not been clearly estab-
heterozygous levels of enzyme activity. Molecular studies lished, but autosomal-recessive inheritance, with a variable
such as the cloning of complementary deoxyribonucleic degree of expression, appears most likely. Most patients
acids (cDNAs) can complement accurate enzyme assays.*” receive the diagnosis before they reach 40 years of age. The
Several specialized substrates used for the diagnosis of earlier in life the disease is found, the more severe it is
MPS are now available commercially. likely to be. Major findings on physical examination are
In contrast to findings in the other lysosomal storage splenomegaly and usually hepatomegaly. Also described, but
diseases, nonmetabolized products may be detected in the occurring less consistently, are abnormalities of the eyes,
urine of patients with MPS.*° The toluidine blue spot test or skin, and nervous system. Involvement of the lung may be
the turbidity test to detect acid mucopolysaccharides is the noted on radiographic examination. Involvement of the
initial screening test. The spot test may be unreliable, how- lymph nodes is not seen.
ever, with up to 32% false-negative test results in patients Significant laboratory findings are usually confined to
with Hurler’s syndrome reported. Also of note is that the the blood. In the peripheral blood, thrombocytopenia is found
urine of normal healthy newborn infants may give false- with great frequency. Consequently, clinical manifestations
positive results, a phenomenon that disappears by 2 weeks of such as epistaxis, gastrointestinal tract bleeding, and purpura
age. Any positive screening test result should be confirmed may be expected. However, there is no correlation of the
by lysosomal enzyme assays. degree of thrombocytopenia with the size of the spleen. Blood
An interesting but somewhat inconsistent finding in the lipid levels are normal. Abnormal liver function study results
peripheral blood of patients with MPS is the presence of large are only rarely seen.
granules in leukocytes, especially lymphocytes. These are The bone marrow aspirate is usually the site of diagnosis.
known as Alder-Reilly bodies (see Fig. 23-6). In polymor- Histiocytes of variable size (20 to 60 um) are present in
phonuclear leukocytes this needs to be distinguished from greatly increased numbers. They contain the blue-to-green
toxic granulation, but the large size of the granules in MPS staining granules that vary in size, shape, and ability to take
usually leaves little doubt. A metachromatic stain, such as up the stain (Fig. 23-7). Thus, not all cells will have the same
toluidine blue, aids in confirmation. These granules are found staining intensity. It is not currently known why the granules
with much greater regularity, however, in histiocytes and stain blue with these stains. The cells also react with the PAS,
lymphocytes in the bone marrow. Sudan black B, and acid-fast stains, but not with toluidine
blue or iron stains.”
PROGNOSIS Most patients with this syndrome do well and have
The prognosis of the MPSs varies somewhat with the type. normal life spans. Splenectomy is not always required;
Patients with Hurler’s syndrome may live only one decade, many patients never have the spleen removed. As previ-
whereas those affected with Hunter’s syndrome may live into ously mentioned, manifestations of the disease at an
their 20s.*’ The theoretical aid of enzyme replacement therapy early age may imply more severe symptoms. The general
has yet to be translated into practical results.

TREATMENT
Bone marrow transplantation has been used successfully
in treating some patients with MPS.*' More studies are
needed, however, to prove that bone marrow transplanta-
tion can be a practical treatment leading to a complete
cure. Prenatal diagnosis is still important, including first
trimester diagnosis by chorionic villus sampling, early
amniocentesis for more sensitive lysosomal enzyme assays,
use of DNA analysis for detecting mutations, and the possi-
bility of preimplantation diagnosis of early embryos after
in vitro fertilization.

Histiocytosis
Sea-Blue Histiocyte Syndrome Figure 25-7 ® Sea-blue histiocytes. Note the abnormally coarse
azurophilic granules present in neutrophils, lymphocytes, and
Although initially described in isolated case reports of young monocytes. (From Hyun, BH, et al: Bone marrow. In: Practical
adults with an enlarged spleen, the syndrome of the sea-blue Hematology. A Laboratory Guide with Accompanying Filmstrip.
histiocyte is a genetic disorder with a benign course. The WB Saunders, Philadelphia, 1975, with permission.)
538 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

characteristics of sea-blue histiocytosis are summarized in

Table 23-19. Case Study
Other Histiocytic Disorders (Eosinophilic A 32-year-old man visited his physician complaining of pain
in his forearms and fatigue. Physical examination revealed
Granuloma, Hand-Schiller—Christian
an enlarged spleen (2 cm) and multiple bruises down the
Disease, Letterer-Siwe Disease) patient’s forearms. His physician ordered the following
laboratory workup:
This group of “histiocytic” disorders represents an abnor-
mal proliferation and accumulation of mature histiocytes, RBC count a x10
or Langerhans’ cells. Langerhans’ cells are large but incon- WBC count 4.9 X 10°7/L
spicuous cells in the skin whose function is to process and Hemoglobin 11.0 g/dL
present antigen to other cells in the area, including lympho- Hematocrit 32%
cytes.2? These cells, as well as histiocytes, are normally MCV 92 fL
MCHC 31%
found in small numbers in the skin and reticuloendothelial
Reticulocytes 0.4%
system. Most patients with these disorders are either Differential:
children or young adults. A favorable outcome may be Segmented neutrophils 52%
expected in most cases, with the exception of Letterer—Siwe Lymphocytes 41%
disease, which may be fatal. These disorders may actually Monocytes 4%
Bands 3%
represent a continuum, from the unifocal and benign
eosinophilic granuloma to the generalized and sometimes
fatal Letterer-Siwe disease.?? The term histiocytosis X A bone marrow aspiration was performed at the left pos-
is generally used to describe these disorders. The major terior iliac crest and revealed a histiocytic-appearing cell
characteristics of histiocytic disorders are summarized in (see Fig. 23-2). These cells stained positive with PAS,
Table 23-20. Sudan black B, and Prussian blue.

QUESTIONS
1. This case history is representative of what lipid storage
disease?
2. What further testing must be done to confirm the
diagnosis?
3. Is “effective erythropoiesis” apparent in this case? Why
Autosomal-recessive, benign genetic disorder or why not?
Striking blue histiocytes with Wright’s stain 4. Classify the anemia according to the red blood cell
Splenomegaly and hepatomegaly (RBC) indices.
Thrombocytopenia 5. What treatment is available to this patient?

Disease Age at Onset Main Site(s) of Involvement Disease

Eosinophilic Children and young adults, Unifocal—skull, rib, femur most common Rare spontaneous healing;
granuloma especially males, often no most require surgical
symptoms until bone removal; occasional
fracture patients develop
recurrence later
Hand-—Schiiller— Usually <5 years old Multifocal—bones, skin, lymphoid 50%; spontaneous recovery
Christian tissue; triad of pituitary, eye, and skull 50%; recovery with
disease involvement is characteristic but chemotherapy
uncommon
Letterer-Siwe Usually <3 years old Generalized—skin, lymphoid tissue, Chemotherapy has
disease bones, +/— bone marrow; more severe improved prognosis,
and extensive than Hand—Schiiller— which was previously
Christian disease considered poor
 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis 539

| y
©. stions ne=
1, Whatdefect
isfound in lipid storage diseases? 2 6. What
are the characteristics of Niemann—Pick cells?
a. Subcellular accumulation of unmetabolized material iin a. Atypical lymphocytes with large vacuoles
lysosomes b. Cytoplasm filled with lipid droplets, inconspicuous
b. Cellular accumulation of metabolites in cytoplasm nucleus
c. Protein accumulation in cellular mitochondria c. Vacuolated histiocytes or foam cells
d. Abnormal sequestration of minerals and trace ele- d. Lymphocytes with Alder—Reilly bodies
ments in cellular nuclear organelles 5 . What is the enzyme deficiency seen in Tay-Sachs disease?
2. What is the enzyme deficiency seen in Gaucher’s disease? a. Sphingomyelinase
a. Sphingomyelinase b. Hexosaminidase A
b. Hexosaminidase A c. B-Glucocerebrosidase
c. B-Glucocerebrosidase d. 8-Galactosidase
Se eOaiae iyidise 8. What are the clinical features of Tay—Sachs disease?
3. Which description best characterizes type I Gaucher’s a. Waxy, jaundiced skin; retarded physical and mental
disease? development; cherry-red spot in macula of eye
a. Found in any ethnic group; multiple neurologic signs, b. Startle reflex; blindness; macrocephaly; no enlarge-
including difficulty in swallowing and manifestations ment of liver, spleen, or lymph nodes
involving brain stem; enlargement of liver and spleen c. Abnormal facial features; deafness; increased body
b. Found primarily in Ashkenazi Jews; enlargement of hair, mental retardation; heart damage; structural
liver and spleen; anemia thrombocytopenia deformities
c. Found in northern Sweden; neurologic disorders, bone d. Splenomegaly; hepatomegaly; eye, skin, nervous
disorders, skin pigment changes system, and lung abnormalities
d. Found in Mediterranean populations; hypermetabolic 9. Which cell is found in Tay-Sachs disease, but is not
manifestations;
risers fever, lethargy, poor musculature, considered diagnostic?
bone deformities
a. Atypical lymphocytes with large vacuoles
4. What are the characteristics of Gaucher’s cells? b. Cytoplasm filled with lipid droplets; inconspicuous
a. Atypical lymphocytes with foamy cytoplasm nucleus
b. Hypersegmented neutrophils with Auer’s rods c. Vacuolated histiocytes or foam cells
c. Large, multilobed monocytes with prominent red d. Lymphocytes with Alder—Reilly bodies
granules ' : 10. Which cell may be found in MPS disorders?
d. Histiocytes with blue, folded cytoplasm a. Large, foamy histiocytes with blue or green granules
5. What is the enzyme deficiency seen in Niemann—Pick b. Neutrophils with toxic granulation
disease? c. Neutrophils with Déhle bodies
a. Sphingomyelinase d. Lymphocytes with Alder—Reilly bodies
b. Hexosaminidase A
c. B-Glucocerebrosidase See answers at the back of this book.
_ d. B-Galactosidase

mw Gaucher’s disease has three clinically recognizable types:

~’) SUMMARY CH the adult or non-neuronopathic form (type I); the infantile,
acute, or malignant neuronopathic form (type II); and the
m Lipid storage diseases range from essentially asympto- juvenile or subacute neuronopathic form (type IID).
matic to severe and incapacitating, resulting in death. m Type I (chronic non-neuronopathic) adult Gaucher’s
mw Gaucher’s disease results from a deficiency of the enzyme disease is the most common type of Gaucher’s disease. It
B-glucocerebrosidase, which leads to an accumulation of occurs frequently as an inherited disorder in the Ashkenazi
unmetabolized substrate glucocerebroside in cells, pre- Jewish population. Clinical features include anemia,
dominantly the monocyte-macrophage system. This accu- thrombocytopenia, massively enlarged liver and spleen,
mulation of glucocerebrosides produces the distinctive and extensive skeletal disease.
Gaucher’s cells.
continued
540 Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis

of the unmetabolized lipid sphingomyelin as well as

cholesterol. Type A is also known as infantile or classic
Niemann-—Pick disease. Type B is also called the chronic or
mw Type II (acute or malignant neuronopathic) infantile adult form. Type C has been described in two forms: infan-
Gaucher’s disease occurs in infancy. Patients rarely sur- tile and juvenile.
vive past the age of 2 years. It is found in all ethnic m Tay-Sachs disease, also known as GM, gangliosidosis, is
groups, although it is uncommon in the Jewish popula- an autosomal-recessive sphingolipidosis that occurs as a
tion. The clinical presentation of type II disease is much result of deficiency of the enzyme hexosaminidase A,
more uniform than that of type I, and is very severe. Fea- with an increase in hexosaminidase B.
tures include difficulty swallowing, opisthotonos, and
other manifestations of brain stem involvement, which are = Mucopolysaccharidoses are rare disorders that constitute
noted in early infancy. a group of lysosomal storage diseases caused by a defi-
ciency in one of the enzymes involved in the breakdown
m Type III (subacute neuronopathic) juvenile Gaucher’s dis-
of mucopolysaccharides,
ease may be present from early childhood to the teenage
years. Two distinct subtypes of type III have been described. w Sea-blue histiocyte syndrome is a genetic disorder with a
Type IIIa usually presents clinically between early child- benign course. The striking blue color of the histiocytes
hood and mid-adult life. Type IIb is a clinically aggressive with Wright’s or May-Griinwald-Giemsa stain gives the
systemic disease, with neurologic involvement character- syndrome its name.
ized by isolated horizontal supranuclear gaze palsy. @ Histiocytic disorders represent an abnormal proliferation
m Gaucher’s disease is the first lysosomal storage disorder and accumulation of mature histiocytes, or Langerhans’
for which enzyme replacement therapy is available. cells.

mwNiefann—Pick disease is caused by a deficiency of the

enzyme sphingomyelinase, with a secondary accumulation

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it I
PART 4
HEMOSTASIS AND INTRODUCTION TO THROMBOSIS

Chapter —

Introduction
to Hemostasis
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA)
Claudia E. Escobar, MT(ASCP)SH
David L. McGlasson, MS, CLS(NCA)

Platelets and Hemostatic OBJECTIVES

Mechanisms
Overview At the end of this chapter, the learner should be able to:
Vascular System i. Define the terms coagulation, fibrinolysis, petechiae, ecchymosis, and hemorrhage.
Primary Hemostasis 2 . List the major and minor systems involved in maintaining hemostasis.
Platelet Structure (Basic
Information) 3 . Describe the events that take place in primary hemostasis.
Platelet Structure 4, Name the three structural zones of platelets.
(Advanced)
5). Describe the composition and functions of the peripheral zone, sol-gel zone, and
Platelet Function
organelle zone.
Overview
Maintenance of Vascular . List steps in platelet plug formation and describe the process of platelet adhesion and
Integrity aggregation.
Platelet Plug Formation
. Describe the events that take place in secondary hemostasis.
Secondary Hemostasis:
. Name the product responsible for stabilization of the hemostatic plug.
Fibrin-Forming
(Coagulation) System . List characteristics for the contact coagulation proteins, prothrombin proteins, and
Classification of Coagulation fibrinogen group.
Factors by Hemostatic
. Define the extrinsic system, intrinsic system, and common pathway.
Function
Classification of Coagulation . Describe the events that take place in fibrinolysis.
Factors by Physical
. Describe the use of the prothrombin time test in monitoring hemostasis.
Properties
Blood Coagulation: The . Describe the use of the activated partial thromboplastin time test in monitoring
“Cascade” Theory hemostasis.
Extrinsic Pathway
. Describe the thrombin-mediated reactions in hemostasis.
(Factor VII)
Intrinsic Pathway (Factors
XII, XI, IX, and VIII)
Common Pathway (Factors
X,-V, Il, and 1)
Thrombin-Mediated
Reactions in Hemostasis
(Advanced Concepts)
544 Chapter 24 =Introduction to Hemostasis

Thrombin-Mediated Platelet
Aggregation
Thrombin Formation: Role
of Extrinsic Pathway
Thrombin Formation: Role
of Common Pathway
Thrombin-Mediated
Anticoagulant Activity
Fibrinogen Conversion and
Fibrin Stabilization via
Thrombin
Thrombin-Mediated Tissue
Repair
Blood Coagulation: A
Cell-Based Model of
Hemostasis
Fibrin-Lysing (Fibrinolytic)
System
Action of Plasmin
Kinin System
Protease Inhibitors
Complement System
Laboratory Evaluation of
Hemostasis
Case Study

Platelets and Hemostatic Mechanisms effect on cellular function, in particular, endothelial cell
function. Mechanical or pathologic disruption of intact
Overview endothelium within the vessel wall initiates localized hemo-
static and thrombotic responses. If the vascular injury
Hemostasis is the complex process by which the body spon-
exceeds the capacity of the platelets mediated by thrombin to
taneously stops bleeding and maintains blood in the fluid state
form a hemostatic platelet plug, then the clinical signs and
within the vascular compartment. The major role of the hemo-
symptoms of hemorrhage occur. Stagnation of blood flow as
static system is to maintain a complete balance of the body’s
tendency toward clotting and bleeding (Fig. 24-1). Normal
hemostasis is both rapid and localized. Hemostasis is
achieved by the highly integrated and regulated interaction of:
1. Blood vessels
2. Platelets
3. Coagulation proteins
4. Fibrinolysis
5. Serine protease inhibitors
Minor ancillary systems involved are also listed in
Table 24—1. Through a complex series of molecular interac-
tions involving cells and biochemicals, a balance between
procoagulant and fibrinolytic activity is achieved. A fine line
separates clot formation in circulating blood from bleeding.
It has long been recognized that an imbalance, either
acquired or inherited, in any one of the many factors that Hemostasis
contribute to hemostasis often leads to hemorrhage or throm-
bosis. Significant changes in blood flow have a profound Figure 24-1 @ Hemostasis: A system in balance.
 Chapter 24 Introduction to Hemostasis 545

platelet plug formed in primary hemostasis. Secondary hemo-

stasis 1s activated by the release of tissue factor from epithe-
lial cells that are exposed to the circulation at the site of
vascular injury. Defects in secondary hemostasis decrease
fibrin production and reduce the stability of the formed clot.**+
Major Systems Minor Systems The cascade of coagulation reactions is a result of complexes
Vascular system Kinin system that form among enzymes, substrates, phospholipid surfaces,
Platelets Complement system and cofactors.
Coagulation system Gradually, the stable clot is dissolved through a process
Fibrinolytic system known as fibrinolysis. Fibrinolysis involves the proteolytic
Serine protease inhibitors
digestion of fibrinogen and fibrin by the enzyme plasmin.
Through the release of platelet-derived growth factor
(PDGF), vascular healing and repair is promoted, completing
a result of arterial disease or mechanical impedance disturbs the process of hemostasis.?
the endothelial cell anticoagulant effect, thus leading to In general, the relative importance of the hemostatic
the formation of a thrombus (clot) and hypercoagulable mechanisms varies with the vessel size (Table 24—2). Basi-
states. ! cally, the larger the area of bleeding, the larger the vessel
Protective mechanisms exist that prevent thrombin for- involved. Ecchymoses, epistaxis, petechiae, gastrointestinal,
mation in normal healthy vessels. Intact endothelial cells and genitourinary bleeding represent mucocutaneous hemor-
within a vessel wall are nonreactive or thromboresistant rhage highly suggestive of qualitative or quantitative platelet
through the interaction of receptors and proteoglycans. Coag- disorders.» Hemorrhage into joint cavities and into the
ulation proteins circulate in blood as inactive enzymes or retroperitoneum are characteristic of factor deficiencies or
zymogens held in balance by anticoagulant and fibrinolytic warfarin (Coumadin) toxicity. Coumadin is a therapeutic
pathways, and platelet procoagulant membrane lipids are anticoagulant. The hemorrhagic condition characteristically
internalized within the cell. These combined mechanisms seen in disseminated intravascular coagulation (DIC) presents
serve to keep hemostasis in balance. with both mucocutaneous and joint bleeding, evidence of both
Hemostasis can be divided into two stages: primary and platelet and coagulation protein deficiencies’ (see Chaps. 25
secondary hemostasis. Primary hemostasis refers to the and 26). Some sources and types of bleeding are listed in
response to vascular injury that produces a platelet plug at the Table 24-3.
site of damage. Primary hemostasis serves to limit bleeding The focus of this chapter is to provide a comprehensive
immediately through the formation of a loose platelet plug by review of present-day knowledge of the biochemistry, func-
adhering to the endothelial wall at the site of injury. Platelets tion, interaction, and regulation of each of the major com-
then release potent coagulant compounds that enhances their ponents that participate in hemostasis. Subsequent chapters
aggregation, forming a more substantial plug. These platelets further define the genetic and molecular mechanism, labora-
then provide a phospholipid surface for activated coagulation tory diagnosis, and current treatment of the various throm-
enzyme complexes.* The interaction of platelets and vascular botic and hemorrhagic disorders of hemostasis.
endothelium is the first step in a series of reactions that lead
to thrombus formation and serve to halt bleeding after vascu- Vascular System
lar injury.2* However, the primary response alone is ineffec-
tive for any duration of time. ADVANCED CONCEPTS
Secondary hemostasis involves the enzymatic activation The blood vessel, with its smooth and continuous endothelial
of the coagulation proteins to produce fibrin from fibrinogen. lining and fibrous coat, is designed to facilitate blood flow as
Soluble fibrin monomers polymerize and are then cross- well as participate in the process of hemostasis. The structure of
linked into insoluble strands that serve to stabilize the loose the vessel wall, which includes the outermost layer (adventitia),

Vessel Relative Sizes General Breach-Sealing Requirements*

Capillary Smallest Generally direct sealing

Venule Mostly fused platelets
Arteriole Mostly tused platelets
Vein Vascular contraction, fused platelets, perivascular and intravascular hemostatic
factor activation
Artery Largest Great vascular contraction, more fused platelets, greater perivascular
and intravascular hemostatic factor activation

*In general, the larger the vessel, the more hemostatic system involvement is required to seal the breach.
546 Chapter 24 = Introduction to Hemostasis

Table 24-3. Some Sources

. and Types of Bleeding _
Source _ Type
1. Contraction of vessels (vasoconstriction) and reflex
Arteriole, venule Panne Pechial toe stimulation of adjacent vessels
(diapedesis or leakage of 2. Diversion of blood flow around damaged vasculature
blood out of small vessels) 3. Initiation of contact activation of platelets with subsequent
Veins Ecchymosis (large, ill-defined adhesion, release reaction, and aggregation
soft tissue bleeding) 4. Contact activation of the coagulation system (both extrin-
Artery Rapidly expanding “blowout” sic and intrinsic) leading to fibrin formation
hemorrhage

the middle layer (media), and the inner layer (intima) is outlined (1) von Willebrand factor (vWF), (2) collagen fibers, and
in Table 244 The endothelial surface of the blood vessel is usu- (3) platelet membrane glycoprotein Ib (GPIb; the receptor
ally inert to platelets and coagulation factors. It is termed a non- for vWF).°
thrombogenic surface because the physical and biochemical The adherent platelet undergoes activation events,
characteristics of the endothelium render the cell surface throm- including shape change, platelet aggregation, and secretion of
boresistant. This 1s achieved through: ADP from the platelet-dense bodies. Secretion of platelet
ADP acts in the recruitment of additional platelets to form the
. Synthesis and secretion of a vasodilator, prostacyclin (PGI,)
primary hemostatic «plug. Activated platelets provide an
. Secretion of tissue plasminogen activator (t-PA)
important phospholipid surface that enhances activation of
. Inactivation and clearance of thrombin
the coagulation proteins in both the intrinsic and extrinsic
. Activity
RWNe of the cofactor thrombomodulin in the thrombin-
system. In addition, platelet secretion of thromboxane A,
dependent activation of protein C
(TXA,), a prostaglandin; and serotonin, further promotes
5. The degradation of proaggregating substances such as
vasoconstriction. Endothelial release of tissue factor (for-
adenosine diphosphate (ADP) and vasoactive amines?
merly known as tissue thromboplastin) during vascular injury,
However, when the endothelial lining is disrupted, the represents one of the body’s major procoagulant abilities.’*
vascular system acts to prevent bleeding by promoting rapid The initiation of the extrinsic pathway of coagulation
vasoconstriction of the injured vessel as well as adjacent requires tissue factor, factor VII/VIla, calcium, and a nega-
vessels. This process diverts blood flow around the damaged tively charged phospholipid membrane. The factor VIIa—
vessel and enhances contact activation of platelets and coag- tissue factor complex is now known to initiate the activation
ulation factors (Table 24—5S). Platelet adhesion occurs almost of both factor X and factor IX. Release of t-PA by the dam-
immediately to the exposed collagen fibers of the subendothe- aged endothelial cell provides the mechanism for clot disso-
lium. The principal mechanism of platelet adhesion involves lution, necessary to re-establish vascular patency.? PDGF,
secreted by platelets, assists in proliferation and repair of
damaged endothelium. The vascular response involved in the
hemostatic mechanisms usually lasts less than 1 minute.
Some of the major substances contained within the endothe-
lium, subendothelium, or both, that play primary roles in
hemostasis are listed in Table 24—6. Other substances known
to bind to endothelial cells that promote coagulation are
Vessel ayer" ) Composinon and Eiancucn listed in Table 24-7.

Thies eee Connective tissue cell ence

Tunica media Elastic tissue and smooth
muscle, controlling Primary Hemostasis
vasoconstriction and
sometimes vasodilation Platelet Structure (Basic Information)
Tunica intima Broad flat endothelial cells
with an underlying Platelets are anucleated cytoplasmic fragments measuring
basement membrane 2 to 4 wm in diameter and originate from bone marrow
supported by a few megakaryocytes. On Wright’s stain, platelets appear as pale
connective tissue cells, blue cells with fine azurophilic granules (Fig. 24-2). During
providing a smooth megakaryocytopoiesis, the interval from megakaryoblast to
nonwettable surface but
facilitating migration of production of platelets is approximately 1 week. In the
cells through the spaces peripheral blood, about 70% of the platelets are circulating,
as needed whereas 30% are sequestered in the microvasculature of the
spleen and serve as functional reserves after their release from
 Chapter 24 Introduction to Hemostasis 547

Substance(s)

Collagen Binds to Satter Toone GPIb/Ila: activates plasma coagulation factors via
intrinsic pathway
Heparin sulfate A glycosaminoglycan that has anticoagulant activity and contributes to the
activation of antithrombin
von Willebrand factor (VWF) Protein synthesized in endothelial cells and megakaryocytes; primarily binds to
platelet membrane receptor GPIb to promote adhesion; also functions as a carrier
for factor VII, providing factor stability in circulation; may also bind to platelet
GPIIb/IIIa in conditions of high shear stress to promote adhesion
PGI, A prostacyclin synthesized by endothelial cells that physiologically inhibits platelet
aggregation and limits thrombus formation beyond the damaged vessel
Tissue factor (TF) A lipoprotein released following vascular trauma that initiates coagulation by
activating factor VII with ionized calcium (extrinsic pathway) and contributes to
the activation of factors X and IX (intrinsic pathway)
Tissue factor pathway inhibitors (TFPIs) glycoproteins found on endothelial surfaces that serves as an anticoagulant by
. inhibiting factors VIla/TF and Xa
Thrombomodulin A large protein found on endothelial surfaces that serves as a cofactor in protein C
activation when bound to thrombin
Tissue plasminogen activator (t-PA) Serine protease secreted by endothelial cells that regulates fibrinolysis; when
bound to fibrin, t-PA activates plasminogen to form plasmin
Plasminogen activator inhibitors (PAIs) Proteins found in endothelial cells that regulate fibrinolysis by neutralizing the
activity of plasmin, plasminogen, and t-PA; associated with risk of thrombosis

High-molecular-weight Cofactor required for efficient activation of the contact phase

kininogen (HMWK or HK)
Factors X and Xa On endothelial cells mediates prothrombin activation
Factor [X/IXa Cell-bound factor [Xa functions in factor VUI-dependent activation of factor X
Prothrombinase complex Complex (Xa, Va, Ca**, and phospholipid) assembled on endothelial cells further
propagates coagulation and triggers intracellular signaling events
Fibrinogen and fibrin Present in healing blood vessel subendothelium (fibrinogen); binds to endothelial cells;
reinforces platelet plug
Fibronectin Functions as an adhesive protein for platelets, promoting spreading

the bone marrow. Platelets survive for 7 to 10 days in circula-

tion and are active in hemostasis.
The normal platelet count ranges from 150,000 to
400,000 per microliter (uL), depending on the methodology
employed. Normal platelet function in vivo and in vitro requires
more than 100,000 platelets per microliter.'° It is unusual for a
patient with a platelet count greater than 20,000/uL to have
major hemorrhages. Spontaneous hemorrhage may occur when
platelet count falls to 10,000 or below.° Assuming normal
platelet function, a platelet count greater than 50,000/uL will
minimize the chance of hemorrhage during surgery.°
Platelets must be adequate in both number and function
to participate optimally in hemostasis. Platelets participate in
hemostasis by:

Figure 24-2 = Normal platelets: Wright-stained blood smear 1. Providing a negatively charged phospholipid surface for
(peripheral blood). factor X and prothrombin activation
548 Chapter 24 Introduction to Hemostasis

bo. Releasing substances that mediate vasoconstriction, platelet

aggregation, coagulation (thrombin generation), and vascu-
lar repair
>) . Providing surface membrane glycoproteins such as
GPIIb and GPIlIla to attach to other platelets via fibrino-
gen, and GPIb to bind to collagen and subendothelium
via VWF."
Platelets achieve these tasks by functioning in shape
change, adhesion, aggregation, and secretion (release). A
basic understanding of the platelet’s ultrastructure and
its organelles is crucial to understanding how the platelet
performs each of its vital functions (Fig. 24-3). Each of
these individual functions can be assessed by clinical test-
ing; however, all functions are required during the forma-
tion of the hemostatic platelet plug, known as primary
hemostasis.

Platelet Structure (Advanced)

The platelet structure is quite distinct, leading to subdivision
into three defined zones, each possessing unique functional
capabilities. These zones are prominently delineated by the
circumferential band of microtubules found in the platelet, as
seen in Figure 244. The three described zones and their con-
tents are summarized in Table 24-8. Impaired cellular function
of the platelet membrane, cytoskeleton, granular constituents,
and secreted proteins often leads to platelet dysfunction and
abnormal hemostasis.

Figure 24-4 @ Internal anatomy of a stimulated platelet. Circum-

ferential band of microtubules (MT) leads to reorganization of the
internal structure of the platelet into three zones. The peripheral
zone (PZ) is the region external to a circumferential band of micro-
tubules (MT). The intermediate zone (IZ) (encircling arrows) includes
the microtubules and the closely adjacent cytoplasmic material. The
central zone (CZ) is internal to the microtubule band and contains
many organelles such as granules (G), dense bodies (DB), dense
tubular system (DTS), lysosomes (Ly), mitochondria (M), and many
profiles of the open canalicular system (OCS). Magnification
* 49,700. (From Barnhart, MI: Platelet responses in health and
disease Mol Cell Biochem 22:115, 1978, with permission.)

PERIPHERAL ZONE
BASIC CONCEPTS The peripheral zone is a complex region
of the platelet consisting of the glycocalyx (an amorphous
MT Microtubules exterior coat), platelet membrane, numerous deeply penetrat-
G Other granules ing surface-connecting channels known as the Open Canalic-
DB Dense bodies
DTS Dense tubular system
ular System (OCS), and a submembranous area of specialized
M Mitochondria microfilaments (see Fig. 24~3).
Gly Glycogen lakes (particles) A number of glycoproteins anchored in the platelet
Lys Lysosomes
OCS Open canalicular system
membrane are present in the glycocalyx and mediate the crit-
Mf Microfilaments ical events of platelet adhesion and aggregation. Specific
EC Exterior coat (glycocalyx) binding of platelet membrane receptors to adhesive macro-
GA Golgi apparatus
molecules such as fibronectin and vitronectin in plasma or
collagen in vascular subendothelium, further facilitates the
Figure 24-3 @ Discoid platelets; (top) summary diagram of the spreading of platelets on the damaged vessel wall and leads to
platelet organelles; (bottom) transmission electron micrograph platelet activation and the formation of large platelet aggre-
(TEM) of cross-sectioned platelets illustrating basic ultrastructure. gates.'° Platelet membrane glycoproteins serve as receptors
 Chapter 24 Introduction to Hemostasis 549

also present on the surface of the platelet membrane, as are

various platelet factors (PFs) that participate in the formation of
fibrin. At least seven PFs have been identified; they are listed in
Table 24-9. PF3 and PF4 seem to be the most important
I. Peripheral zone (stimulus receptor/transmitter region)
a. Glycocalyx
platelet factors. '?
b. Platelet membrane
ADVANCED CONCEPTS A number of glycoproteins present in
c. Open canalicular system (OCS)
d. Submembranous region the glycocalyx are responsible for blood group specificity
. Sol-gel zone (cytoskeletal/contractile region) (ABO), tissue compatibility (human leukocyte antigen [HLA]),
a. Circumferential microtubules and platelet antigenicity.
b. Microfilaments Although the primary receptor for vWF seems to be
. Organelle zone (metabolic/organelle region)
GPIb, in rapidly flowing blood with a high shear stress, VWF
a. Granules
1. a Granules is more likely to bind to GPIIb/IHa.'*
2. Dense granules The membranous surface-connecting system referred to
3. Lysosomes as the open canalicular system (OCS) consists of tubular
4. Glycogen granules invaginations of the plasma membrane that articulate through-
b. Mitochondria
out the platelet even though it is part of the peripheral zone.
c. Dense tubular system
d. Peroxisomes Chemical substances stored in the dense and alpha (a) gran-
ules of the platelet are released to the exterior through the
OCS. The OCS also facilitates the collection of plasma proco-
agulants that aid in fibrin formation by providing increased
and facilitate transduction of activation signals across the surface absorptive area. This membrane system appears to be
platelet membrane in response to various external stimuli. the calcium-regulating mechanism of the platelet.
Adhesion of platelets to exposed collagen within the
SOL-GEL ZONE
subendothelium involves the interaction of the platelet mem-
BASIC CONCEPTS The term cytoskeleton is often used to
brane receptor, GPIb, and vWF, a plasma component.
describe this zone. Within the matrix of the platelet are micro-
Platelet adhesion typically occurs | to 2 seconds following
tubules, microfilaments, and submembranous filaments.
vascular injury. Following adhesion, release of adenosine
Microtubules encase the entire platelet, maintaining its
diphosphate (ADP) and adenosine triphosphate (ATP) from
discoid shape. Microtubules are composed of protein subunits
intracellular platelet granules causes shape change in adja-
called tubulin. In the stimulated platelet (see Fig. 24-4),
cent platelets. Platelet aggregation requires a conformational
contraction of the circumferential band of microtubules
change in platelet membrane receptor GPIIb/IIIa (and GPV),
toward the center of the platelet appears to be responsible for
thus allowing the binding of fibrinogen.'* The binding of the
both the movement of organelles toward the center and their
two symmetric ends of fibrinogen to different platelets pro-
reorganization, which facilitates the secretory process.*
motes platelet-to-platelet interaction, thus leading to the
Microfilaments are randomly interwoven throughout the
formation of a large platelet aggregate. Calcium and/or mag-
cytoplasm of the platelet and are composed of two contractile
nesium, in the form of divalent cations, are also required for
proteins, actin and myosin. Also present is thrombosthenin, a
fibrinogen binding. Platelet aggregation begins approxi-
contractile protein similar to actomyosin.* Actomyosin is
mately 10 to 20 seconds after vascular injury.'®
complexed actin and myosin. Microfilaments can convert
The platelet membrane also includes receptors for sub-
from an unorganized gelatinous state to organized parallel
stances such as ADP, thrombin, epinephrine, collagen, throm-
filaments capable of contraction within seconds as the
boxane A, (TXA,), and serotonin, which play a role in platelet
platelet’s shape changes.
aggregation.'* ADP and TXA, are potent platelet aggregators
when bound to their specific platelet membrane receptors."
These two platelet-derived aggregators are released during
platelet activation in response to thrombin and amplify intra-
cellular events, leading to platelet secretion and platelet
aggregation. The secretion of such substances by activated Coagulation factor V
platelets causes the activation of additional platelets, thus Thromboplastin-like material
augmenting the primary hemostatic response. Platelet thromboplastin*
The platelet membrane, similar to other plasma mem- Antiheparin factor*
branes, contains a phospholipid component. Activated platelets Fibrinogen coagulant factor
Antifibrinolytic factor*
undergo shape change, develop stickiness, and expose platelet Platelet cothromboplastin
membrane phospholipids. Platelet factor 3 (PF3) is known to
*Most important.
move to the outer surface of the platelet membrane, thus allow-
Source: Adapted from Bick, RL, and Murano, G: Physiology of
ing for the assembly of the vitamin K—dependent coagulation hemostasis. In Bick, RL (ed): Hematology: Clinical and Laboratory
factors involved in secondary hemostasis.'* PF3 serves as a Practice. CV Mosby, St. Louis, 1992, pp 1285-1309.
cofactor in the complex. Coagulation factors V and VIII are
550 Chapter 24 Introduction to Hemostasis

ADVANCED CONCEPTS Microtubules disappear from the Dense granules store ADP, ATP, ionic calcium, serotonin, and
center of the platelet after secretion, and reappear in other phosphates.
peripheral areas such as pseudopods. Microtubules appear to Lysosomes appear similar to the azurophilic granules
monitor the internal contraction of platelets, preventing platelet found in granulocytes and contain microbicidal enzymes,
secretion in response to only minimal stimulation and thereby neutral proteases, and acid hydrolases. Proteases may con-
regulating the degree of platelet response to external stimuli. tribute to disruption of the subendothelial structure following
vascular injury. Glycogen granules are also found within the
ORGANELLE ZONE organelle zone and function in platelet metabolism.
BASIC CONCEPTS The organelle region is responsible for The contents of both a and dense granules are released
the metabolic activities of the platelet. Like many other cells, during secretion into the OCS. Secretion promotes the recruit-
platelets possess mitochondria and various cytoplasmic gran- ment of additional platelets to the platelet aggregate at the site
ules. Unlike many cells, platelets are anucleated and do of vascular injury. Platelet secretion is an energy-dependent
not possess either a Golgi body or rough endoplasmic reaction that relies on the metabolic function of mitochondria.
reticulum (RER). The estimated 10 to 60 mitochondria present per platelet require
Generally, the most numerous organelles are the platelet glycogen as their source of energy for metabolism.° In the rest-
granules, which are heterogeneous in size, electron density, ing platelet, ATP (energy) production is generated by glycolysis
and chemical content. Platelets contain three morphologically and the oxidative Krebs cycle. In the activated state, about half
distinct types of storage granules: dense granules, a granules, the ATP production in platelets occurs through the glycolytic
and lysosomes. The a granules are most numerous (20 to pathway. The important platelet-secreted proteins and their
200 per platelet) and store a number of different substances. known role in hemostasis are summarized in Table 24—10.
The content of the a granules along with their major function The dense tubular system (DTS) is another important
in hemostasis are listed in Table 24—10.° structure present in the cytoplasm of the organelle zone of
Dense granules or bodies, are smaller and fewer in num- platelets. Similar to the sarcotubules in skeletal muscle, the
ber (2 to 10 per platelet) and appear as dense opaque granules DTS is derived from the smooth endoplasmic reticulum
in transmission electron microscope (TEM) preparations. (ER) of immature megakaryocytes. The DTS is the site of

Granules

Alpha eianuies
Platelet-Specific Proteins Inhibits heparin; chemotactic; promotes smooth muscle growth for vessel
Beta-thromboglobulin (8-TG) repair
Platelet factor 4 (PF4) Inhibits heparin
Platelet-derived growth factor (PDGF) Promotes smooth muscle growth; involved in atherosclerosis and lipid
metabolism
Thrombospondin Promotes platelet-to-platelet interaction; mediates cell-to-cell interaction
Factor V Once activated complexes with Xa to convert prothrombin to thrombin

Plasma Proteins
Fibrinogen Fibrin formation
von Willebrand factor (vWF) Promotes platelet adhesion
Factor V Cofactor in fibrin formation
Factor VUI Cofactor in fibrin formation
Fibronectin Cellular adhesion molecule; promotes platelet spreading
Albumin Uncertain
High-molecular-weight kininogen (HMWk) Activation of the intrinsic pathway via contact
a.-Antiplasmin Inhibits plasmin
Plasminogen Precursor to plasmin; functions in fibrinolysis

Dense Granules
ADP (nonmetabolic) Promotes platelet aggregation
ATP (nonmetabolic) Uncertain
Calcium Primary and secondary messenger regulates platelet activation/ aggregation
Serotonin Promotes vasoconstriction

Lysosomes
Neutral proteases* Uncertain
Acid hydrolase* Uncertain
Bacteriocidal enzymes* Uncertain

*The specific function of these proteins is uncertain: heemay serve to pices plas activation in response to thrombin.
 Chapter 24 Introduction to Hemostasis 551

prostaglandin and thromboxane synthesis and sequestration

Maintenance of Vascular Integrity
of calcium. It is primarily the release of calcium from the
DTS that triggers platelet contraction and subsequent internal BASIC CONCEPTS
activation of platelets. Platelets are involved in the nurturing of endothelial cells lin-
ing the vascular system. Maintaining the integrity of the ves-
sel wall is a function of the endothelial cells, the connective
Platelet Function tissue of the subendothelium, and platelets. A primary feature
of normal intact endothelium is that it is nonthrombogenic: it
Overview
is resistant to platelets, leukocytes, and coagulation proteins.
Numerous stimuli can trigger platelet activation, which may Platelet and endothelial cell interaction is modulated by the
be transient, reversible, or irreversible. Activation refers to prostaglandin PGI, (prostacyclin).'!° PGI, is synthesized by
several separate responses of platelet function that include endothelial cells from arachidonic acid (a membrane phospho-
adhesion, shape change, secretion, and aggregation. Platelets lipid) and has an antagonistic effect on platelet adhesion and
respond in a graded fashion, depending on the strength and platelet aggregation, thus serving to locally limit the hemosta-
duration of the stimuli as well as the physiologic or patho- tic response. Routinely, circulating platelets survey the vessel
logic state of the platelet. wall for denuded endothelium. Platelets do not adhere to
In the initial stage of activation, platelets form pseudopods normal vascular endothelial cells. However, once the vessel
as they begin to contract. As activation progresses, contraction wall integrity is disrupted, platelets are incorporated into the
and pseudopod formation progress, organelles including the a vessel wall, releasing a substance called platelet-derived
granules and dense bodies are reorganized to the center of the growth factor (PDGF) that nurtures the endothelial cells,
platelet, and further contraction causes the granules to spill maintaining normal vascular integrity and thromboresistant
their contents into the OCS, which shunts the contents to the properties.'° Endothelial disruption, such as a cut in the blood
outside of the platelet.'’ Adjacent platelets are activated vessel, provides binding sites for the adhesive protein vWF,
through receptor contact with the granular contents, amplifying through the platelet GP Ib/IX/V complexes and fibrinogen, as
the activation process. Therefore, it may be said that platelet well as fibronectin through integrin.'? These “sticky” proteins
activation spans shape change, platelet adhesion, granular are thought to participate in the formation of a bridge from
secretion, cytoskeletal reassembly, and platelet aggregation. platelets to subendothelial connective tissue. Figure 24—5
Before proceeding further, the reader should review the shows a scanning electron micrograph (SEM) demonstrating
structure of the platelet in order to visualize and understand platelet adherence at the site of endothelial loss compared
subsequent events that occur in the platelet at the ultrastruc- with the normal smooth contour of the endothelial cell.
tural level during hemostasis (see Fig. 24-3). The basic
events that occur in primary hemostasis after injury to a ves- ADVANCED CONCEPTS
sel are outlined in Table 24-11. PDGF is a mitogen stored in and secreted from the a granule of
the platelet. Specific receptors for PDGF have been isolated on
cultured fibroblasts and smooth muscle cells.'° When vessel
injury occurs and platelets are activated, PDGF is secreted, stim-
Table21d
24 Events that Occur tee ulating endothelial cell migration and proliferation of smooth
a ‘inPlatelets After Vess | muscle growth, thereby mediating wound healing and vascular
repair. PDGF is an important growth factor and is thought to be
linked to the pathologic development of atherosclerosis through
Vasoconstriction heen blood flow in its influence on lipid metabolism. PDGF also demonstrates
damaged vessel; increases chemotactic properties for neutrophils and monocytes, as well
concentration of biochemi-
cals to promote platelet
as fibroblasts and smooth muscle cells. The characteristics of
activation events PDGF help to limit the hemostatic response; thus, the platelet
Platelet shape change Signals intracellular activation, plug remains local to the site of vascular injury.
leading to platelet plug In the absence of platelets, a large number of red cells
formation migrate through the vessel wall, enter the lymphatic drainage,
Platelet aggregation A platelet-to-platelet interaction
mediated by fibrinogen,
and appear as petechiae or purpura in the skin or mucous
Ca**, and platelet membrane membranes. The process of maintenance of normal vascular
activated glycoprotein integrity, involving nourishment of the endothelium by the
IIb/IIIa complex platelet or actual incorporation of platelets into the vessel
Platelet plug Adhesion and aggregation of wall, utilizes a small minority of the platelets in circulation
platelets to the site of injury
Release of chemical constituents
but is nevertheless an important function.
Platelet secretion
contained in various granules;
amplifies platelet response
Stabilization Platelet plug stabilized by Platelet Plug Formation
formation of fibrin mesh
over the platelet aggregates Adhesion and aggregation of platelets to the site of vascular
damage occurs in concert with cellular activation. These
552 Chapter 24 Introduction to Hemostasis

2. A platelet membrane receptor, GPIb

3. Collagen fibers!!!

The adhesion of platelets to collagen facilitates platelet

spreading on the subendothelial surface, which promotes
release of dense granules’ contents, leading to platelet
aggregation. Activation of additional platelets, through the
interaction with either collagen or other mediators (such as
thrombin, ADP, or TXA,), amplifies the response and pro-
vides a positive feedback mechanism that recruits more
platelets into the site.”
Evidence indicates that vWF is synthesized by endothe-
lial cells and megakaryocytes. Once made, vWF is released
into the plasma where it is absorbed onto the surface of the
platelet bound to its receptor, GPIb (Fig. 24-6), or incorpo-
rated into the subendothelium. Platelets thus adhere to the
area of injury at the endothelial lining, acting to arrest the
initial episode of bleeding. In Figure 24-7, a TEM demon-
strates platelet adhesion to subendothelial connective tissue
at the focus of endothelial loss. A decrease in platelet num-
ber as well as platelet dysfunction results in increased
bleeding.

SHAPE CHANGE The interaction of circulating platelets with

external stimuli or agonists results in a series of complex
reactions known collectively as platelet activation that
precede the ultimate activation event of platelet aggregation
and platelet secretion. Platelet activation occurs when an
external agonist interacts with its specific membrane receptor
on the platelet. Signal transduction occurs, transmitting the
signal from outside the cell to inside the cell. Activation of
second messenger pathways within the platelet lead to further
Figure 24-5 @ SEM of platelet adherence at the site of endothelial intracellular biochemical changes, culminating in platelet
loss. Short arrow points to a discoid intact platelet with a single
activation events such as shape change, secretion, cytoskele-
pseudopod; Jong arrow points to an elongated adherent platelet;
top double arrow marks densely adherent platelets appearing as tal reassembly, and platelet aggregation.'® Following vessel
elongated humps fused to the subendothelial layer. (From Cotran, injury and exposure to external stimuli, platelets change
E: Robbins Pathologic Basis of Disease, ed 1. WB Saunders,
Philadelphia, 1979, p 120, with permission.)

cellular events are mediated through specific receptors on the GP 1b attached

to platelet
platelet membrane. The membrane is the key to the interac-
tion of extracellular agonists with intracellular biochemicals.
A series of complex intracellular events occur which lead to vWF bridge
further biochemical and morphological change of the platelet.
The culmination of these processes permits the platelet to Vessel wall break
perform its vital role in hemostasis, assuming there is normal
platelet number and function.
Platelet
membrane
ADHESION Damage to the endothelial monolayer exposes
flowing blood to the subendothelial connective tissue matrix,
which is composed of adhesive molecules (i.e., collagen,
vWF, fibronectin, and thrombospondin). The specific binding
of platelet receptors to these adhesive molecules in plasma or
vascular subendothelium mediates adhesion. Adhesion is a
reversible process. The principal mechanism of adhesion
Platelet membrane Exposed subendothelium
involves three critical components: Glycoprotein Ib receptor

1. vWF, a plasma protein that links the platelet to subendothe- Figure 24-6 @ Pictorial representation of platelet adhesion to
lial binding sites subendothelium through von Willebrand's factor (vWF) bridge.
 Chapter 24 Introduction to Hemostasis NnNnWwW

shape from circulating discs to spheres with pseudopods,

indicating platelet activation (Fig. 24-8).
Many agonists such as collagen, ADP, thrombin, and
TXA, alter the internal levels of cytosolic calcium, thus
promoting shape change.'° The normal discoid shape of
the platelet is defined by the circumferential microtubules.
Increases in cytosolic calcium cause dissolution of the
circumferential microtubules, thereby altering platelet
shape.'® Shape change is thought to represent the most sen-
sitive parameter of platelet activation. TXA, and ADP are
potent platelet agonists when bound to their specific mem-
brane receptors. During platelet activation in response to
thrombin, two platelet-derived agonists (ADP and TXA,)
are released, serving to amplify intracellular events that
result in platelet secretion and platelet aggregation.'* When
platelets become activated, shape change and exposure of
V7
platelet membrane phospholipids such as PF3 occurs. Expo-
sure of PF3 promotes the assembly of vitamin K-dependent
Figure 24-7 @ TEM of platelet adherence to subendothelial con- factors on the platelet membrane surface. Activated
nective tissue at the focus of endothelial loss. (7) Intact platelet with
platelets adhere to exposed collagen, mediated by GPIb and
pseudopod (thin arrow indicates a granule; thick arrow indicates
dense body), (2) partially degranulated platelet, (3) degranulated
vWE. Dependent on the strength of the agonist, shape
platelet “ghost,” (4) internal elastic lamina. (From Cotran, change and signaling platelet activation may be followed
E: Robbins Pathologic Basis of Disease, ed 1. WB Saunders, by platelet aggregation.
Philadelphia, 1979, p 116, with permission.)
AGGREGATION Platelet-to-platelet interaction is known as
aggregation and usually begins 10 to 20 seconds after vascu-
lar injury and platelet adhesion.’ Platelet aggregation is an
energy-dependent process that requires ATP, which is primar-
ily derived from glycolysis. Ionized calcium (Ca’*), fibrino-
gen receptors GPIIb and IIa, and fibrinogen are necessary for
platelet aggregation. Fibrinogen must bind to the activated

Figure 24-8 @ TEM showing disk-to-sphere transformation of an activated platelet. Note progression from (1) disk shape to (2) pseudopod
formation to (3) degranulated ballooned sphere.
nn‘nS Chapter 24 Introduction to Hemostasis

platelet membrane GPIIb-IIla complex.'’? Once fibrinogen

binds to this membrane complex, extracellular Ca**-dependent
fibrinogen bridges form between adjacent platelets, thereby
promoting platelet aggregation. ADP induces the exposure of
fibrinogen receptor sites on the platelet membrane. It should
be noted that normal plasma fibrinogen levels support platelet
aggregation. In the absence of plasma fibrinogen, platelet
fibrinogen stored in the a@ granules, is released, promot-
ing platelet aggregation. Platelets deficient in GPIIb/II]a
(Glanzmann’s thrombasthenia) do not aggregate in response
to platelet-aggregating agents.'’ Platelets will not aggregate in
the absence of membrane glycoproteins, fibrinogen, or diva-
lent calcium.
In vitro platelet aggregation can be stimulated by a vari-
ety of agents (agonists) (Table 24-12). In vitro aggregation
can be visualized as a two-phase process that may be
reversible or irreversible, depending on the strength of the
stimulus. In the first phase, aggregation is initiated by an
agonist, causing the release of small amounts of ADP from
the dense granules. This initial aggregation wave is referred to
as primary or reversible aggregation.'’ At this point in the
aggregation process, small aggregates are formed with low
ambient concentrations of ADP. ADP at this concentration is
only a weak platelet agonist, and therefore these aggregates
may dissociate into individual platelets if the process does not
proceed normally. Primary aggregation involves contraction
Figure 24-9 ™@ TEM of an activated and a degranulated platelet.
of the circumferential microtubules, and the reorganization/
A. Early aggregation of activated platelet (the primary wave of
centralization of platelet organelles. aggregation, a reversible process). B. Degranulated platelet (the
The second phase, or secondary wave, of aggregation secondary wave of aggregation, an irreversible process). (From
is dependent on the activation stimulus being strong enough Barnhart, MI: Platelet responses in health and disease. Mol Cell
to evoke the secretion of platelet granule contents, particu- Biochem 22:117, 1978, with permission.)
larly non-metabolic ADP from the dense granules, as a
consequence of a stronger more complete contraction of the
circumferential microtubules. The beginning of secondary Various laboratory methods are employed to evaluate
aggregation actually defines the activation event of secre- platelet aggregation. One method of aggregometry, optical,
tion. Ultrastructural analysis shows the internal reorganiza- utilizes a spectrophotometer in which platelet-rich plasma and
tion of organelles is more severe, and degranulation is exogenous aggregating agents are added in a cuvette. As
evident by the lack of density of the granules with TEM platelets aggregate, decreasing optical density results in
(Fig. 24-9). Biochemical studies have confirmed the increased light transmittance. The change in density (percent
release of substances such as ADP, serotonin, and transmittance) is recorded, creating typical aggregation
epinephrine. Secondary aggregation is often considered to “patterns” for each aggregating agent. The pattern produced is
be irreversible. analyzed for reaction time, shape change, primary aggrega-
tion, release reaction, and secondary aggregation. Another
method: Lumiaggregometry, employs both optical density
and fluorescence in measuring platelet aggregation and simul-
taneous release of ATP; firefly luciferin-luciferase reacts with
the ATP producing characteristic wave patterns as the release
ADpP* (low, optimal, and high concentrations) reaction occurs. Figure 24-10 depicts a typical biphasic
Collagen* response of in vitro platelet aggregation to ADP, as recorded
Epinephrine* by an aggregometer. It should be noted that aggregation is an
Thrombin*
energy-dependent process that requires and exhausts the ATP
Ristocetin*
Serotonin resources of the platelet (see Chap. 33 for discussion of the
Arachidonic acid principles, instrument methods, and interpretation of platelet
Immune products aggregation).
Snake venoms A third method, whole blood aggregometry, measures
*More commonly used. the ability of the platelets to respond to agonists while in
ADP = adenosine diphosphate. whole blood suspension using the electrical impedance prin-
ciple. Small parallel electrodes are inserted into a cuvette
 Chapter 24 Introduction to Hemostasis NnNnNn

both primary and secondary aggregation (depending on the

60 amount secreted) and serves to amplify the process. Amplifi-
cation of the initial aggregation of platelets (a reversible
50 phenomenon) results in secondary aggregation and the recruit-
ment of many other platelets into a large platelet mass. The
transformation of irreversibly aggregated platelets into a mass
40
of degenerative platelet material without membranes is termed
viscous metamorphosis (Fig. 24-11). The biochemical con-
30 tents of the platelet lysosomes are also released during secre-
tion. It is thought that these enzymes function in viscous
Transmission
%
20 metamorphosis and later dissolution of the platelet plug at the
site of vascular injury.
10 Specific platelet-secreted proteins are released from « and
A dense granules as well as from platelet lysosomes following
ADP appropriate stimulation. Four platelet-specific proteins secreted
re) \

from the a granule have been well characterized to date and are
Figure 24-10 @ A typical biphasic response of in vitro platelet currently used as ‘markers of platelet activation.” These include
aggregation to ADP, as recorded via an aggregometer. beta-thromboglobulin (B-TG), platelet factor 4 (PF4), throm-
bospondin, and PDGF, which confirm the degranulation of
platelet a granules.'’ Because B-TG, PF4, thrombospondin, and
containing citrated whole blood in a 1:1 saline dilution. The PDGF are proteins virtually absent from normal plasma and
electrodes produce a small electric current running through the found in small concentrations within platelet a granules, these
whole blood suspension. As platelet aggregation occurs, proteins specifically mark platelet activation.' A number of
the platelets collect on the electrodes, reducing or impeding clinical conditions such as arteriosclerosis, cerebrovascular dis-
the magnitude of the current. The change is directly propor- ease, cardiopulmonary bypass, shock, venous thrombosis, and
tional to the degree of platelet aggregation. The peak ampli- DIC are associated with increased plasma levels of these mark-
tude is measured in ohms. The whole blood aggregation ers, thus signifying platelet activation.
pattern resembles a platelet-rich plasma (PRP) aggregation
tracing but is measured in ohms. However, you rarely see STABILIZATION OF THE HEMOSTATIC PLUG The last
secondary aggregation demonstrated and the peak amplitude stage involved in arresting bleeding after vessel damage is the
is much lower than in a PRP tracing. One distinct advantage formation of a stable platelet plug. This stabilization is
of whole-blood aggregation is that it virtually eliminates achieved through the activation of the coagulation cascade
the need for PRP preparation, reducing the blood volume and formation and deposition of fibrin (the end product of
required for testing, and the platelet count does not have to be coagulation) on the platelet aggregates. Exposure of collagen
standardized. The operator pipettes an aliquot of properly within the subendothelium of the damaged vessel (via the
mixed whole blood into the cuvette, adds an equal volume of intrinsic pathway) and the release of tissue factor (via the
physiologic saline pre-warmed to 37°C, and a stir bar. After extrinsic pathway) directly initiate fibrin formation. Thus,
placing the cuvette into the reaction well, the electrodes are fibrin interweaves through and over the initial platelet
placed into the mixture and the aggregating agent is added. plug, compressing it into place at the site of the vessel injury.
The aggregation pattern is then measured.'* Recent studies What originally starts as a small gelatinous mass gradually
have also shown that whole blood aggregation may be more
sensitive, and reflect the true nature of platelet aggregation in
vivo than PRP methods, and may be more sensitive to aspirin
elfecise a0

SECRETION (THE RELEASE REACTION) Platelet secretion

(release) and secondary aggregation are intimately related and
occur almost simultaneously; therefore, the discussion of the
two topics is difficult to separate. As stated previously, the
beginning of secondary aggregation actually defines the acti-
vation event of secretion. For secondary aggregation to occur
there must be secretion, particularly ADP, from the dense gran-
ules. A sufficiently strong stimulus is necessary for the secre-
tion, or release reaction, to occur. The release reaction from
dense granules involves the secretion of ADP, serotonin, and
calcium. During the various stages of aggregation, platelet-
secreted proteins are released from cellular organelles, thus Figure 24-11 @ TEM of viscous metamorphosis.
serving as markers of platelet activation. ADP is responsible for
556 Chapter 24 Introduction to Hemostasis

increases in size. In pathologic conditions, thrombus forma- Vascular Injury

tion may often occlude the lumen of a vessel, producing
ischemia to the affected organ or tissue.° During thrombus for-
mation, thrombin, plasminogen, tissue plasminogen activator Exposed
1. ADHESIONS Subendothelium
(t-PA), and antiplasmin are all incorporated into the clot.'” As
thrombin is incorporated into the clot, it is protected from
degradation by its inhibitors: antithrombin (AT) and heparin
cofactor I]. Thrombin, now trapped within the meshwork #
<—_—_—_ 2. AGGREGATION
oF
of the clot, activates factor XIII (fibrin-stabilizing factor), ———————

10-20 sec.
resulting in the cross-linking of fibrin strands and, thus, clot Platelet Release Thrombin
stabilization.
Fibrinolysis, the complex series of enzymatic reac-
tions that promote clot dissolution, is simultaneously acti- |
as 1-3 min.
3. PLUG FORMATION

vated during the process of clot formation. As previously Fibrin Formation

mentioned, plasminogen, the precursor of the proteolytic
enzyme plasmin, is incorporated into the fibrin clot. ¢ 4. CONSOLIDATION
Endothelial cells of the damaged vessel wall secrete t-PA, 3-5 min.
which catalyzes the conversion of plasminogen to plas- Retraction
min.?! Release of both endothelial cell t-PA and its
inhibitor, plasminogen activator inhibitor (PAI), is medi- q 5. FIBRIN STABILIZATION
ated by thrombin. Any excess plasmin is controlled by its 5-10 min.
inhibitor, antiplasmin. Thus, fibrinolysis is initiated in
Figure 24-12 ™ Sequence of events in hemostatic plug formation.
the final phase of thrombus formation. It is important to
(1) Platelet adhesion to exposed subendothelial connective tissue
mention that thrombin also stimulates the secretion of structures. (2) Platelet aggregation by ADP, thromboxane A,, and
endothelial cell substances as well as the secretion of thrombin recruitment through transformation of discoid platelets into
platelet proteins that promote tissue repair and play a major reactive spiny spheres that interact with one another through calcium-
role in wound healing. Clot dissolution, therefore, provides dependent fibrinogen bridges. (3) Contribution of platelet coagulant
activity to the coagulation process, which stabilizes the plug with a
a critical means by which formed thrombi are removed
fibrin mesh. (4) Consolidation of the platelet mass to provide a dense
from the vascular system, blood vessels are repaired, and thrombus. (5) Fibrin polymerization and fibrin stabilization by
blood flow returns to normal. Protective mechanisms pre- factor XIII. (From Thompson, AR, and Harker, LA: Manual of
vent thrombin formation in healthy intact blood vessels. Hemostasis and Thrombosis, ed 3. FA Davis, Philadelphia, 1983,
A review of the sequence of events involved in platelet with permission.)
plug formation and the approximate time involved in each
stage are provided in Figure 24-12. Effect of Aspirin on Platelet Plug Formation
Aspirin is a widely used antithrombotic agent for the clini-
Advanced Concepts cal treatment of arterial thrombi. Aspirin exerts a perma-
Platelet Activation nent yet limited effect on platelet aggregation by inhibiting
Binding of ADP to the platelet membrane activates phospho- the action of the cyclo-oxygenase enzyme and production
lipase, an enzyme that cleaves the phospholipids present in of TXA,.”* Platelets are anucleated cells; thus, they lack the
the platelet membrane, freeing fatty acids such as arachidonic ability to synthesize new mRNA or protein. Exposure of
acid. Arachidonic acid is converted to prostaglandin endoper- platelets in the peripheral circulation to a relatively small
oxides in the cytoplasm by prostaglandin synthetase, com- dose of aspirin (60 to 325 mg) results in the irreversible
monly known as cyclo-oxygenase. These endoperoxides are inactivation (acetylation) of the platelet enzyme cyclo-
converted to TXA,, a potent platelet aggregator, mediator oxygenase, and inhibition of TXA, synthesis for the life
of platelet secretion, and a promoter of vasoconstriction span of the circulating platelet.” It is this quality that gives
(Fig. 24-13). With its in vivo half-life of 3 seconds, TXA, aspirin its tremendous therapeutic effect on platelet activa-
activity is limited in time because it hydrolyzes spontaneously tion. Megakaryocytes, however, are capable of synthesizing
within the platelet to an inactive form, |1-dehydrothromboxane cyclo-oxygenase, and newly formed platelets show normal
B, (11-DHTB,).*' As TXA, is generated with subsequent enzyme activity. Aspirin also inhibits endothelial cell
aggregating effects on platelets, calcium, sequestered in the cyclo-oxygenase consistent with the fact that they are
dense tubular system of the platelet, is extruded in the sol-gel genetically identical cellular products. However, endothe-
zone. Thrombin, also a potent platelet aggregator, can induce lial cells possess the organelles necessary to synthesize
secretion of all platelet granules. Serotonin, secreted by the cyclo-oxygenase, thereby regenerating enzyme activity as
dense bodies of the platelet, is a weak aggregating agent as the level of circulating aspirin decreases.
the sole stimulus, but amplifies the aggregating effect of other The effect of aspirin on platelet cyclo-oxygenase is
agonists such as ADP. Serotonin serves as an important vaso- highly sensitive yet quite limited. Platelets activate and
constrictor and a potent stimulator of smooth muscle PGI, aggregate in response to stimuli such as thrombin and colla-
production. gen. There are several other intracellular signaling pathways
 Chapter 24 Introduction to Hemostasis = 557

STIMULATED NUCLEUS

MEMBRANE
PHOSPHOLIPID
Phospholipase Az

ARACHIDONIC ACID
Cyclo- oxygenase

PROSTAGLANDIN
ENDOPEROXIDES
Thromboxane
Synthetase

iyTHROMBOXANE A:
~y (TXAg),
NORMAL

AGGREGATED PLATELETS ENDOTHELIAL CELL

@INHIBITS G@)INDUCES Platelet Aggregation

Figure 24-15 @ Synthesis of prostaglandins in platelets and endothelial cells during platelet plug formation.

that mediate platelet activation and aggregation, but only This system is mediated by many coagulation proteins
one, TXA, synthetase, is inhibited by aspirin.*? Thus, aspirin (coagulation factors), normally present in the blood in an
has a modest effect on platelet function in vivo, causing inactive state. The coagulation factors and their most
moderate prolongation of the bleeding time, moderate inhi- commonly used designations are listed in Table 24-13. Low
bition of platelet aggregation, increase in the PFA-100® clo- levels of the coagulation factors in secondary hemostasis may
sure time [epinephrine/collagen cartridge (Dade-Behring)]** be associated with bleeding that is generally delayed when
and reduction of the aspirin-resistance units using the compared to that observed in defects in the primary hemosta-
arachidonic-acid cartridge with the VerifyNow™™ (Accu- tic mechanism. Symptoms of defective secondary hemostasis
metrics) analyzer. Despite its limited effect on platelet may include soft tissue bleeding, hematomas, retroperitoneal
function, aspirin provides a clinically significant antithrom- bleeding or hemarthrosis. Good examples of these problems
botic effect. are the hemophilias, that are associated with factor VII and
IX deficiencies (see Chap. 26). Secondary hemostasis is the
Secondary Hemostasis: Fibrin-Forming phrase used to encompass the coagulation factors’ role in the
hemostatic mechanism.
(Coagulation) System
Most of the coagulation factors are designated by Roman
The fibrin-forming (coagulation) system is that system numerals. The numerical system adopted assigns the number
through which coagulation factors interact to eventually form to the factors according to the sequence of discovery and not
a fibrin clot. The purpose of fibrin clot formation (secondary to the point of interaction in the cascade. Some factors are
hemostasis) is to reinforce the platelet plug (primary hemosta- routinely referred to by their common names, such as fibrino-
sis). Secondary hemostasis may be started by the release of gen and prothrombin, whereas others are more commonly
tissue factor from epithelial or endothelial cells that are referred to by Roman numeral (such as factor XI, plasma
exposed to the circulatory system at the site of a vascular thromboplastin antecedent factor).
injury. Defects in secondary hemostasis decrease fibrin pro- Activation of a factor is designated by the addition of
duction and reduce the stability of the formed clot. a small “a” next to the Roman numeral in the coagulation
558 — Chapter 24 Introduction to Hemostasis

Table 24-15 Nomenclature eee

of Coagulation Factors —
Factor I Fibrinogen
Factor II Prothrombin Substrate
Factor Il Tissue thromboplastin (tissue factor) Fibrinogen (factor I)
Factor IV Ionized calcium (Ca**) Cofactors
Factor V Labile factor (proaccelerin) Labile factor (factor V)
Factor VI Not assigned* Factor VIII:C (antihemophilic factor, coagulant portion)
Factor VII Stable factor (serum prothrombin Enzymes
conversion accelerator [SPCA] Serine proteases
proconvertin) Ila, Vila, [Xa, Xa, XIa, Xa, prekallikrein
Factor VUI Antihemophilic factor A(AHF), Transaminase
factor VIII:C (coagulant portion) Factor XIIa
Factor 1X Christmas factor (plasma thromboplastin
component (PTC),
Antihemophilic factor B)
Factor X Stuart—Prower factor Except for factor XIII (fibrin-stabilizing factor), all the enzymes
Factor XI Plasma thromboplastin antecedent (PTA) are serine proteases when they are in their activated form. These
Factor XII Hageman factor (contact factor)
proteases have serine as a portion of their active enzymatic site
Factor XII Fibrin-stabilizing factor (FSF), plasma
transglutaminase and function to cleave peptide bonds.
HMWK or HK High-molecular-weight kininogen Factor XII] (fibrin-stabilizing factor) is the only member
(Fitzgerald factor) of the transaminase subgroup. It functions to create cross-
PK Prekallikrein (Fletcher factor) linkages between the fibrin monomers formed during the
*The factor VI designation was dropped because a substance origi- coagulation process to produce a stable fibrin clot.
nally thought to be factor VI was found to be a precursor to factor
V, and to avoid confusion, factor VI has not been reassigned.
Classification of Coagulation Factors
by Physical Properties
cascade (e.g., XII — XIla) unless convention dictates other-
On the basis of their physical properties, the coagulation pro-
wise. For example, most references incorporate “thrombin”
teins may also be divided into three other groups:
into the coagulation cascade rather than its alternate designa-
tion, factor Ila. Some of the common names are derived from 1. The contact proteins
the original patients who exhibited symptoms leading to eluci- 2. The prothrombin proteins
dation of that factor deficiency and an understanding of the role 3. The fibrinogen or thrombin-sensitive proteins
of that factor in the cascade (e.g., Christmas factor, Hageman
Refer to Table 24—15 for details on each group.
factor). Other common names describe the action of the factor
The contact protein group includes factor XII (Hageman
in the coagulation system (e.g., fibrin-stabilizing factor).
factor), factor XI (plasma thromboplastin antecedent),
All the coagulation proteins are produced in the liver.
prekallikrein (Fletcher factor), and high-molecular-weight
The von Willebrand factor portion of the factor VII complex
kininogen (HMWK;; Fitzgerald factor). These proteins are
is produced in other body sites as well; namely, endothelial
involved in the initial phase of intrinsic system activation.
cells and megakaryocytes.
Although deficiencies of these coagulation proteins are asso-
ciated with markedly abnormal laboratory tests, an isolated
Classification of Coagulation Factors factor XI deficiency usually is associated with a mild bleed-
by Hemostatic Function ing disorder (severe bleeding has been reported in some
cases). Interestingly, problems with thrombosis have been
In terms of general hemostatic function, the coagulation reported in patients with factor XII (Hageman factor) and
factors can be divided into three categories: substrate, cofac- prekallikrein (Fletcher factor) deficiencies.
tors, and enzymes (Table 24-14). The prothrombin group (see Table 24-15) generally
Factor I, fibrinogen, is regarded as the main substrate of consists of low-molecular-weight proteins that include
the blood coagulation system because the formation of a fib- factors II (prothrombin), VII (stable factor), [IX (Christmas
rin clot from fibrinogen is the final product. Cofactors are pro- factor), and X (Stuart-Prower factor). Each member of this
teins that accelerate the enzymatic reactions involved in the group contains a unique amino acid: gamma carboxy-
coagulation process. Some examples of blood coagulation glutamic acid, which is necessary for both calcium binding
cofactors are V (labile factor) and VIII:C (antihemophilic and attraction of these coagulation factors to the surface of
factor, or AHF). activated platelets, where the formation of a fibrin clot
The last general category of blood coagulation factors is occurs. Vitamin K is required for the carboxylation of the
the enzyme category. Enzymes involved in coagulation can be glutamic acid residues, therefore, this group is also known
subdivided into two groups: serine proteases and transaminases. as the vitamin K—dependent coagulation proteins. These
 Chapter 24 Introduction to Hemostasis 559

Contact Group Prothrombin Group Fibrinogen Group

Factors XII, XI, PK, HMWK WU WAU 1D OS I, V, VIL, XII

Consumed during coagulation No No (except I) Yes
Present in serum Yes Yes (except ID) No
Present in stored plasma Yes No*
Adsorbed by BaSO, No No
Present in adsorbed plasma NGS
Vitamin K—dependent No No

*Factors V and VIII are not present in stored plasma because of their labile nature, but factors I and XIII are present. PK = prekallikrein;
HMWK = high-molecular-weight kininogen; BaSO, = barium sulfate.

factors may also be selectively removed from plasma by Blood Coagulation: The “Cascade” Theory
adsorption on barium sulfate (BaSQO,).
Drugs that act as antagonists to vitamin K (such as war- The process of blood coagulation involves a series of bio-
farin [Coumadin], commonly used for oral anticoagulant ther- chemical reactions that transforms circulating substances into
apy) inhibit this vitamin K-—dependent reaction, which is an insoluble gel through conversion of soluble fibrinogen to
required for functionally active coagulation factors of the pro- fibrin. This process requires plasma proteins (coagulation
thrombin group.'® Factors II, VII, [X, and X, proteins C and factors) as well as phospholipids and calcium.
S, and protein Z, are still synthesized by the liver, but are non- Blood coagulation leading to fibrin formation can be
functional because they lack the specific receptors for calcium separated into two pathways, extrinsic (Fig. 24-14) and
and cannot enter into the formation of an enzyme-substrate intrinsic (Fig. 24-15), both of which share specific coagula-
complex.” Therefore, patients who are nutritionally vitamin tion factors with the common pathway (Fig. 24-16).*° Both
K-deficient also exhibit decreased production of functional extrinsic and intrinsic pathways require initiation, which
prothrombin proteins. These dysfunctional factors are known leads to subsequent activation of various coagulation factors
as “Proteins Induced by Vitamin K Absence or Antagonists” in a cascading, waterfall, or domino effect. Useful demonstra-
(PIVKAs). tions can be derived from the waterfall or domino concept.
Acquired deficiencies of the vitamin K—dependent coagu- According to the cascade theory, each coagulation factor is
lation factors are relatively common because the body does not converted to its active form by the preceding factor in a series
contain appreciable stores of vitamin K. Clinical situations in of biochemical chain reactions. Ca** participates in some of
which a vitamin K deficiency can develop include: patients the reactions as a cofactor. Each reaction is promoted by the
who have just had surgery and are receiving parenteral feeding; preceding reaction, and if there is a deficiency of any one of
patients who are receiving high doses of intravenous antibi- the factors, the consequences listed in Table 24-16 result.
otics; and patients suffering from liver disease. Table 24-16 Eventually, both the extrinsic and intrinsic systems lead
lists the consequences of a factor deficiency. to the common pathway with generation of the enzyme
The fibrinogen group (see Table 24—15) consists gener- thrombin, which converts fibrinogen to fibrin (Figs. 24-17
ally of high-molecular-weight proteins that include factors I and 24-18). The term extrinsic is used because this pathway
(fibrinogen), V (labile factor), VIII:C (antihemophilic factor), is initiated when tissue factor, a substance not found in blood,
and XIII (fibrin-stabilizing factor). During coagulation, gen- enters the vascular system (see Fig. 24-14). The tissue factor
erated thrombin acts on all the factors in the fibrinogen group. includes a phospholipid component that is the source of
Thrombin enhances the activity of factors V and VIII:C required phospholipid in the extrinsic system (Fig. 24-19).
by converting these proteins into active cofactors, that are Phospholipid provides a surface for interaction of various fac-
involved in the assembly of macromolecular complexes on tors. The phospholipids required in the intrinsic pathway are
the surface of activated platelets. Thrombin converts fibrino- provided by the platelet membrane. In the intrinsic pathway,
gen to fibrin and also activates factor XIII.
Factors V and VIII:C are the least stable factors,
because their activity is relatively labile to degradation and
denaturation. Therefore, testing for factor V and VIII:C
should be rapid, unless appropriate storage measures are
taken. In addition to their presence in plasma, these factors ¢ Coagulation cannot proceed at a normal rate
¢ Initiation of the next subsequent reaction is delayed
are also found within platelets. Some factors within the
¢ The time required for the clot to form is prolonged
fibrinogen group have been reported to increase with ° Bleeding from the injured vessel continues for a longer
inflammation, pregnancy, and with the use of oral contra- time (or there may be a physiologic tendency toward
ceptives. Other physical properties of the coagulation thrombosis present if the patient is deficient in factor XII)
factors are summarized in Table 24-17.
 “UOSOUTUTY JYSIOM-IV[NOOTOWI-YSIYy = YMINH ‘Uo [eyeid = Yd
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Introduction to Hemostasis

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Chapter 24
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 Chapter 24 Introduction to Hemostasis 56]

all the factors necessary for clot formation are intrinsic to the the VIa- tissue factor complex can activate factor [IX to [Xa
vascular compartment because they are all found within the in the intrinsic pathway. In the laboratory, the prothrombin
circulating blood (see Fig. 24-15). time (PT) test is used to monitor the extrinsic pathway (for a
review of the procedure, see Chap. 33).
Extrinsic Pathway (Factor VII)
In the extrinsic pathway, factor VII is activated to factor VIIa Intrinsic Pathway (Factors XIl, XI, IX,
in the presence of Ca** (factor IV) and the tissue factor and VIII)
(factor III), which is released from the injured vessel wall
(see Fig. 24-14). Only factor VIa, Ca**, and factor III (tissue BASIC CONCEPTS
factor) are needed to activate factor X to Xa. Following exposure to negatively charged foreign substances
Figures 24-17 and 24-18 show that the extrinsic path- such as collagen, subendothelium, or phospholipids, activation
way provides a means for very quickly producing small of factor XII involving “contact factors” and factor XI initiate
amounts of thrombin, leading to fibrin formation. In addition, clotting through the intrinsic pathway (see Fig. 24-15).

Extrinsic Pathway
P nat OrA A} wing.
‘ Pen, 25

Tissue Factor ~Bea

Ca‘* platetels yn
Vil
Tapatahize) Vila

ppeioes
athwa

Extrinsic System (VII)

Break in Vessel Wall
releases
(Tissue Factor (TF))
Tissue Thromboplastin (11)

A Vila
TF
complex

Activates

Common Pathway Intrinsic Pathway

K—-> Xayvetc! Xi ala ele.

Figure 24-14 m The extrinsic pathway and the role of factor Vila in activation of factor X and IX. (From American Bioproducts Company.
Modified with permission.)
562 Chapter 24 Introduction to Hemostasis

Intrinsic Pathway

AE: aaHMWK
~~ Kallikrein

ee Xil
Tapa dligem 4 Xila
pathway Teen alice)
pP athwa y

F XII
|
Contact with Collagen

F Xila
HMWK F IX
Prekallikrein
FX! ——— > F Xia —> | CA
Common Pathway
F lXa Ca'PF 3] Activates (X———— Xa) etc.
Villa
Intrinsic System
(Factors XIl, XI, IX, VIII)

Figure 24-15 @ The intrinsic pathway and its role in activation of factor X. HMWK = high-molecular-weight kininogen; PF3 = platelet
factor 3. (From American Bioproducts Company. Modified with permission.)

Once generated, factor XIla, in the presence of Fitzger- (Table 24-18). Factor VII complex consists of two main por-
ald factor (HMWK) and Fletcher factor (prekallikrein), con- tions, factor VIII:C (the procoagulant protein) and vWF (the
verts factor XI to XIa. Factor XIa is capable of activating carrier protein).
factor XI without HMWK, but the activation takes place It should be noted that factor VIII requires enhancement
much more slowly.”’ by the generated enzyme thrombin to amplify its activity.?’ In
The next reaction in the intrinsic pathway is the activa- the laboratory, the activated partial thromboplastin time
tion of factor [IX to factor [Xa by factor XIa, in the presence (APTT) test is used to evaluate the intrinsic pathway. During
of Ca**. Activated factor [X (IXa) participates, along with the laboratory testing, the intrinsic pathway is initiated in vitro by
essential cofactor VIII:C, in the presence of ionized calcium activation on negatively charged surfaces, such as glass or
and PF3, a source of phospholipids, to activate factor X, kaolin; or chemically by ellagic acid (for a review of this pro-
which leads to the generation of thrombin and formation of cedure, see Chap. 33).
fibrin. The complex consisting of factor [Xa-factor VIIla- The factor VIII complex, which consists of several com-
phospholipid-Ca** has been called the tenase complex ponents, comprises the largest protein involved in the coagu-
because it activates factor X (Fig. 24-20). '” lation cascade. The major portion of this protein complex is
The macromolecular complex of factors [Xa, VIIIa, considered to be the carrier protein called von Willebrand
X, PF3, and Ca’* assembles on the surface of the activated factor (vWF), although this is not the portion active in the
platelet (providing the phospholipid) during the intrinsic coagulation cascade. A smaller subunit or protein that is
pathway of blood coagulation.*’ This surface provides a associated with factor VIII is responsible for the clotting or
protective environment that facilitates the enzymatic reac- procoagulant activity of factor VII (VIII:C). The C in the
tions of the coagulation cascade without interference from expression VIII:C stands for “coagulant.” It is factor VIII:C
the physiologic anticoagulants normally present in plasma. that is functionally active in the coagulation cascade.
In regard to the intrinsic pathway, it is also important to The vWF portion of the complex carries the VIII:C pro-
be familiar with the properties of the factor VIII complex coagulant portion. The vWF portion may exhibit a stabilizing
 Chapter 24 Introduction to Hemostasis 563

monomer, and helps to stabilize the fibrin monomer by convert-

ing factor XIII to XIIla, which cross-links the fibrin monomers
to form stable fibrin polymer. Thrombin also has other actions
(see later discussion of Thrombin-Mediated Reactions in Hemo-
stasis [Advanced Concepts]). Because the common pathway
contains the factors X, V, I, and I, these factors may be moni-
tored by both the PT and the APTT.
From the extrinsic system, factor VIIa in the presence of
Fibrinogen tissue factor and ionized calcium converts factor X to Xa.
\—FpA From the intrinsic system, the tenase complex (factor [Xa in
Fibrin Monomer the presence of factor VHI:C, phospholipid [PF3], and Ca’*)
converts factor X to Xa (see Figs. 24-17 and 24-18).
Fibrin Polymers After the formation of factor Xa, this activated factor,
along with cofactor V, in the presence of Ca** and phospho-
Fibrin Clot
lipid (PF3), converts factor H, prothrombin, to the active
enzyme thrombin (Ila). The phospholipid is present to pro-
vide surfaces so that prothrombin and factor X can be bound
by bridges of ionized calcium.'* The association of factor Xa,
factor V, phospholipid, and Ca’* is called the prothrombinase
complex (or the prothrombin activator) because it enzymati-
cally converts the substrate prothrombin to the enzymatically
active thrombin (Fig. 24—21).'’ This additional macromolecu-
lar complex of factors Xa, Va, Ca**, PF3 (the platelet mem-
Prothrombin (II)
brane phospholipid), and prothrombin also assembles on the
PAL,
Catt surface of activated platelets.
DE tamer Peet
Va Activation of thrombin is slow, but once generated, it fur-
XI ther amplifies coagulation (Table 24-19). Thrombin acts on
Thrombin (Ila) ——- {
fibrinogen to form fibrin monomers. Fibrinogen 1s composed
Fibrinogen ____, Soluble _ Xia, Stable of three pairs of polypeptide chains (two alpha [a] chains, two
(1) Fibrin Fibrin beta [8] chains, and two gamma [y] chains). Thrombin cleaves
Monomer Polymer a portion of each of the a and £ polypeptides to form fib-
rinopeptides A and B. Most of the a and 6 chains are still left
| on the fibrinogen molecule. After this cleavage of fibrinopep-
Common Pathway Fibrin Clot
tides A and B, the remainder of the fibrinogen molecule is
(Factors X, V, I, I) called the fibrin monomer (Fig. 24-22).
By using the PT and APTT test results in the laboratory,
Figure 24-16 @ The common pathway and formation of fibrin one can identify defects or deficiencies as occurring in the
clot. PL = phospholipid source. (From American Bioproducts intrinsic, extrinsic, or common pathways of blood coagulation
Company. Modified with permission.) with the exception of factor XIII functional deficiency. Using
Table 24—20, the reader can practice interpretation of these
tests in identifying possible factor deficiencies. However, the
effect over factor VIII:C by protecting it from proteolytic most common cause of a prolonged PT or APTT is anticoag-
activity.'' Because of VWF’s extremely large size and its abil-
ulant therapy.
ity to bind to the platelet membrane GPIb and IIb/IIIa recep-
tors, it appears to have a role in anchoring the platelet plug to
the vessel breach."’
Thrombin-Mediated Reactions
in Hemostasis (Advanced Concepts)
Common Pathway (Factors X, V, II, and 1)
Each of the four major components of coagulation, and the
The terms intrinsic and extrinsic pathways really no longer fit various substances produced by each component, help to regu-
with the current understanding of hemostasis; however, they are late the basal state of hemostasis by which blood remains fluid
still used to identify the coagulation factors.* The conmmon and cell surfaces remain nonthrombogenic. Vascular damage
pathway begins with the activation of factor X by the intrinsic initiates physical, cellular, and molecular changes to platelets,
system, the extrinsic system, or both (see Figs. 24-16 and endothelium, and circulating coagulation proteins. When the
24-17). Factor Xa, in the presence of factor V, Ca**, and phos- inert proenzymes of coagulation become activated, inhibitors
pholipid (PF3), converts prothrombin to its active form, throm- irreversibly bind to these activated factors, thus controlling the
bin. Thrombin then takes the following actions: it feeds back to hemostatic response. Numerous pathways exist that ultimately
activate factors VIII and V, converts fibrinogen to soluble fibrin lead to the generation of thrombin, and consequently fibrin
564 = Chapter 24 Introduction to Hemostasis

INTRINSIC SYSTEM

Contact Collagen
‘a -e oeaa
XII alte Xlla MWK
HMWK PK
X| —— Xla
|Catt 2
IX ——-> IXa
t
AO) SSN

Blood Coagulation: The Cascade Theory

enzymes which activate

— — — — amplifiers
cofactors: (V and VIII)

Lab Tests

APTT Monitors: PT Monitors:

XII, XI, IX, VIII (Intrinsic) VII (Extrinsic)
X, V, Il, | (Common) X, V, Il, | (Common)

Figure 24-17 @ Blood coagulation: The “cascade” theory of coagulation. The extrinsic system, the intrinsic system, and the common
pathway and the appropriate laboratory tests for evaluation of each. HMWK = high-molecular-weight kininogen; PF3 = platelet factor 3;
PK = prekallikrein; PT = prothrombin time; APTT = activated partial thromboplastin time.

formation, following vascular injury. Thus, thrombin has a cen- Thrombin Formation: Role of Extrinsic
tral role in the bioregulation of hemostasis in both normal and Pathway
pathologic conditions. The following discussion provides an
overview of thrombin-mediated mechanisms in hemostasis. Thrombin’s main duty is to cleave fibrinopeptides A and B
The various roles of thrombin in hemostasis are summarized in from the alpha and beta chains of the fibrinogen molecule,
Table 24—21. thus triggering fibrin polymerization. Also thrombin amplifies
the coagulation mechanism by activating cofactors V, VIII
Thrombin-Mediated Platelet Aggregation and factor XI.'’ Formation of thrombin occurs by way of the
extrinsic pathway and tissue factor. Tissue factor, a membrane
Thrombin is considered to be a potent platelet-aggregating glycoprotein, is released from cells and tissues and binds to
agent. The binding of thrombin to specific platelet membrane factor VII in circulation, thus activating factor VII to factor
receptors initiates cellular events leading to platelet secretion Vila. The TF—VIla complex then activates either factor LX or
and aggregation. Thrombin promotes secretion of serotonin, a X. The prominent pathway is thought to be activation of
vasoconstrictor; and TXA,, a platelet-aggregating agent. As factor IX to promote coagulation and fibrin formation.
a result of thrombin-induced secretion, vessels constrict,
limiting blood flow, and platelets aggregate. The hemostatic Thrombin Formation: Role of Common
plug grows in size, eventually being enmeshed within fibrin. Pathway
Activated platelets provide surface phospholipids for the
assembly of coagulation factors and further thrombin Factors X, II (prothrombin), I (fibrinogen), and cofactor V
generation. are critical to the formation of a thrombus in circulation.
 Chapter 24 Introduction to Hemostasis 565

Coagulation Cascade
Intrinsic Pathway Extrinsic Pathway

@e gpgHMWK
oe << & PK Tissue Factor eo.
Ss : Kallikrein
Cat platelets a
Vg
LO 7 in-vitro 7 vil
v@ pathway in-vivo _Mila
7athway

Fibrinogen
Laey.\
Ee Xxill
mleldim(oyatelint=\g

Fibrin Polymers

Fibrin Clot

QD
ae |

Fibrinolytic Pathway
»

Figure 24-18 @ An overview of coagulation cascade, the intrinsic and extrinsic pathways, and the interaction between the two. The fibrinolytic
pathway and its action on fibrinogen and fibrin. HMWK = high-molecular-weight kininogen; PK = prekallikrein; FoA = fibrinopeptide A. (From
Diagnostica-Stago, Inc., with permission.)

Activated factor [Xa (via factor VIIa) factor X, Ca**, and thrombin from prothrombin at a faster rate. Increased throm-
thrombin-activated VIIla assemble on membrane platelet bin concentrations serve to amplify the activation of cofactors
phospholipid and catalyze the activation of factor X. Newly V and VIII, which, in turn, leads to enhanced thrombin forma-
generated factor Xa, membrane-bound thrombin-activated tion from its precursor prothrombin.
factor Va, Ca?*, and factor II (prothrombin) assemble on the
platelet membrane, forming the prothrombinase complex. Thrombin-Mediated Anticoagulant Activity
This complex catalyzes the conversion of prothrombin to
thrombin. Thus, this thrombin-modulated pathway provides a Antithrombin (AT), formerly known as AT III, is the main
positive feedback mechanism to amplify the generation of physiologic inhibitor of thrombin, factors Xa, [Xa, XIa, and
566 Chapter 24 Introduction to Hemostasis

Phospholipid, Ca*t -Dependent Reactions

of Extrinsic and Common Pathway.
Factor Factor Factor
Vila Xa X

Factor Factor Factor

TF Vila IX or X
Complex"

Factor Factor Factor

©
"Prothrombinase @
TF Tissue Factor Complex" %$$6666666066666
|
Gla = gamma-carboxy-glutamic acid
Catt Calcium ions
a activated factor Factor
PL Phospholipid source Thrombin (Ila)

Figure 24-19 m Platelet membrane phospholipid provides a surface for the interaction of coagulation factors and the formation of “tenase
complex” and “prothrombinase complex.”

XIa, activated protein C, and kallikrein (Fig. 24—23). In the

presence of heparin, the inactivation of thrombin and factor
Xa by AT is significantly increased. AT is consumed in the
inhibition of thrombin. I. Smaller protein subunit
1. Nomenclature
The role of protein C (a vitamin K—dependent factor) as
VIII (referring to the procoagulant portion)
an anticoagulant is related to the presence of thrombin, VIII:C (for coagulant)
thrombomodulin, and protein S.** Protein C is activated by 2. Components
thrombin and endothelial cell thrombomodulin. The forma- a. VIII:C/Ag—antigenic determinant of VIII, measured
tion of the thrombin-thrombomodulin complex accelerates by immunoassays with human antibodies to VII
b. VIII:C—procoagulant property of normal plasma
the activation of protein C. Activated protein C exerts an
measured in the APTT test as coagulant activity
anticoagulant effect by inactivating cofactors Va and VIIIa, 3. Characteristics
thus slowing the rate of thrombin formation.*? It is important a. Inherited as sex-linked recessive
to note that activated protein C does not inhibit the other b. Acts as a cofactor in a complex with factor IXa,
regulatory roles of thrombin in hemostasis. The inhibition of Ca**, and PF3 to activate factor X to Xa
If. Major protein portion
cofactors Va and VIIa by protein C is enhanced by the
1. Nomenclature
presence of protein S, another vitamin K—dependent factor. a. VWE (von Willebrand factor)
Activated protein C is also inhibited by other plasma b. VUI:vWF
proteins. The role of activated protein C is to turn off the . Components
amplification pathway of thrombin generation. It is also a. VWWF:Ag—antigenic determinant on vWF that is
detected by using heterologous antibodies to vWF
b. Ristocetin cofactor (VIII:Rco)-the property of nor-
Factor X mal plasma vWF that supports ristocetin-induced
INTRINSIC EXTRINSIC agglutination of washed normal platelets
Factor IXa . Characteristics
Factor Vila
Cat* Cat+ a. Usually inherited as autosomal dominant
Phospholipid (PF 3) b. Responsible for platelet adhesion
Factor VIII:C Me es c. Responsible for ristocetin-induced aggregation of
platelets
“Tenase” Complex “Tenase” Complex d. Stabilizes VIII:C when bound to vWF during circu-
lation, and functions in protection of VIII:C from
proteolytic inactivation or removal from the
Factor Xa circulation

Figure 24-20 @ Activation of factor X at the beginning of the APTT = activated partial thromboplastin time.
common pathway and the “tenase” complex.
 Chapter 24 Introduction to Hemostasis 567

Prothrombin Table 24— 20 Interpretation. |

Factor Xa
A _ of Coagulation Test.
Factor V Results oan é
Phospholipid (PF 3)
Cant
Test Battery _ Results Possible Cansess:

“Prothrombinase” APTT eeotral ; Vien K Rereer

Complex Abnormal . Liver disease
Normal . Inhibitor present
Thrombin
. Factor deficiency in
Figure 24-21 i Conversion of prothrombin to thrombin by common pathway
prothrombinase complex. (X,V,ID
Abnormal . Factor deficiency in
Normal the intrinsic pathway
. Lupus anticoagulant
. Specific factor
inhibitor (VIII inhibitor)
Normal . Factor deficiency in the
Abnormal extrinsic pathway
. Specific factor inhibitor
Abnormal . Factor deficiency (I)
Abnormal . Severe liver disease
¢ Converts fibrinogen to fibrin
Abnormal EDIC
¢ Activates factor XUI
. Potent inhibitor
¢ Enhances activity of factors V and VII
. Hypofibrinogenemia or
¢ Induces platelet aggregation
_dysfibrinogenemia _

Pr = Sonoibe time; APTT = activated partial thromboplastin time;

TT= thrombin time; DIC = disseminated intravascular coagulation.

THROMBIN ACTION interesting to note that the reactions that lead to thrombin
on formation from its precursor prothrombin are regulated in
FIBRINOGEN
Thrombin Cleaves part by thrombin-mediated mechanisms (see Chap. 28 for a
+4 detailed discussion of anticoagulant therapy).
alpha chain be
sc chains mine ae _ [eae
gamma chains Cia
> Fibrinogen Conversion and Fibrin
thrombin
Stabilization via Thrombin
= EEE a
ome =al Thrombin proteolytically cleaves first fibrinopeptide A, then
sr) o
fibrin
fibrin monomer
peptides
fibrinopeptide B from the @ and B chains of fibrinogen,
A,B respectively, forming a soluble fibrin monomer. Fibrin
spontaneous polymerization monomers polymerize in an end-to-end and side-to-side
manner held together by weak hydrogen bonds. Factor XIII
is activated to factor XIIa by thrombin (in the presence of
hydrogen bonds Ca**) and catalyzes the cross-linking of glutamine and lysine,
weak fibrin polymer thus polymerizing soluble fibrin monomers to an insoluble
fibrin meshwork.

Thrombin-Mediated Tissue Repair

Thrombin has numerous effects on the cells that play a role in
tissue repair and wound healing. Shortly after thrombin gen-

= = = Pe eration, there is increased vascular permeability and increased

adhesion of leukocytes to endothelial cells mediated by vari-
ous adhesion molecules secreted by platelets and endothelial
Cross-Linked Fibrin Polymer cells. Neutrophils and monocytes undergo chemotaxis in
(Stable Fibrin Clot) response to thrombin. Thrombin mediates the release of a
potent smooth muscle mitogen (PDGF, or platelet-derived
Figure 24-22 @ Thrombin activity on fibrinogen. growth factor) as well as stimulates the proliferation of
568 — Chapter 24 Introduction to Hemostasis

Antithrombin (AT) and Protein C Pathways

(Thrombin)
Inactive
AT Complexes

/ Indicates an activation pathway

_” Indicates an inhibition pathway

Figure 24-23 @ The inhibitor pathway of coagulation. AT = antithrombin; PC = protein C; APC = activated protein C; PS = protein S;
C4b-BP = C4b-bound protein; TM = thrombomodulin.

fibroblasts, smooth muscle cells, and endothelial cells, thus

aiding in vascular repair.

Blood Coagulation: A Cell-Based Model

of Hemostasis
A cell-based model of hemostasis has been proposed to
eee
©

Boe replace the traditional “cascade” model of coagulation.*°*!

According to this model, specific cellular surface receptors for
Procoagulant
the coagulation proteins promotes hemostasis which occurs in
¢ Induces platelet activation and aggregation three overlapping phases: (1) initiation, (2) amplification and
¢ Activates cofactor VIII to VIIa
¢ Converts fibrinogen to fibrin (3) propagation.*”*! Basically, two cell surfaces are involved in
¢ Activates factor XIII to XIIa the cell-based model; namely, a tissue factor (TF) bearing cell
¢ Via autocatalysis converts prothrombin to thrombin (such as a fibroblast or a white blood cell) and platelets.*?
Coagulation Inhibitor Phase 1, initiation, occurs on a tissue factor-bearing cell.
During initiation, factor VIIa bound to tissue factor activates
* Binds with antithrombin III to inhibit serine proteases
(XIla, XIa, Xa, [Xa, and kallikrein) X to Xa, as well as IX to [Xa. The Xa/Va complex which forms,
¢ Promotes endothelial cell release of t-PA results in the formation of small amounts of thrombin (IIa).
* Binds to thrombomodulin to activate protein C (inhibits Va The initiation phase is rapidly shut down by the tissue factor
and VIIIa) pathway inhibitor (TFPI). Phase 2, amplification, occurs when
Tissue Repair platelets and the cofactors are activated in order to set the stage
¢ Induces cellular chemotaxis
for large scale thrombin generation. The small amount of
* Stimulates proliferation of smooth muscle and endothelial thrombin (Ila) generated from phase | activates platelets, caus-
cells ing release of factor V from the a granules of the platelet,
which is activated to Va; which in turn, activates factor XI to
 Chapter 24 Introduction to Hemostasis 569

XIa. In addition, factor Va cleaves factor VIII from von The fibrinolytic system is mediated mainly by the
Willebrand’s factor (vWF) activating it to VIIa. Recent studies enzyme plasmin, which acts primarily on fibrin to produce
have shown that activated platelets can synthesize TF.’***° lysis of the clot. Plasmin is generated from the inactive zymo-
Phase 3, propagation, occurs when large amounts of gen called plasminogen. Plasminogen is activated to plasmin
thrombin are generated on the platelet surface. The factor XIa by t-PA and other substances (see Chap. 27).
generated from the amplification phase binds to the activated A number of plasmin inhibitors exist to keep fibrinolysis
platelets and subsequently activates factor X to Xa. The acti- from getting out of control. In addition to plasmin, plasmino-
vated platelet surface protects FXla from an inhibitor by a gen and plasminogen activators, inhibitors of plasmin are a
specific protease. FXIa can efficiently activate FLX to F[Xa on part of the fibrinolytic system.*” Some inhibitors of plasmin
the platelet surface, inducing an explosive thrombin forma- are listed in Table 24-22.
tion by activating X to Xa, which moves directly into a pro- It is important to realize that some of the same substances
tected complex with factor Va. This results in a burst of that initiate or enhance clot formation also initiate clot degrada-
thrombin generation as the Xa/V complex activates prothrom- tion. For example, in tissue, both tissue thromboplastin (initiator
bin to thrombin (Fig. 24-24). of extrinsic pathway of fibrin formation) and t-PA (which acti-
The extrinsic or tissue factor (TF) pathway works on the vates plasminogen) are released with endothelial damage. t-PA
initiating cell during phase 1. The prothrombin time (PT) is produced by vascular endothelial cells and selectively binds
assays the factors in the extrinsic/initiation pathway. The to fibrin as it activates fibrin-bound plasminogen. Because cir-
intrinsic pathway works on the platelets during the amplifica- culating plasminogen is not activated by t-PA, this biologic sub-
tion and propagation stage. The APTT assays the factors in stance is efficient in dissolving a clot without causing systemic
the intrinsic/platelet surface pathway. fibrinolysis and serves as an ideal therapeutic fibrinolytic agent.
Biologic t-PA has been successfully produced by recom-
binant deoxyribonucleic acid (DNA) technology and is cur-
Fibrin-Lysing (Fibrinolytic) System rently available. Inhibitors of t-PA also exist and contribute to
The fibrin-forming and fibrin-lysing systems are intimately controlling its actions.*? It is also important to note that
related. Activation of coagulation also activates fibrin lysis. thrombin generates both fibrin and plasmin formation.
Fibrinolysis, the physiologic process of removing unwanted
fibrin deposits, represents a gradual progressive enzymatic
cleavage of fibrin to soluble fragments. These fragments are
then removed from the circulation by the fixed macrophages
of the mononuclear phagocytic system (MPS). This action of
the fibrinolytic system re-establishes blood flow in vessels ¢ «,-Antiplasmin (a rapid inhibitor of plasmin)
occluded by a thrombus and facilitates the healing process * a,-Macroglobulin (a slower inhibitor of plasmin)
following injury. For a detailed description of the fibrinolytic ¢ Others (see discussion in text of protease inhibitors)
system refer to Chapter 27.

CELL-BASED MODEL OF HEMOSTASIS

Free vWF
1. Initiation (Small amount
of thrombin)

Tissue-factor bearing
Villa
cell (fibroblast)
IX
Platelet

2. Amplification

lla
(Large amounts
of thrombin)

Activated platelet

Figure 24-24 m Cell-based model of hemostasis. Phase 1 = initiation; phase 2 = amplification; phase 3 = propagation.
570. Chapter 24 Introduction to Hemostasis

Action of Plasmin The action of plasmin on cross-linked, stabilized fibrin

(fibrinolysis) yields similar products: a Y-Y fragment, com-
Plasmin is a broad-spectrum endopeptidase (proteolytic posed of a central cross-linked D-D ___domain flanked on each
enzyme) that acts nonspecifically, but with a stronger affinity side by E-domains; the E-domains are subsequently cleaved
for fibrin.*’ Plasmin, however, cannot distinguish between the leaving only the central cross-linked D-D domains and
proteins fibrin or fibrinogen. The action of plasmin on fibrino- E-domains as fragments. These products are collectively
gen (fibrinogenolysis) begins by cleaving portions of each of known as either fibrin(ogen) degradation products (FDPs)
the a and B polypeptide chains (a larger portion than that or fibrin(ogen)-split products (FSPs).'’ The D—D fragment,
cleaved by thrombin), and a smaller portion of each of the two called D-dimer, is separately detectable by a monoclonal
y polypeptide chains. The remaining molecule is called the X antibody immunoassay to provide evidence of cross-linking.
monomer (which is still thrombin-clottable), composed of a Pathologic degradation of fibrinogen, as occurs when free
central E-___domain bonded on each side to a D-___domain . plasmin is present, yields D and E fragments but no detectable
As fibrinogenolysis progresses, a D-___domain is cleaved D-dimer. The various fragments may be detected by either
from the X monomer forming a Y fragment (no longer clot- quantitative or semiquantitative immunoassays, revealing
table by thrombin), and subsequently, smaller D and E frag- fibrinolytic activity."
ments.'’ Therefore, the final fibrinogen-degradation (split) The term fibrin(ogen) means that the FDPs can come from
products are two D fragments and one E fragment generated either fibrinogen or fibrin. Early FDPs include X monomer and
from one molecule of fibrinogen (Fig. 24—25). Y fragments; late FDPs include D and E fragments. These frag-
ments are important clinically because they can increase vascu-
lar permeability and interfere with thrombin-induced fibrin
formation. In patients with certain disease conditions, when
plasmin is activated, FDPs are measured (see Chaps. 27 and 28
PLASMIN ACTION for further information). In the presence of disseminated
on
FIBRINOGEN intravascular coagulation (DIC) the FDP’s and D-dimer prod-
ucts may both be positive. The quantitative D-dimer test is also
a Plasmin Cleaves ae
useful as a negative predictor in ruling out deep vein thrombo-
sis or pulmonary emboli (see Chap. 28).
In addition to its action on fibrin and fibrinogen, plasmin
also destroys factors V, VII, and other coagulation factors.
The actions of plasmin are summarized in Table 24-23.

(M.W. 340,000) -
Fibrinopeptide
plasmin cleaves
AandB Kinin System
The kinin system, important in inflammation, vascular perme-
ability, and chemotaxis, is activated by both the coagulation
and fibrinolytic systems (see Chap. 27).
Prekallikrein (PK) and high-molecular-weight kininogen
(HK) are additionally needed to enhance or amplify the contact
thrombin clottable factors involved in the intrinsic system (Fig. 24-26). Specifi-
cally, factor XI]a in the presence of HK converts prekallikrein
plasmin cleaves
to kallikrein. Kallikrein feeds back to accelerate the conversion
AlYva
ddd of factor XII to XIIa, speeding up intrinsic system processes.

Table 24-25 The Actions of Plasmi

not clotted by thrombin * Destroys fibrinogen and fibrin
* Produces fibrinogen degradation products, which increase
vascular permeability and interfere with thrombin-induced
fibrin formation
* Produces D-dimer, a degradation product specifically
derived from cross-linked stabilized fibrin polymer
* Destroys factors V, VIII, IX, XI, and other plasma proteins
31V1
dds * Indirectly enhances or amplifies conversion of factor XII to
XIa
* Enhances or amplifies conversion of prekallikrein to
kallikrein, liberating kinins from kininogen
* Cleaves C3 into fragments (see later discussion of comple-
Figure 24-25 @ Plasmin activity on fibrinogen. FDP = fibrin ment system)
degradation product.
 Chapter 24 Introduction to Hemostasis 57]

Y, Intrinsic
YA XI >Xla— coaguiation |

HMW |Kininogen Prekallikrein

(Fletcher)
_Very slow
slow: MW
[Fibrinolysis]
“normally” Kila ee

System
: (C, cleaved into} |
HMW (Fitzgerald) C;, and C5,)
Kallikrein > ;
Kininogen
| Plasminogen

Kininogens

LMW HMW
Kininogen Kininogen
(Fitzgerald)
“a v
: a Bradykinin Kinins formed 27
Se Kallidin

Figure 24-26 @ Interrelationship of coagulation, fibrinolytic, kinin, and complement systems. HMW = high molecular weight; LMW = low
molecular weight.

The activation of factor XII acts as the common link clotting factors, play an important role as mediators of both
between many aspects of the hemostatic mechanism, includ- immune and allergic reactions. The reactions in which com-
ing the fibrinolytic system, the kinin system, and the comple- plement participates take place in the blood or in other body
ment system (see Fig. 24-25 and Chap. 27). fluids. The most important biologic role of complement is the
production of cell membrane lysis of antibody-coated target
cells. Two independent pathways of activation of the comple-
Protease Inhibitors ment cascade may occur along with a common cytolytic path-
Because the fibrinolytic system is activated when coagula- way. These are designated the classic and alternate pathways
of complement activation (see Chap. 13 for a review of the
tion is activated, extra fibrin is degraded and eliminated
along with some of the coagulation factors. However, complement system).
enzymes such as plasmin and kallikrein still circulate until Plasmin activates complement by cleaving C3 into C3a
and C3b. Cl esterase inhibitor inactivates complement and
they are eliminated by:
also has a role in hemostasis, as described earlier.
1. Liver hepatocytes (which have an affinity for activated en-
zymes),
2. MPS cells (which pick up particulate matter), or
3. Serine protease inhibitors present in plasma.”
Serine protease inhibitors attach to various enzymes and
inactivate them. Some important serine protease inhibitors are ¢ Antithrombin III
listed in Table 24—24. ¢ a,-Macroglobulin
¢ a,-Antiplasmin
* a,-Antitrypsin
Complement System ¢ Cl esterase inhibitor
¢ Protein C inhibitor
The complement system is composed of approximately 22 ¢ Protein S inhibitor
serum proteins that, working together with antibodies and
572. Chapter 24 Introduction to Hemostasis

Both the coagulation system and the fibrinolytic system The bleeding time has been utilized as a screening tool
are interrelated with the complement system.** The interrela- to determine the risk of bleeding in many clinical settings;
tionship of the coagulation, complement, and fibrinolytic however, there is now a large body of literature that
systems is discussed in Chapter 27. has shown no correlation with risk of bleeding in these
instances.
Recent development of an automated platelet function
Laboratory Evaluation of Hemostasis analyzer (PFA-100) has made the bleeding time almost

The diagnosis of any hemostatic disorder is made by the

obsolete. This instrument allows the patient’s blood to be
systematic evaluation of information obtained in the history drawn in a citrated tube. The citrated blood is then run
through a cartridge with either ADP and collagen or epi-
and physical examination, along with the appropriate labora-
nephrine and collagen. The cartridge has a small aperture
tory testing. Diagnostically, the most valuable data from a
and the instrument measures the amount of time required for
patient’s history include:
closure of the aperture by a small platelet plug. In some
* Documentation of the physical appearance, site, severity, instances, the collagen/epinephrine cartridge is run first, and
and frequency of bleeding episodes if abnormal, the collagen/ADP cartridge is run; however,
¢ A reliable patient and family history of bleeding disorders often the laboratories run both cartridges as a platelet func-
¢ An accurate drug history tion screen (see Chap. 33).
¢ Other contributing or underlying illnesses The PT and APTT are both variations of plasma recalci-
fication times accelerated by the addition of a thromboplastic
Bleeding disorders present themselves differently, depend-
substance. The PT reagent contains thromboplastin and cal-
ing on the causative problem. Two general rules apply: first,
cium chloride that, when added to patient plasma, initiates
patients with platelet disorders usually exhibit petechiae and
mucous membrane bleeding. In general, this is because a defect rapid formation of a fibrin clot. The APTT reagent contains
of primary hemostasis is present, resulting in the formation of a phospholipid substitute, activator, and calcium chloride to
defective platelet plug. Second, patients with coagulation initiate fibrin clot formation.
As mentioned previously, the PT test measures the
defects may develop deep spreading hematomas and bleeding
into the joints with evident hematuria. In general, this is factors of the extrinsic and common pathways of coagulation
(factors VII, X, V, II, and I). Factor VII is the only factor listed
because a defect of secondary hemostasis is present, resulting
that is restricted to the extrinsic system, as factors X, V, I,
in the inadequate fibrin reinforcement of a functionally normal
platelet plug. and I are part of the common pathway (see Figs. 24-17 and
Alteration of any aspect of the hemostatic mechanism 24-18). The PT test is ideally used to detect early vitamin K
may cause abnormal bleeding in a wide variety of familial deficiencies, because factor VII has the shortest half-life of
and acquired clinical disorders. These defects may be classi- the coagulation factors and is vitamin K—dependent. The PT
fied into three broad categories that can be diagnostically test 1s used in conjunction with the International Normalized
approached by a systematic laboratory evaluation. These Ratio (INR) to monitor subjects on oral anticoagulant therapy.
include vascular and platelet disorders, coagulation factor Any abnormalities of these factors, a vitamin K defect, liver
deficiencies or specific inhibitors, and fibrinolytic disorders. disease, or the presence of inhibitors will result in an
Although many laboratories differ in their approach to a abnormally prolonged PT.
bleeding disorder, a general profile of laboratory tests is usu- The APTT test measures factors of the intrinsic and
ally established. This profile can often be used as a means of common pathways of blood coagulation (XII, Fletcher,
differentiating various hemostatic problems. Laboratory Fitzgerald, XI, IX, VII, X, V, I, and I). It should be noted
screening tests routinely ordered to assess hemostatic dys- that factors XII, XI, LX, VIII, Fletcher, and Fitzgerald are
function are listed in Table 24-25 (see Chap. 33). limited to the intrinsic system. Deficiencies or inhibitors of
any of these factors will result in an abnormally prolonged
APTT. Both the PT and APTT tests will show prolonged
results with an abnormality of the shared factors of the
common pathway (X, V, II, and I). Usually the APTT will
only be prolonged in fibrinogen (factor I) deficiencies if the
level is less than 100 mg/dL. A factor abnormality refers
to a deficiency of that factor in plasma for any one of the
following reasons:
* Platelet count 1. Decreased synthesis
* Peripheral blood smear examination 2. Synthesis of a dysfunctional factor molecule
* Ivy bleeding time (IBT) or platelet function assay 3. Excessive destruction of factors through acquired disorders
(PFA-100)
¢ Prothrombin time (PT) 4. Inactivation of factors through circulating inhibitors
* Activated partial thromboplastin time (APTT) The interpretation of the PT and PTT test results is sum-
¢ Thrombin time (TT)*
marized in Table 24—20.
*Included less often. Neither the PT nor the APTT screens for factor XIII
activity. The PT and APTT assays test for the initial conversion
 Chapter 24 Introduction to Hemostasis 573

of fibrinogen to fibrin. Cross-linked stabilized fibrin, which 1. Screening tests for vascular or platelet dysfunction (such as
develops later through mediation of factor XIIa, does not bleeding time, PFA-100, platelet aggregation using platelet-
have an impact on the PT or APTT. Special testing to assess rich plasma or whole blood, and PF3 assay)
factor XIII activity must be done (see Chap. 33). 2. Tests for coagulation (such as factor assays)
PT and APTT testing has been reported in a variety of 3. Special tests (e.g., for fibrinolytic disorders, tests for deter-
ways, such as patient seconds and control seconds. Past mination of FDPs, D-dimers, plasminogen, t-PA, or ELISA
reporting, using ratios for PT testing and percentage, have assays for detection of fibrin monomers)
largely been abolished. The International Normalized Ratio
The reader may refer to subsequent chapters for a detailed
(INR) is the method of choice for PT reporting, because it
discussion of vascular and platelet-related disorders (see
adjusts for source-related thromboplastin sensitivity differ-
Chap. 25), plasma clotting factor defects (see Chap. 26), inter-
ences and testing methodology through use of a mathematical
action of systems involved in hemostasis (see Chap. 27), throm-
exponent, the International Sensitivity Index (ISI).*? The ISI
bosis and anticoagulant therapy (see Chap. 28), and laboratory
is unique to each batch of thromboplastin/instrumentation
methods (see Chap. 33).
combination and is furnished by the manufacturer.
Numerous articles state the method for reporting
INR values. INR standardizes PT reporting worldwide by
adjusting all reported values to a World Health Organiza-
tion international reference thromboplastin standard. Case Study
Therefore, all PT results reported by INR methodology are
A 4-year-old boy was brought to the emergency department
theoretically comparable. Using the INR values facilitates
by his parents. The boy had fallen off of the monkey bars in a
optimal oral anticoagulant therapy in patients at risk for
park and hit his thigh against one of the bars during the fall.
thrombosis, especially those on warfarin who travel exten-
His thigh became swollen and painful. There was a history of
sively, and require frequent monitoring. Thromboplastins bleeding in the male family members. They had been labeled
with a low ISI (less than 1.2) correlate better to recombi- as “bleeders” in their family history. Coagulation studies were
nant thromboplastin, which replaced human-brain thrombo- performed that showed a normal PT, prolonged APTT, and
plastin for standardizing INR values by the World Health normal platelet count and bleeding time.
Organization.*° Because the PT was normal, all extrinsic and common path-
The thrombin time is a measure of the ability of thrombin way deficiencies were ruled out. The abnormal APTT led the
to convert fibrinogen to fibrin and is particularly useful laboratory technologist to believe the child had a problem with
in the evaluation of circulating anticoagulants (pathologic a factor deficiency in the intrinsic pathway (VIII, IX, XI, and
XI). Factor XII deficiency was ruled out, because it does not
inhibitors). The thrombin time is prolonged in the following
present with bleeding and patients with this disorder are more
conditions:
likely to develop thromboses. Factor assays were performed
1. Hypofibrinogenemia and dysfibrinogenemia for VIII, LX, and XI, to rule out any deficiencies. Results of the
2. Treatment with heparin factor IX and XI assays were normal. However, factor VIII
3. Circulating FDPs assays revealed abnormal results. VWF antigenic assays were
also performed to rule out von Willebrand’s disease.
4. Pathologic circulating inhibitors
The results of all the assays confirmed a diagnosis of hemo-
Table 24-26 can be used as a general guide toward cate- philia A resulting from deficiency of factor VIII. Hemophilia
gorizing bleeding disorders into the groups previously listed, A is an X-linked, recessive disorder passed from the mother,
using the suggested screening tests. who is the carrier, to her male children. Because of inheritance
Additional laboratory testing is designed to narrow patterns, male children have a 25% chance of being affected,
depending on what X gene is inherited. Female children will
down the abnormality to one of these specific areas. As a
be carriers if they inherit the affected gene. More information
result, laboratory testing can be divided into the following
on hemophilia A and other factor deficiencies can be found in
categories: Chapter 26.

Test Vascular Disorder Quantitative Platelet Disorder Qualitative Platelet Disorder Factor Deficiency

Platelet N AbN N

N N N
N N N
AbN AbN AbN
PFA-100 AbN AbN AbN

*Dependent on the factor deficiency: see Table 23-20 for specific information.
N = normal: AbN = abnormal: PT = Prothrombin time: APTT = activated partial thromboplastin time: IBT = Ivy bleeding time.
574 Chapter 24 Introduction to Hemostasis

OD reectonn
1 . Which of the following is true concerning the organelle _ ®¢, Factors VII, X, V, IL, I
zone? d. Factors XII, XI, IV, PF3, VIII
a. Responsible for metabolic activities of the platelet . What events take place in the extrinsic system?
b. Contains dense granules, a granules, and glycocalyx a. Activation of factor X to Va
c. Contains the dense tubular system which is the site of b. Acceleration of intrinsic pathway by enhancement of
prostaglandin synthesis, calcium release, and platelet the activity of factors XII and XI
relaxation c. Activation of factor XII to XIa to initiate clotting
d. Contains the OCS to deliver stored products to the d. Activation of factor VI to VIla in the presence of
platelet surface Ca** and factor I
. What events are involved in the normal formation of a
to
. Which of the following factors are unique to the intrinsic
platelet plug? system?
a. Adhesion, activation, fibrinolysis, secondary hemostasis a. Factors XII, XI, IX, VIII, X, V, I, I, Fletcher, Fitzgerald
b. Aggregation, coagulation, release reaction, lupus anti- b. Factors XII, XI, LX, VIII
coagulant c. Factors VII, X, V, I, I
c. Release action, adhesion, lupus anticoagulant, sec- d. Factors III, VII, X, V, IL, I
ondary hemostasis
d. Activation, adhesion, aggregation, release reaction ee What factor is not found in the common pathway?
a. Factor X
3. What product is responsible for stabilization of the b. Factor V
hemestatic plug? ; PE3
a. TXA, d. Factor XII
b. PF3
c. Fibrin . Which of the following is a function of thrombin?
d. GPIb a. Conversion of fibrinogen to fibrin
b. Activation of factor XIII to stabilize fibrinolysis
. Which of the following are classified as contact group c. Conversion of factor VIT to XIla
proteins? d. Enhancement of factor V, VIII, and XI activity
a. Factors I, VU, IX, X
b. Factors XII, XI, PK, HMWK 135 Which of the following is a function of plasmin?
c. Factors I, V, VII, XI a. Cleavage of T-PA
d. Factors I, II, V, X b. Destruction of fibrin
c. Conversion of XII to Xla
. Which of the following are classified as prothrombin
d. Inhibition of XIa
group proteins?
a. Factors II, VU, IX, X 14. What is the purpose of the PT test in monitoring
b. Factors XII, XI, PK, HMWK hemostasis?
c. Factors I, V, VUI, XU a. Measures factors of the extrinsic pathway
d. Factors I, If, V, X b. Detects platelet decrease or dysfunction
c. Detects presence of aspirin
. Which of the following are classified as fibrinogen group
d. Monitors heparin therapy
proteins?
a. Factors II, VU, Ix, X i). What is the purpose of the APTT test in monitoring
b. Factors XII, XI, PK, HMWK hemostasis?
c. Factors I, V, VII, XI a. Monitoring heparin anticoagulation
d. Factors I, If, V, X b. Detects deficiency of factors for both intrinsic and
extrinsic pathways
. Which of the following factors are dependent on vitamin
c. Measures circulating FDPs
K for synthesis?
d. Detects platelet dysfunction
a. Factors IL, VII, IX, X
b. Factors XII, XI, PK, HMWK
See answers at the back of this book.
c. Factors I, V, VIII, XIII
d. Factors I, Il, V, X
. Which of the following factors are unique to the extrin-
sic system?
a. Factors XII, XI, X, IV, VIII, V, Il, I
b. Factors Ill, VIT
 Chapter 24 Introduction to Hemostasis 575

~')] SUMMARY C w Platelet aggregation (platelet-to-platelet interaction) is an

energy-dependent process that requires adenosine triphos-
phate (ATP), primarily derived from glycolysis.
m Hemostasis is the complex process by which the body
spontaneously stops bleeding and maintains blood in the m Four platelet-specific proteins secreted from the a gran-
fluid state within the vascular compartment. ules are currently used as “markers of platelet activation.”
These include b-thromboglobulin, platelet factor 4,
= Hemostasis can be divided into two stages: primary and
thrombospondin, and PDGF.
secondary. Primary hemostasis is defined by platelet
adhesion to exposed collagen within the endothelium of @ During thrombus formation, thrombin, plasminogen, tis-
the vessel wall. Secondary hemostasis involves the enzy- sue plasminogen activator, and antiplasmin are incorpo-
matic activation of the coagulation proteins to produce rated into the clot.
fibrin from fibrinogen, thereby stabilizing the fragile clot w Aspirin exerts a permanent yet limited effect on platelet
formed during primary hemostasis. aggregation by inhibiting the action of the cyclo-oxyge-
= Biochemical characteristics of the endothelium render the nase enzyme and production of thromboxane A2 (TXA2).
cell surface thromboresistant by (1) synthesis and secre- @ Coagulation factors are designated by Roman numerals.
tion of a vasodilator prostacyclin (PGI2); (2) secretion of Activation of a particular factor is designated by a lower
tissue plasminogen activator {t-PA); (3) inactivation and case “a.”
clearance of thrombin; (4) activity of the cofactor throm-
w Coagulation factors may be divided into three categories:
bomodulin in the thrombindependent activation of protein
substrate, cofactors, and enzymes. On the basis of physi-
C; and (5) the degradation of proaggregating substances
cal properties, coagulation proteins may be divided into
such as adenosine diphosphate (ADP) and vasoactive
three groups: contact proteins, prothrombin proteins, and
amines.
fibrinogen or thrombin-sensitive proteins.
w The principal mechanism of platelet adhesion involves (1)
= Blood coagulation leading to fibrin formation can be sep-
plasma, (2) collagen fibers, and (3) platelet membrane
arated into three pathways: extrinsic, intrinsic, and the
glycoprotein GPIb (the receptor for von Willebrand’s fac-
common pathway.
tor [VWF]).
m@ The role of protein C (a vitamin K—dependent factor) as
m Platelets measure roughly 2 to 4 um in diameter. Normal
an anticoagulant is related to the presence of thrombin,
platelet count ranges from 150,000 to 350,000 cells/uL.
thrombomodulin, and protein S.
Platelets participate in hemostasis by (1) providing a neg-
atively charged phospholipid surface for factor X and gw Fibrinolysis is the physiologic process of removing
prothrombin activation; (2) release of substances that unwanted fibrin deposits.
mediate vasoconstriction, platelet aggregation, coagula- m The kinin system, important in inflammation, vascular
tion (thrombin generation), and vascular repair; and permeability, and chemotaxis, is activated by both the
(3) providing surface membrane glycoproteins such as coagulation and fibrinolytic systems.
GPIb and [Ia to attach to other platelets via fibrinogen. mwThe complement system is composed of approximately
gw Within the matrix of the platelet are microtubules, micro- 22 serum proteins that, working together with antibodies
filaments, and submembranous filaments. and clotting factors, play an important role as mediators of
mw The initial stage of platelet activation is as follows: both immune and allergic reactions.
platelets form pseudopods, organelles including a gran- g A patient’s history should include (1) physical appearance,
ules and dense bodies are reorganized to the center, and site, severity, and frequency of bleeding episodes;
contraction causes the granules to spill their contents into (2) patient and family history; (3) drug history; and
the open canalicular system (OCS). (4) contributing or underlying illnesses.
m Platelet-derived growth factor (PDGF) is a mitogen stored g Factor abnormality may occur as a result of decreased
in and secreted from the a granule of the platelet. synthesis, dysfunctional factor molecule(s), excessive
destruction of factors, or inactivation of factors.
m Platelet adhesion involves three components: (1) vWF,
(2) glycoprotein Ib (GPIb), and (3) collagen fibers. @ Thrombin time may be prolonged because of hypofib-
= Activation of second messenger pathways within the platelet rinogenemia and dysfibrinogenemia, treatment with
heparin, circulating fibrin degradation products (FDPs),
leads to intracellular biochemical changes, culminating in
and pathologic circulating inhibitors.
platelet activation events such as shape change, secretion,
cytoskeletal reassembly, and platelet aggregation.
576 Chapter 24 Introduction to Hemostasis

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 Chapter —

Disorders of Primary
Hemostasis
Quantitative and Qualitative Platelet
Disorders and Vascular Disorders
Darla K. Liles, MD
Charles L. Knupp, MD

Introduction OBJECTIVES
Laboratory Evaluation At the end of this chapter the learner should be able to:
of Disorders of Primary
Hemostasis 1. Describe the laboratory tests that may be utilized in the evaluation of quantitative and
qualitative platelet disorders.
Quantitative Platelet
Disorders: . 2. Describe the pathophysiologic processes that cause thrombocytopenia. List the
Thrombocytopenia thrombocytopenic disorders caused by each process.
Deficient Platelet Production
ee
Abnorinal. Distribution of 3. Define 1 -medi
efine immune-mediated :
thrombocytopenia
Platelets 4 . List conditions that are associated with autoimmune and alloimmune thrombocytopenia.
Increased Destruction of ! bea ese
Secs SSUUCHO ORO P). Describe how the diagnosis of idiopathic thrombocytopenic purpura (ITP) is made.
6. Compare ITP and thrombotic thrombocytopenic purpura (TTP).
Quantitative Platelet
3
Disorders: Thrombocytosis . Compare thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic
Primary Thrombocytosis syndrome (HUS).
Reactive Thrombocytosis
8. List the characteristics of the inherited platelet membrane defects and
Qualitative Platelet Disorders compare Bernard-Soulier syndrome to Glanzmann’s thrombasthenia.
Congenital Disorders of
Platelet Function 9. Describe the types of von Willebrand disease and how they differ in the
von Willebrand Disease management of bleeding.
Acquired Qualitative Platelet 1(. Differentiate von Willebrand disease from Bernard—Soulier syndrome and
Disorders hemophilia A.

Vascular Disorders 11. Describe the pathophysiology responsible for storage pool and platelet release
Primary Purpura
defects and how it relates to laboratory studies used for diagnosis of these disorders.
Secondary Purpura
12. Differentiate between reactive and primary thrombocytosis and describe the
Vascular and Connective
hemostatic problems expected.
Tissue Disorders
Case Study 1
Case Study 2
Case Study 3
Case Study 4

Sve
578 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

Introduction The laboratory tests that aid in the evaluation of disorders of

primary hemostasis are listed in Table 25—3 (see Chap. 33),
The diagnosis of any hemostatic disorder is made by the sys- The automated platelet count is one of the most important
tematic evaluation of information obtained in the history and initial tests to evaluate a bleeding tendency because acquired
physical examination, along with the appropriate laboratory quantitative platelet disorders are the most common disorders
testing. Bleeding disorders present themselves differently, of primary hemostasis. Visual inspection of the peripheral
depending upon the cause of the disorder. smear can be used to confirm the automated platelet count
Disorders of primary hemostasis include abnormalities and reveal platelet morphology. The peripheral smear should
that clinically result in bleeding because of defects in the for- be carefully inspected for evidence of large or dysplastic
mation of an adequate platelet plug. These include quantita- platelets. Direct inspection should also assess for platelet satel-
tive and qualitative platelet disorders, and defects of the blood litism or platelet clumping, which may cause a falsely low
vessel wall. These disorders can be inherited or acquired. In automated platelet count termed pseudothrombocytopenia.
inherited disorders, the patient will have a history of child- When pseudothrombocytopenia is suspected, a “true” auto-
hood bleeding such as frequent gingival bleeding or a history mated count can often be obtained by performing a platelet
of easy spontaneous bruising. The acquired disorders usually count in alternative anticoagulants such as citrate or heparin.
will not have any history of a childhood bleed and will pre- This phenomenon does not result in clinical bleeding. It is usu-
sent with signs of bleeding in adulthood. It is always impor- ally a result of ethylene diaminetetraacetic acid (EDTA)-
tant to rule out a history of drugs or other underlying diseases dependent cold agglutinins.'? These antibodies have recently
which may cause the bleeding episode. Despite the number of been shown to be IgG antibodies that are EDTA dependent and
disorders that one encounters in primary hemostasis, the clin- bind to the platelet membrane glycoprotein IIb.’
ical manifestations are typically limited to skin (petechiae) The bleeding time has been utilized as a screening test to
and mucosal bleeding (Table 25—1). Bleeding, such as spon- evaluate platelet and vascular disorders. There are several
taneous hemarthrosis and hematomas of deep structures, is a standard methods for performing bleeding times, as described
typical feature of defects in secondary hemostasis (coagula- in Chapter 33; however, the Ivy bleeding time is the most
tion protein deficiency states), and is usually not seen in commonly utilized method for performing this test. Templates
platelet disorders or vascular defects (see Chap. 26). To are utilized to allow uniformity of the incision to improve the
understand platelet quantitative and qualitative disorders, it is reproducibility of this test. Bleeding times may be abnormal
important to review the normal platelet physiology and func- with qualitative or quantitative platelet disorders, certain
tion, which is provided in Chapter 24. This chapter reviews vascular defects, and von Willebrand’s disease. The bleeding
the etiology, pathophysiology, clinical manifestations, and time must be interpreted with caution in the context of a care-
laboratory tests used to identify the various quantitative and fully obtained history and other platelet tests, especially an
qualitative platelet disorders as well as the numerous vascular automated platelet count. A medication history is essential,
disorders. because ingestion of aspirin and nonsteroidal analgesics may
cause a prolonged bleeding time.* The bleeding time has been
utilized as a screening tool to determine the risk of bleeding
Laboratory Evaluation of Disorders in many clinical settings; however, there is now a large body
of Primary Hemostasis of literature that has shown no correlation with risk of bleed-
Although many laboratories differ in their approach to a ing in these instances.*
bleeding disorder, a general profile of laboratory tests is usu- Recent development of an automated platelet function
ally established. This profile can often be used as a means of analyzer (PFA-100) has made the bleeding time almost obso-
differentiating various hemostatic problems (Table 25-2). lete. This instrument allows the patient’s blood to be drawn in a
citrated tube. The citrated blood is then run through a cartridge
with either ADP and collagen or epinephrine and collagen. The
cartridge has a small aperture and the instrument measures the
amount of time required for closure of the aperture by a small
platelet plug. In some instances, the collagen/epinephrine car-
tridge is run first, and if abnormal, the collagen/ADP cartridge is
run however, often the laboratories run both cartridges as a
platelet function screen. Both the ADP and epinephrine closure
Ecchymosis times are prolonged in patients with Von Willebrand’s disease,
Petechiae inherited qualitative platelet disorders and quantitative platelet
Purpura disorders (thrombocytopenia when the platelet count is less than
Mucosal bleeding 100,000/uL).° The PFA-100 can be used instead of the bleeding
Epistaxis
Gingival bleeding time to screen patients for these disorders. Aspirin and other
Gastrointestinal bleeding platelet inhibitors have been shown to have variable effects
Menorrhagia on the PFA-100 closure times. In addition, lower hemat-
Hematuria ocrits (<35%) and increased sedimentation rates will also
affect the PFA-100 closure times, usually prolonging them.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 579

Vascular Disorder Quanutanye Platelet Deon Qualitative. Platelet Disorder

Platelet count N AbN

N N
N N N
AbN AbN AbN
PEA- 100 Assay _AbN_ AbN- ADN
N = normal: AbN = = Lunia PT = Pasthnombinttime: APTT =Ettvaied a fEiinboplisinthtime: IBT=
ie bleedingtime:
PFA= platelet function analyzer.

Because von Willebrand disease mimics the bleeding platelet surface and release defects can be identified with this
diathesis of platelet disorders, assays to exclude this diagno- test. Drugs and disorders such as hepatic or renal disease can
sis are an integral part of the evaluation. To determine the interfere with platelet function and make interpretation of this
presence or absence of von Willebrand disease, factor VIII test difficult. Lumi-aggregation or °'Cr release assays may be
coagulant activity, von Willebrand’s antigen (VWF:Ag), von useful to specifically measure platelet release.
Willebrand’s activity by ristocetin-induced platelet aggrega- Bone matrow aspiration and biopsy are useful in deter-
tion, and von Willebrand multimers must be assayed mining the etiology of quantitative platelet disorders. The
together. Further discussion of these tests is provided later in bone marrow specimen can be used to assess adequacy of
this chapter in the section on von Willebrand disease. megakaryocytes, overall cellularity, and to identify infiltrative
Platelet antibody testing determines the amount of processes such as myelodysplastic syndromes, malignancy, or
immunoglobulin G (IgG) bound on the platelet surface fibrosis.
by various methodologies. Increased amounts of platelet-
associated IgG are often found in immune-mediated throm-
bocytopenias, but this finding is not specific enough to Quantitative Platelet Disorders:
establish a diagnosis of an immune origin. Flow cytometry Thrombocytopenia
can be used to measure platelet surface glycoproteins and
Platelets must be present in adequate numbers to maintain
platelet-bound IgG.
normal hemostasis. Platelet production remains relatively
Platelet aggregation assesses platelet function by mea-
constant for an individual over time. The body senses the
suring the response of the platelet to various stimuli such as
total platelet mass, related both to number and mean volume
epinephrine, adenosine diphosphate (ADP), collagen, throm-
of platelets, and regulates this tightly. Thrombopoietin (TPO),
bin, and ristocetin. The procedure for performing platelet
a growth factor produced by the liver and to a lesser degree
aggregation is described in Chapter 33. Abnormalities of the
by the spleen, is responsible for this regulation. When the
platelet mass is normal, TPO is cleared from plasma by TPO
receptors on megakaryocytes. With thrombocytopenia, the
overall clearance is low and the plasma concentration of TPO
increases to boost megakaryocyte and platelet production.
ay peaBroce” The average platelet count ranges from 150 to 400 10°/L of
of Primary Hemostasis whole blood. Thrombocytopenia is defined as a platelet count
below the lower limit of normal, although clinical signs and
Platelet count
Peripheral blood smear symptoms of thrombocytopenia typically are not manifested
Ivy bleeding time or PFA-100 until the platelet count falls below 100 x 10°/L and usually
Von Willebrand studies not until the platelet count falls below 50 X 10°/L. Overt
FVUI:C spontaneous hemorrhage is not usually seen until the platelet
vWE antigen
count falls to less than 20 X 10°/L. Platelet counts less than
vWF activity
Platelet antibody testing 10,000 may result in life-threatening hemorrhage and may
Flow cytometry require emergency platelet transfusions. Quantitative platelet
Platelet glycoprotein analysis disorders are the most commonly encountered group of
Platelet-associated IgG platelet abnormalities and can be simply divided into two
Platelet aggregation studies
categories, thrombocytopenia and thrombocytosis. Thrombo-
Lumiaggregometry
*1Cr release cytopenia results from three distinct mechanisms: deficient
_Bone marrow aspiration aud biopsy platelet production, abnormal platelet distribution (splenic
sequestration), or increased destruction (Table 25-4). Qualita-
“EVIL C= acter VIE: = yon Willebrand factor;
VWF =
PFA= platelet function analyzer. tive platelet defects may coexist with quantitative platelet
defects to increase bleeding risk.
580 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

and leukopenia) and a blood smear characterized by nucleated

Table 25-4 Classification red blood cells, teardrop-shaped cells (dacrocytes), and
of Disorders Causing immature granulocytes. These characteristic findings on the
Thrombocytopenia peripheral smear are often referred to as a myelophthisic
picture and should make one highly suspicious of an infiltra-
Deficient Platelet Production tive process within the bone marrow. Bone marrow aspiration
Myelophthisic (marrow infiltrative processes)
and biopsy are indicated to confirm a diagnosis of marrow
Leukemia
Lymphoma aplasia or infiltration when these findings are present.
Multiple myeloma
Metastatic carcinoma INEFFECTIVE THROMBOPOIESIS
Myelofibrosis
Ineffective erythropoiesis is associated with normal to
Aplasia
Aplastic anemia (Fanconi’s anemia) increased marrow cellularity but peripheral blood cytope-
Amegakaryocytic thrombocytopenia nias. Megaloblastic anemia associated with vitamin B,, or
Drug effect (chemotherapy) folic acid deficiency is commonly associated with thrombo-
Radiation therapy cytopenia as a result of impaired DNA synthesis. Serum
Ineffective erythropoiesis
Pernicious anemia (vitamin B,, deficiency)
lactate dehydrogenase (LDH) levels are high as a result of
Folic acid deficiency intramedullary death of hematopoietic progenitors. Throm-
Alcohol ingestion bocytopenia is generally mild. Platelet life span has
Myelodysplasia been reported as being normal to only slightly decreased.
Paroxysmal nocturnal hemoglobinuria Myelodysplastic syndromes may simulate vitamin deficien-
Congenital disorders
cies but do not respond to vitamin replacement. Chromoso-
May-—Hegglin syndrome
Thrombocytopenia with absent radii (TAR) syndrome mal abnormalities may be present. Paroxysmal nocturnal
Bernard—Soulier syndrome hemoglobinuria, a rare disorder with increased cellular sen-
Abnormal Platelet Distribution sitivity to complement, also is associated with cytopenias
Hypersplenism (splenomegaly) resulting from intramedullary cellular destruction
Hemangiomas (Kasabach—Merritt syndrome)
These disorders are discussed in detail in Chapters 7, 8,
Increased Platelet Destruction
Immune (primary) and 19.
Idiopathic thrombocytopenic purpura (ITP) Alcohol has a direct toxic effect on the marrow, thereby
Posttransfusion purpura producing thrombocytopenia in the absence of a folic acid or
Neonatal isoimmune purpura vitamin B,, deficiency. Mild thrombocytopenia and acquired
Drug-induced thrombocytopenia
Heparin-induced thrombocytopenia and thrombosis
platelet function defects appear to improve after the use of
Immune (secondary) alcohol is stopped.
Lymphoproliferative disorders
Systemic lupus erythematosus/collagen vascular disorders
Viral infection (mononucleosis, measles, HIV)
CONGENITAL DISORDERS
Microangiopathic thrombocytopenia A number of inherited disorders produce thrombocytopenia;
Thrombotic thrombocytopenic purpura (TTP) however, all are rare. Patients with Thrombocytopenia with
Hemolytic uremic syndrome (HUS) absent radius TAR syndrome have absent radii in addition to
Disseminated intravascular coagulation (DIC) the thrombocytopenia. Eczema and immunodeficiency are
Pregnancy-associated thrombocytopenia
Gestational thrombocytopenia associated with Wiskott—Aldrich syndrome. Bernard—Soulier
Preeclampsia—eclampsia and HELLP syndrome syndrome and May—Hegglin anomaly do not have associated
skin or skeletal defects. Some of these disorders also exhibit a
HIV= human Guninedenciacy virus; HELLP = Hnclfes
elevated liver enzymes and low platelet count. qualitative abnormality in platelet function and are discussed
under qualitative platelet disorders later in this chapter. A list
of congenital disorders and their associated abnormalities is
provided in Table 25-5.

Deficient Platelet Production

Abnormal Distribution of Platelets
Impaired platelet production resulting in thrombocytopenia
may be caused by many disorders. These disorders produce Normally the spleen pools approximately one-third of the
megakaryocytic hypoplasia often with erythroid and granulo- platelets produced by the marrow. When the spleen enlarges,
cytic hypoplasia, resulting in pancytopenia. These platelet more platelets can be sequestered in the spleen, leading to
disorders may occur spontaneously (aplastic anemia) or as a thrombocytopenia. Many conditions, including liver cirrhosis,
result of injury to the bone marrow (radiation or chemother- hematologic malignancies, and portal vein thrombosis, can
apy). Replacement of marrow hematopoietic tissue as a result cause hypersplenism. Typically, the platelet count remains
of infiltrative processes such as myelofibrosis, leukemia, greater than 50 X 10°/L in patients with hypersplenism.
Hodgkin’s and non-Hodgkin’s lymphoma, and metastatic Kasabach-Merritt syndrome, a rare disorder, results in platelet
cancer results in pancytopenia (anemia, thrombocytopenia, sequestration in giant hemangiomas.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 581

with more chronic form, which is treated like the more common
Table 25-5 Congenital Disorders. adult form of ITP, except that splenectomy is often avoided
_
| Associated with— because of the infectious complications of splenectomy in
ROUSE: - Decreased Platelet young children.
ee - Production | a
Adult ITP
eDisonden: mssociated Abnormalities In adults, ITP commonly presents in the 20- to 50-year-old
group, as a chronic disease process with a greater predilection
Alport’s stuns Giant aieicn
Thrombocytopenia for women. Occasionally patients will have an acute immune
Deafness thrombocytopenia after a viral illness or exposure to drugs,
Nephritis but this is uncommon. In most cases of adult immune throm-
Chédiak-Higashi Partial oculocutaneous bocytopenia, there is usually not a recent history of drug
syndrome albinism
exposure or infectious illness that can be related to the onset
Increased susceptibility to
pyogenic infections
of thrombocytopenia. Platelet counts are typically less than
Storage pool defect of dense 30 X 10°/L in patients who present with bleeding manifesta-
granules tions. Clinically, patients present with mucosal bleeding
Hermansky-Pudlak Platelet deficiency of typical of a primary hemostatic defect, such as menorrhagia,
syndrome nonmetabolic ADP epistaxis, easy bruisability, or petechiae (Figs. 25-1 and
Oculocutaneous albinism
May-Hegglin anomaly Thrombocytopenia 25-2). Some patients are diagnosed while still asymptomatic
Giant platelets based on a low platelet count on a routine complete blood
Dohle bodies count obtained for other reasons. These patients usually have
TAR syndrome Multiple skeletal and cardiac a platelet count greater than SO < 10°/L. Adult ITP does not
abnormalities usually remit spontaneously.
Storage pool defect
Wiskott-Aldrich Disorders of dense granules
syndrome Recurrent pyogenic infections Common Clinical Findings
Eczema The bone marrow is characterized by increased or normal
Thrombocytopenia numbers of megakaryocytes (Fig. 25—3). Platelet life span is
shortened and circulating platelets are morphologically large
on the smear, reflecting the early release from the marrow in
Increased Destruction of Platelets response to the peripheral destruction. There are also changes
in the splenic microcirculation that cause a reduction of
PRIMARY IMMUNE-MEDIATED THROMBOCYTOPENIAS platelet transit time in the spleen and increased destruction of
This group of thrombocytopenias all have an immune-mediated the antibody-coated platelets.’ The bleeding time in ITP may
mechanism by which there is increased platelet destruction that not be as prolonged as the bleeding time in other disorders
can be primary (idiopathic) or secondary to an underlying associated with the same degree of thrombocytopenia, sug-
disease (see Table 25-4). gesting that the younger platelets in ITP may be more effec-
tive at maintaining hemostasis.
IDIOPATHIC THROMBOCYTOPENIC PURPURA Idiopathic Because ITP is a diagnosis of exclusion, other causes of
or immune thrombocytopenic purpura (ITP) is one of the thrombocytopenia must be considered and eliminated first.
most common disorders causing severe isolated thrombocy- Drugs that may cause thrombocytopenia must be stopped, and
topenia and is caused by an autoantibody to the patient’s
platelets. There is not a specific test that readily confirms the
diagnosis of ITP, so it is typically a diagnosis of exclusion.
ITP can present in children and adults; however, there are
important differences between the two groups when compar-
ing the long-term prognosis.°

Childhood ITP
Young children may present with an immune thrombocytopenia
that typically develops acutely with a |- to 2-week duration,
usually with bruising or petechiae. Serious bleeding is uncom-
mon. Most children present with an initial platelet counts of less
than 20 X 10°/L. Bone marrow aspiration and biopsy is often
performed to exclude the diagnosis of acute leukaemia. This dis-
order is usually self limited. Spontaneous remissions, with or
without therapy, occur in the majority of these patients. How-
ever, immunoglobulin G and corticosteroids are often used to Figure 25-1 @ Oral cavity of a patient with idiopathic thrombocy-
decrease the period of thrombocytopenia. A minority of children topenic purpura (ITP).
582 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

thereby increasing the platelet life span and ameliorating the

thrombocytopenia. The aim of therapy is to give high-dose
daily prednisone sufficient to obtain a satisfactory increase in
the platelet count initially, and then to reduce the dosage to a
level that maintains the platelet count at a hemostatic level.
Approximately 70% to 90% of patients treated initially
respond favorably to steroids. However, sustained responses
are usually seen in only about 25%. Most patients also usually
respond to intravenous immunoglobulin G. The response is
usually transient and must be repeated to sustain a remission.
Guidelines for the treatment of ITP recommend the use of
corticosteroids initially unless there is active bleeding, but
recommend the addition of immunoglobulin G if bleeding is
present.* There does not appear to be a more rapid increase of
the platelet count with both of these agents compared to either
alone. Anti-D globulin can be used in some individuals with
ITP who are Rh-positive. Its mechanism of action is probably
similar to that of immunoglobulin G but has the benefits of
being a short infusion over 5 minutes and not requiring the
Figure 25-2 m Petechial bleeding of the lower extremities in a volume necessary to administer immunoglobulin G. There is
patient with ITP.
often an associated mild hemolysis of red cells associated
with the administration of anti-D globulin; therefore, the
considefation given to a bone marrow aspiration and biopsy dosage should be adjusted according to the hemoglobin level
to exclude a primary bone marrow disorder. Antiplatelet anti- at time of treatment. The response to anti-D globulin is high-
body tests, which measure IgG bound to the platelet surface, est prior to splenectomy, with only rare responses reported in
have been utilized by some to confirm the diagnosis of ITP; patients who have failed to respond to splenectomy. The time
however, these tests are not specific. Total IgG measurement to response with any of these initial treatments varies with
on the platelet surface may simply correlate with platelet size, each individual, although most responses are seen within a
as platelet size tends to increase in disorders of increased week or two of initiation of therapy. Thrombocytopenia usu-
platelet destruction. Measurement of antibodies specific for ally reoccurs when these treatments are stopped.
platelet surface glycoproteins IIb/IIIa and Ib/LX may provide Splenectomy is the long-term treatment of choice for ITP,
greater specificity but still are not diagnostic. after an initial response to steroid therapy or immunoglobulin G.
Significant improvement following splenectomy is obtained in
Treatment 50% to 80% of patients. The benefit of splenectomy results from
Splenectomy and corticosteroids are the conventional thera- the removal of the organ responsible for the autoantibody pro-
pies used to treat patients with ITP.*° Patients with ITP duction and the sequestration of moderately sensitized platelets.
are initially treated with corticosteroids to rapidly increase the Many other agents have been utilized in patients who do
platelet count and improve hemostasis. Corticosteroids are not respond to splenectomy, or who relapse after splenectomy."°
thought to reduce autoantibody production and to suppress Unfortunately, the studies of efficacy of these treatments
splenic sequestration of moderately sensitized platelets, have involved relatively small numbers of patients. Some
of the agents that have been attempted include danazol, dap-
sone, colchicine, vitamin C, chemotherapeutic agents such as
vincristine, cyclophosphamide, or combination chemotherapy,
immunosuppressive therapy such as azathioprine, mycopheno-
late mofetil, or cyclosporine A, and plasma exchange with or
without extracorporeal immunoadsorption of plasma by staphy-
lococcal protein A columns. The next most effective agent when
there has been failure of response to splenectomy has not been
clearly defined, and the decision about which treatment modal-
ity to utilize varies according to the treating physician. There are
now many reports which describe excellent responses with
monoclonal antibody treatment using the anti-CD 20 antibody
(Rituximab).'' Rituximab is rapidly becoming the preferred
treatment for individuals refractory to primary treatment
because of its superior efficacy to other regimens in numerous
case reports.
Figure 25-3 @ ITP, bone marrow aspirate. Note the increased In patients who are actively bleeding, antifibrinolytic
number of megakaryocytes with normal cellularity (M/E 3:1). agents such as epsilon (€)-aminocaproic acid (EACA) can be
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 583

used to help control bleeding until the platelet count can be To confirm the presence of a platelet-specific antibody, a panel
corrected. These agents are safe to use either before or after with Pl‘'-positive cells should be run. The patient’s platelets
splenectomy and are most effective at sites of high fibrinolytic should also be phenotyped but the patient’s own blood type
activity (i.e., urinary tract, nose, and mouth). Platelet transfu- will not be evaluable until after recovery. Confirmation that
sions are usually not effective because transfused platelets are the patient is PI‘! negative provides additional support for the
usually rapidly destroyed; however, in some patients a platelet diagnosis. In some cases of PTP, isosensitization to HLA anti-
response may occur and bleeding may improve.'? gens found on platelets occurs, as well as the appearance of
platelet-specific antigens other than PI‘', making serologic
POSTTRANSFUSION PURPURA (PTP) In this disorder, sud- typing difficult.
den onset of thrombocytopenia occurs approximately | week Treatment with platelet transfusions is usually not
after transfusion of blood or blood products containing effective as the alloantibodies destroy even PIl*!-negative
platelets (Fig. 25-4). The majority of cases are a result of an platelets. Plasmapheresis without exchange and intravenous
alloantibody directed against the platelet antigen PI4', also IgG infusions have each been effective means of treating the
referred to as HPA-1a.'* The PI“! antigen is found in approx- hemorrhagic complications associated with PTP. In a num-
imately 97% of the normal population; the 3% of people who ber of cases, patients have had repeated episodes of PTP
lack the PI*' (HPA-1a) antigen on their platelets are consid- following re-exposure to PI‘'-positive blood, but some do
ered at risk for developing PTP. It is believed that posttrans- not. Pl‘'-negative blood is indicated for all subsequent
fusion purpura (PTP) results from an anamnestic immune transfusions when possible because patients are considered
response from prior exposure to the antigen. Most reported at risk for recurrence of PTP with subsequent transfusions.
cases have been in middle-aged women who have had chil-
dren. It is believed that primary immunization occurs during ISOIMMUNE NEONATAL THROMBOCYTOPENIA Similar
pregnancy, when PI*'-positive fetal platelets sensitize a to the pathogenesis of erythroblastosis fetalis, isoimmune
Pl*'-negative mother (see the following discussion of isoim- neonatal thrombocytopenia results from immunization of the
mune neonatal thrombocytopenia). Other mechanisms for the mother by fetal platelet antigens and placental transfer of
development of PTP have been suggested.'* Why these alloan- maternal antibody.'® Isoimmune neonatal thrombocytopenia
tibodies destroy the patients PI*!-negative platelets is not clear. is most often caused by maternal alloantibodies to the PI*'
Recycling of antigen from destroyed platelets onto the (HPA-la) antigen: however, as in posttransfusion purpura,
patient’s platelets may explain this phenomenon. Rare reports isoimmune neonatal thrombocytopenia has been reported
of cases of PTP have occurred in association with other only rarely with other platelet antigens such as Pl**, Bak’,
platelet antigens, including Pl**, Bak*, Bak”, Pen*, and Br*.'° Bak”, Br*, and Br®.'> It is an uncommon disorder, generally
Complement fixation, release of °'Cr, or '*C-serotonin affecting the first-born child. Based on gene frequency of the
have been some of the reliable laboratory tests used to detect PL“! (HPA- 1a) antigen in fathers, there is a high probability
and measure anti-Pl*! (HPA-1a) antibodies in PTP. Currently, that a Pl*'-negative mother will have a Pl*!-positive child.
direct and indirect laboratory tests have been developed Once isoimmune neonatal thrombocytopenia has developed,
to increase specificity and sensitivity in the detection of there appears to be an increased risk of the next child being
platelet antibodies. These tests employ some of the following affected, because most fathers are homozygous for PI*'.
techniques: enzyme-linked immunosorbent assay (ELISA), A large percentage of PI*'-negative mothers who give birth
Western blot followed by ELISA or radioimmunoassay to an affected child are phenotype-positive for the HLA-B8
(RIA), platelet suspension immunofluorescence, and immuno- antigen. It has been suggested that the HLA-B8 antigen serves
precipitation of radiolabeled glycoprotein Hla (GPIIIa). to protect from immunization, which accounts for the rela-
tively low incidence of isoimmune neonatal thrombocytope-
nia, despite the frequency of the PI*! antigen and the chance
for maternal sensitization. The relationship of ABO incompat-
ibility to symptomatic isoimmune neonatal thrombocytopenia
is unclear.
Infants who develop isoimmune neonatal thrombocy-
topenia appear normal at birth but within hours develop scat-
tered petechiae and purpuric hemorrhages, with platelet
counts below 30 X 10°/L. Intracranial hemorrhage is the pri-
mary cause of mortality in these infants. Characteristically, in
this disorder the platelet count begins to decrease shortly
after birth with low levels reached several hours later.
Therapy is aimed at preventing intracranial hemorrhage
and keeping platelet counts at hemostatically safe levels. Cae-
sarean delivery is usually performed to prevent intracranial
hemorrhage from birth trauma when the disorder is suspected
prior to delivery. Corticosteroids or intravenous IgG may be
Figure 25-4 m Posttransfusion purpura (PTP). used prior to delivery. Postnatal treatment is not necessary if
584 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

the infant is asymptomatic and the platelet count remains after initial exposure to heparin; however, in individuals who
above 30 X 10°/L. When the infant manifests clinical signs of have been previously exposed to heparin it can develop within
bleeding and the platelet count falls below 10 < 10°/L, com- | to 3 days after re-exposure. Patients with myocardial infarc-
patible platelet transfusions utilizing maternal platelets or tion and cardiogenic shock or those who have undergone major
P]*!-negative donor platelets is the preferred treatment. vascular surgery may be particularly susceptible. Venous or
arterial thrombosis results in an incidence of morbidity and mor-
DRUG-INDUCED IMMUNE THROMBOCYTOPENIA Drugs tality of 20% of the patients who develop HITTS (see Chap. 28).
may also cause thrombocytopenia. Quinine has been recog- Pathologically, this syndrome develops secondary to anti-
nized as one of the most frequent causes of drug-induced body produced to platelet factor 4-heparin complex with
thrombocytopenia since the first report in 1928.'° The drug immune complex Fe receptor-induced activation of platelets.'”°°
appears to act as a hapten, eliciting an antibody response Laboratory confirmation of HITTS is difficult. Origi-
when complexed with a larger carrier molecule. Antibody nally the test available to confirm HITTS was platelet aggre-
binding with drug appears to be the initial step in the forma- gation test utilizing donor platelets mixed with patient serum
tion of the complex. Cellular binding or adsorption of the after the patient has been off heparin for 8 hours or longer.
antibody—drug complex to the platelet membrane via the Heparin is added to the patient samples at low and high con-
antibody Fab region results in platelet injury and splenic centrations, and platelet aggregation is observed in the
sequestration. Removal of these platelets by the spleen results heparin sample compared to a control sample without
in thrombocytopenia. heparin. In patients with HITTS the aggregation test should
The list of drugs that have been implicated to cause show aggregation of platelets at the low concentration of
immune drug purpura is rather extensive. However, many of heparin but not at the high concentration of heparin or the
these reports do not provide sufficient evidence to prove causa- sample without heparin. This test is approximately 40% sen-
tion. The drugs most frequently cited are quinine, quinidine, sitive but 90% specific for confirming the diagnosis of
salicylates, thiazides, and sulfa drugs.'’ When possible, any sus- HITTS. Platelet release assays increase the sensitivity of
pect drug should be stopped in a thrombocytopenic patient. detection. Aggregation and release assays are not readily
Drug-induced immune purpura appears to occur more available in many hospitals. Newer tests utilizing flow cytom-
frequently in the elderly population as a result of the etry or ELISA are even more sensitive for the diagnosis of
increased usage of medication; however, cases have been HITTS and can be performed in the presence of heparin.
reported in children and young adults. Purpura occurs approx- ELISA testing is usually more readily available for clinical
imately 7 days after initial use of the drug but may occur laboratories. However, this method may detect the presence
within 3 to 5 days owing to an anamnestic response on of antibody that has not caused the syndrome in patients with
re-exposure to the drug. It is estimated that 1 in 100,000 indi- thrombocytopenia resulting from other causes. Using a suffi-
viduals using prescription medication per year will require ciently high optical density cutoff has led to much greater sen-
hospitalization as a result of a drug-induced blood disorder. sitivity and has resulted in use of ELISA as the most common
The frequency for users of quinine and quinidine is approxi- laboratory method used to diagnose the disorder.?!
mately | in 1000. The disorder is generally self-limiting In patients with suspected HITTS, heparin must be dis-
because the platelet count returns to normal once the drug has continued. The platelet count should return to normal within
been removed from circulation. Re-administration of a drug 4 to 6 days. Alternative anticoagulation is recommended with
known to cause purpura should be avoided. or without thrombotic complications in any individual in
whom the diagnosis of HITTS is strongly suspected. In some
HEPARIN-INDUCED THROMBOCYTOPENIA instances, patients have been given warfarin without intra-
AND THROMBOSIS venous anticoagulation; however, recent reports have sug-
Heparin therapy is associated with the development of two dis- gested warfarin may increase the risk for skin necrosis and
tinct types of thrombocytopenia. One type develops early in venous limb gangrene in this syndrome.?? In patients who
treatment and is benign. The platelet count rarely falls below have life- or limb-threatening thrombosis, there are a limited
100 < 10°/L, and there are no resultant bleeding or thrombotic number of choices for intravenous anticoagulation that all
complications. The second type is associated with severe throm- have potential hemorrhagic risk.% In the past, Dextran has
bocytopenia and paradoxically thrombotic episodes instead of been administered; however, there is no laboratory measure
hemorrhagic complications (see Chap. 28). Initially, it may be other than clinical observation to measure its efficacy.
difficult to distinguish between these two types of thrombocy- Dextran coats circulating red blood cells, complicating type
topenia based on laboratory values alone. and crossmatch for red cell transfusion products. Some data
In the second type of thrombocytopenia, platelet counts as now suggest that Dextran may be detrimental; therefore it is
low as 20 * 10°/L occur in association with arterial and venous no longer used. Newer agents that are approved for treatment
thrombosis; this type has been termed heparin-induced throm- in this setting include the direct thrombin inhibitors lep-
bocytopenia and thrombosis syndrome (HITTS). The actual thirudin and argatroban.*> Lepthirudin is the recombinant
incidence of this syndrome is not well defined, but it probably form of the leech salivary gland protein hirudin. Argatroban
occurs in fewer than 1% of patients receiving heparin.'’ It has is a synthetic peptide direct thrombin inhibitor. Bivalirudin,
been reported more often with bovine heparin administration a synthetic derivative of hirudin, has been used off-label
than with porcine heparin. HITTS typically develops 4 to 7 days and has a somewhat shorter half-life than lepthirudin and
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 585

argatroban. Fondaparinux, which is the synthesized pen- The pathogenesis of HIV-related thrombocytopenia
tasaccharide portion of the heparin molecule, does not appears to be heterogeneous. An immune etiology is suggested
appear to cross-react with heparin in the manner that by studies which have shown a diminished in vivo platelet sur-
low-molecular-weight heparins can in promoting the clini- vival. Detection of platelet-associated IgG and circulating
cal syndrome. The bleeding that may occur with these treat- immune complexes that contain antiplatelet glycoprotein anti-
ments may not be easily corrected. Fortunately, the direct bodies provides support for immune destruction in HIV-related
thrombin inhibitors have short plasma half-lives so their thrombocytopenia. The pattern of IgG subclasses found in HIV-
effects resolve relatively quickly. Fibrinolytic therapy may infected patients, as well as the level of anti-[gG immune com-
play a treatment role to dissolve thrombus, especially in plexes on platelet surfaces in HIV disease, are significantly
individuals who develop neurovascular compromise related different from patients with “classic ITP” (Table 25-6).
to their thrombosis. Immunohistochemical markers show increased CD8+ T cells
in the spleens of patients with HIV-related immune thrombo-
ACQUIRED SECONDARY IMMUNE-MEDIATED cytopenic purpura. However, the finding that thrombocytope-
THROMBOCYTOPENIA nia does not frequently occur in infants of HIV-infected
LYMPHOPROLIFERATIVE DISORDERS/COLLAGEN mothers with thrombocytopenia suggests an immune etiology
VASCULAR DISORDERS Lymphoproliferative disorders may not be the principal cause. Viral infection of hematopoi-
such as Hodgkin’s disease and non-Hodgkin’s lymphoma, as etic cells, altered marrow microenvironment or dysfunction
well as other hematologic malignancies such as chronic lym- of the reticuloendothelial system contribute to ineffective
phocytic leukemia, have been reported with an ITP-like thrombopoiesis in HIV-related thrombocytopenia. Develop-
thrombocytopenia associated with decreased platelet survival. ment of marrow fibrosis or marrow involvement by AIDS-
In systemic lupus erythematosus (SLE), roughly 14% of the related lymphoma may also lead to thrombocytopenia. These
patients develop thrombocytopenia resembling ITP during the factors may be more important in producing HIV-related
course of the disease. The hematologic manifestations of thrombocytopenia.
SLE, which include immune-mediated thrombocytopenia and Treatment with antiretroviral therapy alone is often
thrombocytopenia secondary to bone marrow suppression, effective.** HIV-related thrombocytopenia which is more severe
may precede the other clinical manifestations of the disease. or is associated with bleeding is treated similarly to ITP. Intra-
Thrombocytopenia resulting from either etiology in SLE venous IgG and anti-D globulin have been used successfully.
responds well to corticosteroid therapy. Corticosteroids may be effective but have the potential to
increase the risk of infection in these immunosuppressed indi-
VIRAL INFECTIONS Viral infections may also transiently viduals. Splenectomy may also be effective. As there seems to
impair megakaryopoiesis without a reduction in marrow be increased morbidity with use of Rituximab, when added to
cellularity. Chronic viral infections such as human immun- standard chemotherapy for the treatment of HIV-related non-
odeficiency virus (HIV) or hepatitis may lead to marrow Hodgkin’s lymphoma, its use in HIV-related thrombocytopenia
hypocellularity and, subsequently, to thrombocytopenia in cannot be recommended.
affected individuals.
Thrombocytopenia as a result of acute viral, bacterial, or MICROANGIOPATHIC THROMBOCYTOPENIA
parasitic infections has been well documented. An immuno- THROMBOTIC THROMBOCYTOPENIC PURPURA Throm-
logic mechanism appears likely in the development of throm- botic thrombocytopenic purpura (TTP) is a rare and often
bocytopenia as a result of infection.*°’’ Viral infections such fatal syndrome associated with thrombocytopenia and
as mononucleosis, mumps, and rubeola may be complicated microangiopathic hemolytic anemia (Fig. 25-5). Despite being
by severe thrombocytopenia. In bacterial sepsis; thrombocy- recognized since 1924, the pathogenesis of this syndrome
topenia may be present with or without disseminated intravas-
cular coagulation (DIC). Malaria is frequently associated with
thrombocytopenia as a result of increased destruction and
splenic sequestration.

HIV-RELATED THROMBOCYTOPENIA An array of hemo-

static complications have been described in association
HIV infection. The most common hemostatic abnormality Characteristics Acute Chronic

in these patients is thrombocytopenia. The incidence of throm- Age at onset Child Adult
bocytopenia appears to vary according to the stage of the dis- Previous infection Yes No
ease. There appears to be a correlation between CD4+ T cell Platelet count Usually Usually 30—50,000
depletion, viral load in plasma and the occurrence of thrombo- = 20,000
Bleeding episode Abrupt Slow
cytopenia. Many affected patients also have active hepatitis Duration of Transient Prolonged
infection. Most patients with HIV-related thrombocytopenia thrombocytopenia
do not have significant bleeding Hemorrhagic complications Spontaneous Yes No
may occur but are difficult to predict based solely on the remission
platelet count.
586 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

The exact pathogenic mechanisms responsible for this

disorder remain uncertain. Endothelial cell damage, possi-
bly secondary to drugs or infectious agents, appears to
be critical in the development of vascular injury. Once
endothelial cell damage occurs, inhibition of fibrinolysis
and deficiency of prostacyclin (PGI,) synthesis may then
lead to platelet aggregation and thrombosis. The large von
Willebrand’s multimers that are secreted by damaged
endothelial cells may then persist in circulation and appear
to promote development of microvascular thrombosis. The
presence of platelet microparticles may increase the throm-
botic tendency. A genetic predisposition or an underlying
se tS disorder may be necessary for expression of the disease but
Pe
G20? s this has not been proven except for the familial variant of
the disorder.
Figure 25-5 ®™ Microangiopathic hemolytic anemia. Note the
In the rare inherited familial variant of TTP, there is now
presence of schistocytes (arrows) and nucleated red cell (top border).
evidence that the protease in vascular endothelial cells
that is responsible for cleaving von Willebrand’s multimers
remains poorly understood. Both inherited and sporadic (ADAMTS 13) is defective or absent.*° This disorder is
acquired nonfamilial forms of the syndrome occur. This disor- caused by ADAMTS 13 mutations with deficient production
der was first described as a pentad of signs and symptoms that of the protease. This form of TTP usually appears in infancy
included thrombocytopenia, microangiopathic hemolytic ane- or early childhood and is treated with periodic plasma infu-
mia, fever, neurologic abnormalities, and renal dysfunction.” sions to maintain remissions by replacing the ADAMTS 13
As TTP is recognized more readily, it has become clear that deficiency in plasma.
thrombocytopenia and microangiopathic hemolytic anemia, In the more common nonfamilial acquired cases, autoan-
characterize this disorder, and the fluctuating neurologic abnor- tibody against the ADAMTS 13 protease is present which
malities, fever, and renal dysfunction occur less frequently. The inhibits the protease and results in an acquired deficiency.*!
diagnosis should be suspected in any individual who presents The incidence of TTP is thought to be approximately 4 to
acutely with thrombocytopenia, and consideration should be 11 cases per million..* Women appear to be affected more
given to initiation of appropriate treatment even in the absence often than men, with a female-to-male ratio of 3:2. The peak
of other signs or symptoms. Hyaline microthrombi are the char- incidence occurs between the third and fourth decade of life.
acteristic pathologic feature and are found in multiple organs. Patients who are pregnant, have viral infections, ingest certain
When the diagnosis of TTP is in question, a biopsy of superfi- drugs such as ticlopidine or clopidogrel, or have autoimmune
cial arterioles and capillaries from the skin or gingiva is often disorders appear to be predisposed to development of TTP.
helpful to confirm the diagnosis. However, the microthrombi are TTP may also occur as a complication of antineoplastic
not solely diagnostic for TTP, as they occur in other microangio- chemotherapy or allogeneic stem cell transplantation and
pathic processes such as DIC (Fig. 25-6). The coagulation prognosis tends to be poor with standard treatments.
screening tests and the D-dimer assay are normal in TTP, in con-
trast to DIC, where they are abnormal (see Chap. 27). Clinical Symptoms
Patients with TTP initially present with nonspecific symp-
toms of malaise, weakness, fatigue, fever, or abdominal
pain.’ Neurologic dysfunction and renal abnormalities
are part of the classic pentad but need not be present to
make the diagnosis. The degree of severity of these signs
and symptoms can be quite variable; fever is typically less
than 38.3°C, and the renal abnormalities may be as mild as
proteinuria or microscopic hematuria. Overt renal failure
requiring dialysis is uncommon in TTP, in contrast to
hemolytic uremic syndrome (HUS). Neurologic manifesta-
tions can be as mild as headaches or as severe as coma,
seizures, or obtundation. Abdominal pain, pancreatitis, and
gastrointestinal bleeding may also be associated findings. Any
organ system may become involved in TTP; however, symp-
tomatic pulmonary and cardiac involvement are unusual.
Pericarditis is rare in TTP, but diffuse cardiac ischemia
Figure 25-6 @ Renal biopsy from a patient with thrombotic mimicking pericarditis with diffuse ST segment elevation
thrombocytopenic purpura (TTP) showing glomerular deposits of on electrocardiogram (ECG) can be seen and is typically
platelet-fibrin microvascular occlusion. fatal despite appropriate treatment.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 587

Laboratory Results In patients who do not respond to plasma exchange or

Laboratory features of TTP are those of a severe microangio- who relapse on plasma exchange, there 1s no standard treatment
pathic hemolytic anemia with schistocytes, reticulocytosis, and intervention. The literature has case reports of many interven-
nucleated red blood cells on the peripheral blood smear. Signs tions effective in small numbers of patients. Splenectomy has
of hemolysis are reflected by increases in LDH and indirect been considered the next best therapeutic intervention once
bilirubin, decreased haptoglobin levels, hemoglobinemia patients fail plasma exchange or for patients prone to relapses
and hemoglobinuria. There is severe thrombocytopenia and of the disorder. The majority of patients undergoing splenec-
evidence of decreased peripheral platelet survival despite tomy have also received corticosteroids and blood products,
megakaryocytic hyperplasia in the bone marrow. However, which may also increase the response rate. Antiplatelet drugs
early in the course of TTP, schistocytes and nucleated red such as aspirin, dipyridamole, sulfinpyrazone, and dextran have
blood cells may not be prominent, and severe thrombocytope- been used. Often these antiplatelet drugs are used in variable
nia may be the predominant finding. The bleeding time is combinations, making their efficacy difficult to evaluate.
increased, while the prothrombin time (PT), activated partial Because patients initially present with thrombocytopenia,
thromboplastin time (APTT), fibrinogen level and D-Dimer antiplatelet agents may increase the risk of hemorrhage.
are generally normal in patients with TTP. Slight elevations Immunosuppressive drugs, such as vincristine, and intravenous
in the fibrinogen degradation products (FDPs) have been synthetic prostacyclin have been reported to induce remission.
reported. ADAMTS 13 levels are severely depressed in most Heparin therapy has been used in the past but has shown no
individuals with TTP and may predict a better potential for benefit. There are increasing reports of benefit from the use of
response than for individuals with normal ADAMTS 13 levels. Rituximab for refractory TTP. Other recent reports indicate
Presence of an autoantibody to the ADAMTS 13 protease is patients with relapsed TTP may respond to cyclosporine A
consistent with an acquired TTP. The clinical course of a therapy.
patient does not always parallel the change in the ADAMTS
13 levels. Individuals may rapidly normalize ADAMTS NONIMMUNE THROMBOCYTOPENIA
13 levels in plasma but remain clinically affected by TTP HEMOLYTIC UREMIC SYNDROME HUS and TTP have
while others may have a remission and still have low plasma often been considered different ends of the spectrum of a
ADAMTS 13 levels _ single disorder. HUS is characterized clinically by microangio-
pathic hemolytic anemia, thrombocytopenia, and renal failure.
Treatment Renal vascular damage may initiate intravascular hemolysis
Before the development of newer treatment modalities such with subsequent red cell ADP and platelet membrane phospho-
as plasma exchange, the mortality in TTP exceeded 90%. lipid release. Renal damage may also promote coagulant activ-
With the development of plasma exchange, there has been a ity, resulting in fibrin deposition and endothelial damage but no
significant improvement in the survival of patients with TTP; consumption of coagulation factors. ADAMTS 13 protease
however, the mortality in patients diagnosed with this syn- levels are usually not as severely depressed in HUS, in contrast
drome remains high at approximately 20% to 40%.* Plasma to TTP.
exchange is presently the only treatment modality with HUS resembles TTP pathologically but unlike TTP,
proven efficacy in this disorder and should be instituted as which typically affects adults, HUS is more commonly seen
soon as the diagnosis is made.** The frequency, volume of in the pediatric population. After viral and bacterial infec-
plasma utilized, and type of plasma for exchange has not been tions, HUS usually takes a mild course. The prognosis of
standardized, although most experts support daily exchanges adult onset HUS is worse than that of childhood-onset HUS,
of at least one complete plasma volume utilizing fresh frozen with both a higher mortality rate and higher incidence of
plasma or cryoprecipitate-poor plasma. The length of time long-term morbidity from permanent renal damage.» The
plasma exchange should be continued is also not clearly pathologic thrombi in HUS are almost always limited to the
defined, although most clinicians support continuation of the glomerular capillaries and afferent arterioles of the kidney.
plasma exchange until the platelet count and LDH level have Fever, hypertension, and renal failure are common findings,
both normalized. Corticosteroid therapy when used as the whereas neurologic manifestations are usually less common
sole therapeutic agent is usually ineffective, except perhaps in and less severe. Severe neurologic problems or evidence of
very early relapses. However, corticosteroids in addition to systemic thrombi are more likely to suggest the diagnosis of
plasma exchange may offer some benefit, although the TTP. The hereditary form of HUS, which is more common in
amount of corticosteroids needed and the length of therapy adults, is associated with an unfavorable prognosis.
have not been clearly defined. Recent reports suggest greater
benefit with cyclosporine A added to plasma exchange ther- Treatment
apy with fewer relapses after stopping plasma exchange. Therapeutic management of HUS is usually conservative.
Patients should be monitored closely after discontinuation of Supportive therapy may include hemodialysis, antihyperten-
plasma exchange for evidence of relapse, because relapse is sive therapy, anticoagulants, blood transfusions, antiplatelet
common. Platelet transfusions should be avoided unless the drugs, corticosteroids, and antifibrinolytic agents. Unlike
patient is overtly bleeding, because platelet transfusions may TTP, plasma exchange is not always necessary in HUS and is
accelerate the microangiopathy and have been temporally usually reserved for adults with the familial form of the
related to deterioration and death. disorder. Table 25—7 compares TTP and HUS.
S588 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

and may result in seizures and renal dysfunction; it also has

‘Table 25-7 See tea: _ Ta, a greater association with thrombocytopenia. A variant of
and HUS — eee eclampsia termed HELLP syndrome is also described.’
These disorders are thrombotic microangiopathies with clini-
Characteristics TTP HUS cal manifestations resulting from platelet activation and con-
sumption. The more severe forms of these syndromes must be
Age at onset Adult Children
Organ involvement Multiple Kidney differentiated from TTP or HUS. Preeclampsia and eclampsia
Hematologic typically occur with the first pregnancy and usually occur in
abnormalities the last trimester, whereas TTP and HUS may occur at any
Microangiopathic HA Yes Yes time in pregnancy. Delivery of the fetus usually is effective in
Thrombocytopenia Yes Yes
reversing the eclamptic disorders. Aspirin appears to be use-
Neurological BES Rare
abnormalities ful in treating preeclampsia. However, when these disorders
Renal complications Renal Renal failure are severe or serious complications have developed, plasma
abnormalities exchange therapy is used because it may be difficult to differ-
entiate eclampsia from TTP and HUS.

DISSEMINATED INTRAVASCULAR COAGULATION Acute Quantitative Platelet Disorders:

DIC is caused by many illnesses, sepsis, obstetric emergen-
cies, and severe trauma, and may cause bleeding. Chronic
Thrombocytosis
DIC may be seen with cancer and may result in thrombosis Thrombocytosis, defined as platelet count above the normal
rather than bleeding. Thrombocytopenia is usually seen in range, may be either a reactive process or due to a primary
acute DIC; however, the platelet count may be normal or ele- myeloproliferative disorder (MPD). Reactive thrombocyto-
vated in chronic DIC. In all instances, there appears to be sis, unlike thrombocytosis from a primary MPD, is rarely
accelerated platelet destruction in combination with coagula- associated with bleeding and thrombotic complications, and
tion factor consumption. DIC must be differentiated from the platelet count usually does not exceed 1000 x 10°/L.
other causes of microangiopathy causing thrombocytopenia The morphology of platelets in a reactive process is usually
because treatments differ. Pathogenesis and treatment of DIC normal in contrast to those in a myeloproliferative disorder,
is discussed in Chapter 27. where the platelets are often large and dysplastic. Tests
of platelet function are also typically normal in reactive
PREGNANCY-ASSOCIATED THROMBOCYTOPENIA thrombocytosis. Bone marrow examination may demon-
Thrombocytopenia may occur in pregnancy owing to coinci- strate increased numbers of megakaryocytes; however, in
dental development of disorders discussed earlier in this contrast to MPDs, the megakaryocytes are neither clustered
chapter, or it may occur as a consequence of the pregnancy.*° nor dysplastic.
Disorders resulting from pregnancy include gestational
thrombocytopenia and thrombotic microangiopathies such as
preeclampsia-eclampsia and HELLP syndrome (hemolysis, Primary Thrombocytosis
elevated liver enzymes, low platelet count).
Because all of the myeloproliferative syndromes are charac-
GESTATIONAL THROMBOCYTOPENIA Mild thrombocy- terized by an autonomous proliferation of a pluripotent stem
topenia with platelets counts of 50 to 80 x 10°/L may occur cell, they can all be associated with thrombocytosis. This pro-
in about 8% of normal pregnant women. This most commonly liferation of platelets appears to be independent of the influ-
develops in the third trimester of pregnancy and does not ence of thrombopoietin. Bleeding, thrombosis, and platelet
cause bleeding in the mother or infant. No treatment is neces- function defects can also be seen with any of the MPDs. (The
sary for this disorder. The platelet count returns to normal MPDs as they relate to specific platelet defects are discussed
after delivery. Gestational thrombocytopenia shares features later in the section on acquired qualitative platelet disorders.)
with ITP but the finding that infants born to mothers with Patients with essential thrombocythemia typically have the
gestational thrombocytopenia do not have neonatal thrombo- highest platelet counts of all of the MPDs, often exceeding
cytopenia indicates this may not be immune mediated. Gesta- 1000 X 10°/L. Idiopathic myelofibrosis and chronic myeloge-
tional thrombocytopenia must be differentiated from other nous leukemia (CML) are also associated with milder degrees
thrombocytopenic disorders of pregnancy such as eclampsia, of thrombocytosis. Based on observations of patients with
HELLP syndrome, ITP, or TTP, because these disorders may reactive thrombocytosis and normal platelet function, in
cause harm to the mother or the fetus and require treatment to which bleeding and thrombotic complications are uncommon,
prevent maternal or fetal morbidity and mortality. thrombocytosis alone is not the sole factor in the development
of bleeding and thrombosis in the myeloproliferative syn-
PREECLAMPSIA-ECLAMPSIA AND HELLP SYNDROME dromes. These complications are suggested to result from
Preeclampsia is a relatively common disorder of pregnancy qualitative platelet defects. However, lowering of extreme
that causes hypertension, elevation of uric acid, and thrombo- thrombocytosis with hydroxyurea or thrombocytapheresis
cytopenia. Eclampsia is a more severe form of the disorder may improve the bleeding tendency.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 589

Reactive Thrombocytosis 1. Platelet surface membrane defects,

2. Platelet release or secretion defects,
Reactive thrombocytosis may be attributed to many causes 3. Platelet coagulant activity defects and
and may be either a transient or chronic process. Inflamma- 4. Defects associated with abnormal _platelet-coagulant
tory cytokines such as interleukin-6 and -11 are thought to be protein interactions shown in Table 25-8.
responsible for producing this response. Thrombopoietin
levels in plasma are reduced due to high platelet counts. Some disorders also exhibit thrombocytopenia in addi-
Thrombocytosis is a common finding associated with tion to platelet dysfunction. Other cutaneous, skeletal, and
acute hemorrhage. The platelet count is generally elevated other congenital abnormalities may also be present. Various
within a day or so after hemorrhage as a result of increased congenital disorders as they relate to abnormal platelet func-
marrow stimulation. Similar responses may be seen follow- tion are listed in Tables 25—5 and 25-8.
ing therapeutic phlebotomy for polycythemia vera and
hemochromatosis. PLATELET MEMBRANE DEFECTS
Iron-deficiency anemia has classically been associated As noted in Chapter 24, the platelet surface includes a glyco-
with mild thrombocytosis that does not usually exceed calyx containing various glycoproteins that function as recep-
750 xX 10°/L. This associated thrombocytosis may help tors to bind molecules or proteins and transmit signals to the
to differentiate iron deficiency from other causes of red interior of the platelet to effect platelet reactions. Glanzmann’s
blood cell microcytosis that are not typically associated thrombasthenia and Bernard-Soulier syndrome are congenital
with thrombocytosis. Repletion of iron stores corrects the disorders with surface glycoprotein deficiencies that result in
platelet count to normal. platelet dysfunction. The laboratory abnormalities in these
Thrombocytosis is also associated with underlying syndromes are noted in Table 25-9.
malignancy, and with chronic inflammatory or infectious
processes. GLANZMANN’S THROMBASTHENIA Glanzmann’s throm-
Thrombocytosis can be seen postoperatively after almost basthenia is a rare autosomal-recessive disorder of platelet
any surgical procedure but is most common and pronounced function caused by an absence or deficiency of the membrane
after splenectomy. Within the first 2 weeks, platelet counts GPIIb/IIla complex. GPIIb/IIIa mediates the binding of
rise and then decline to a higher normal or slightly above nor- fibrinogen, von Willebrand’s factor (VWF), and fibronectin to
mal level over a period of months after splenectomy. Platelet activated platelets. GPHb/IIa also serves to connect adhesive
counts may initially increase to as much as two to six times proteins to contractile proteins of the platelet after activation,
preoperative levels. Platelet survival has been documented to thereby facilitating clot retraction. These two proteins exist
be normal. It has been suggested that the thrombocytosis seen within the platelet membrane as heterodimers. Calcium is
after splenectomy is caused by increased platelet production, required for stabilization of the complex. GPIIb/IIla must
because elimination of the splenic pool can account for no exist as a complex in order to function as a ligand to bind
more than a 50% rise in the platelet count. fibrinogen. Both megakaryocytes and platelets express the
Certain drugs may also induce thrombocytosis or assist in GPIIb/GPIIa complex.
platelet recovery after an episode of thrombocytopenia. Admin- The gene that codes for GPIIb and GPIIla is located on
istration of epinephrine will cause a rapid but transient increase chromosome 17. Gene deletion and gene rearrangement of
in platelet count secondary to platelet release from the spleen. GPIlla may account for the phenotypic defects seen in Glanz-
Recovery of marrow suppression from alcohol or chemothera- mann’s thrombasthenia.
peutic agent use will often result in thrombocytosis. Recombi-
nant interleukin-11 (Oprelvekin) may be administered to aid in Clinical Manifestations
platelet recovery after chemotherapy administration to prevent Clinical manifestations of Glanzmann’s thrombasthenia are
quite variable, ranging from minor bruising to severe and
bleeding. This cytokine does not help in other thrombocy-
topenic states. Recombinant TPO was studied in early clinical potentially fatal hemorrhages, with the severity of bleeding
trials but caused thromboembolic problems, premature death consistent within single families.** Although uncommon,
Glanzmann’s thrombasthenia occurs with greater frequency
and permanent bleeding problems and is no longer being tested.
Clinical trials of an experimental oral agent, SB 497115, which
activates the TPO receptor on platelets and increases the platelet
count in healthy volunteers are being conducted in patients with
thrombocytopenic states.
Platelet Membrane Defects
Glanzmann’s thrombasthenia
Qualitative Platelet Disorders Bernard—Soulier syndrome
Platelet Release (Secretion Defects)
Congenital Disorders of Platelet Function Storage pool deficiency (granule defects)
Primary secretion defects (enzymatic pathway defects)
Congenital disorders of platelet function are rare and can be Platelet Coagulant Defects
classified based on the platelet function or response that is von Willebrand Disease
abnormal. This classification currently includes:
590 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

table 25-9 Comparison

of Glanz
"Syndrome
Glanzmann’s Thrombasthenia Bernard-Soulier Syndrome

Platelet count Normal Decreased

Platelet morphology Normal Giant platelets
Bleeding time Prolonged Prolonged
Platelet aggregation
ADP Abnormal Normal
Thrombin Abnormal Abnormal
Collagen Abnormal Normal
Epinephrine Abnormal Normal
Ristocetin Normal Abnormal
Clot retraction Abnormal Normal
Platelet glycoprotein defect GPIIb/IHa GPIb/IX

than Bernard—Soulier syndrome, another surface glycoprotein 2. Normal platelet count and normal platelet morphology,
abnormality. Glanzmann’s thrombasthenia appears to cluster in 3. Prolonged bleeding time,
ethnic populations where consanguinity is prevalent. Bleeding 4. Absent platelet aggregation to ADP, thrombin, collagen,
is most commonly from mucosal surfaces and includes easy and epinephrine, with normal platelet agglutination to
bruisability, epistaxis, spontaneous gingival bleeding, prolonged ristocetin (Fig. 25-7);
bleeding from minor cuts, and menorrhagia. Gastrointestinal 5. Flow cytometric assessment of GPIIb/IIIa surface protein,
hemorrhages are less common. Facial petechiae and subcon- revealing deficiency or absence;
Junctival hemorrhages may be seen in infants associate with 6. Normal lumi-aggregation and °'Cr release; and
crying. The formation of deep hematomas and _ recurrent 7. Abnormal clot retraction.
hemarthroses that are typical features in hemophilia are not
Treatment
present in Glanzmann’s thrombasthenia.
Prevention of bleeding with good dental care and avoidance
Diagnostic Criteria of antiplatelet drugs is important. Patients with Glanzmann’s
Criteria for diagnosing Glanzmann’s thrombasthenia include: thrombasthenia who present with severe bleeding episodes
require platelet transfusions to replace dysfunctional platelets.
1. An autosomal-recessive trait with clinical manifestations Supportive therapy should be used judiciously, because
expressed in homozygotes only; patients may develop alloantibodies to GPIb and GPIIla on

transmittance
Percent

30
8 10 12 14
Time (min)

Figure 25-7 m This graph depicts the lack of platelet aggregation to e pinephrine, 10 um (blue); ADP, 5 um
(black); collagen, 2 ug/mL (red);,
and arachidonic acid, 0.5 g/mL (green)—typical of a patient with Glan zmann’s thrombasthenia.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 591

the transfused platelets or anti-HLA antibodies and become (which is the receptor for vWF) is the cause of the aggregation
refractory to platelet transfusions. Antifibrinolytic agents such defect. 25-The differences in platelet aggregation tests with
as EACA may help in controlling hemorrhage, especially various platelet agonists among von Willebrand’s disease,
from the nose and mouth where fibrinolytic activity is promi- Bernard—Soulier syndrome, and Glanzman’s thromboasthenia
nent. Topical measures such as thrombin or pressure packing are depicted in Table 25-10. Crossed immunoelectrophoresis
may be effective. DDAVP has been used but has not been or flow cytometry of platelet membrane glycoproteins should
helpful. Estrogen therapy in the form of birth control pills is demonstrate a decrease in GPIb, GPIX, and GPV to confirm a
especially useful in controlling menorrhagia. Recombinant diagnosis of Bernard—Soulier syndrome.
factor VIIa has been successfully used but thromboembolism Heterogeneity among the glycoprotein abnormalities in
has occurred in one reported case after discontinuation of the Bernard-Soulier indicates that there are multiple genetic
recombinant factor VIIa treatment. defects. Heterozygotes present with recognizable abnormalities
such as occasional large platelets on the peripheral smear, with
BERNARD-SOULIER SYNDROME Bernard—Soulier syn- a history of bleeding yet few clinical problems. Homozygotes
drome is a rare autosomal-recessive bleeding disorder caused present with abnormal platelet function and morphology,
by a deficiency of the platelet GPIb/IX complex. GPV, which thrombocytopenia, and hemorrhagic disease.
is associated with the GPIb/IX complex on the platelet sur-
face and is a thrombin substrate, has also been documented to Diagnostic Criteria
be deficient in Bernard—Soulier platelets. Gene mutations of Rather specific criteria for the diagnosis of this bleeding syn-
GPIb and GPIX, but not GPV, appear to be the cause of this drome have been established. These criteria are:
disorder. An acquired form of the disorder has been reported
1. An autosomal trait with clinical manifestations expressed
due to autoantibodies to these platelet glycoproteins. As dis-
in homozygotes (or double heterozygotes with combined
cussed in Chapter 24, GPIb/IX plays a major role in various
genetic abnormalities of GPIb, GPV, and GPIX)
hemostatic events. GPIb/IX is the platelet receptor involved in
2. Normal platelet count or moderate thrombocytopenia
the vWF-dependent contact adhesion of unactivated platelets
(despite a normal number of marrow megakaryocytes)
to exposed subendothelium at high shear rates and for the
3. A significant number of giant or large platelets present on
binding of platelets to fibrin. It also serves as a high-affinity
the peripheral blood smear and prolonged bleeding time in
thrombin-binding site and regulates platelet shape and reac-
excess of the degree of thrombocytopenia
tivity. Because the platelet membrane is attached to the
4. Absent platelet agglutination in response to human vWF
cytoskeleton via GPIb, the loss of normal membrane—
and ristocetin
cytoskeletal function may account for the abnormal platelet
5. Normal platelet aggregation in response to ADP, collagen,
morphology seen in Bernard—Soulier platelets.” Bleeding in
and epinephrine, with reduced aggregation in response to
Bernard—Soulier syndrome is due to a combination of hemo-
thrombin
static defects including thrombocytopenia, abnormality of
6. Normal factor VIII coagulant activity (FVII:C) and vWF
platelet-von Willebrand’s factor interaction important for
antigen
platelet adhesion, abnormality of platelet-thrombin interac-
tion, and abnormality of platelet coagulant activity.
Treatment
Affected individuals who present with active bleeding should
Clinical Manifestations and Laboratory Results
be treated with red blood cell transfusions to replace blood
Patients with Bernard—Soulier syndrome present with the typ-
loss and an antifibrinolytic agent such as EACA. Antifibri-
ical symptoms of a primary hemostatic disorder with varying
nolytic agents do not correct the defect but only allow the pri-
severity. Gingival bleeding, epistaxis, purpura, menorrhagia,
mary hemostatic plug to remain intact at the site of injury.
and gastrointestinal bleeding are the typical hemorrhagic
DDAVP has been reported to shorten the bleeding time in
manifestations. Symptoms occur early in life and have a ten-
some individuals*® but not in others. Estrogen therapy may be
dency to decrease with age.
helpful in controlling bleeding.*°*! In instances in which
Laboratory abnormalities in patients with Bernard—
bleeding is not controlled by these measures, platelet transfu-
Soulier syndrome include normal or moderately reduced
sions may be necessary; however, these should be used judi-
platelet counts with large irregularly shaped platelets noted on
ciously to avoid alloimmunization to GPIb. There has been
the peripheral smear, and a prolonged bleeding time. The
successful use of recombinant factor VIla to treat bleeding in
platelets seen on peripheral blood smear may be large enough
this disorder
to resemble lymphocytes. Normal numbers of megakary-
ocytes are found on bone marrow examination. Platelet
aggregation is normal with ADP, epinephrine, and collagen, PLATELET RELEASE (SECRETION) DEFECTS
and is reduced with thrombin. Ristocetin-induced platelet As described in Chapter 24, platelet glycoprotein receptors
aggregation is absent in Bernard—Soulier syndrome, similar to transmit signals intracellularly to cause release of platelet
von Willebrand’s disease. However, the deficient ristocetin- granule substances and platelet shape change. Phospholi-
induced aggregation defect is corrected by the addition of pases (A and C) are liberated from the internal surface of
normal plasma in von Willebrand’s disease, but not in the platelet membrane in response to platelet agonists
Bernard-Soulier syndrome, where the deficiency of GPIb/IX and cause an increase in intracellular calcium levels. This
592 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

Epinephrine Arachidonic Acid Ristocetin Ristocetin + VWF

vWD = +

BSS +
GT = -

vWD = von Willebrand’s disease; BSS = Bernard-Soulier syndrome; GT = Glanzmann’s thrombasthenia.

produces centralization of granules and fusion of granule Laboratory features of storage pool deficiencies include
membrane with the open canalicular system to transport a prolonged or normal template bleeding time. Aspirin inges-
granule proteins to the environment to allow adhesion tion may cause a marked prolongation of the bleeding time.
and aggregation. The disorders of platelet release primarily Plasma coagulation screening tests and factor assays are
involve absence of platelet granules or defective enzymatic normal in these disorders. Platelet aggregation testing in
pathways. 8 storage pool deficiency demonstrates normal primary wave
aggregation but absent or reduced secondary wave aggrega-
STORAGE POOL DEFICIENCIES (GRANULE DEFECTS) tion in response to ADP and epinephrine. Collagen-induced
Storage pool deficiencies are classified according to an analy- aggregation is reduced at lower but not high collagen concen-
sis of the granule proteins and the morphological appearance trations. High concentrations of thrombin do not produce nor-
of the platelets. Most commonly, a decrease in platelet dense mal release, in contrast to platelet secretion defects. Platelet
granules is present, with decreased amounts of secretable aggregation testing is variable in a storage pool deficiency.
ADP, adenosine triphosphate (ATP), calcium, and serotonin ADP and epinephrine-induced aggregation are often normal.
(delta [5] storage pool deficiency). In other patients, alpha (a) Collagen and thrombin-induced aggregation are more fre-
granules and dense granules are decreased, and decreases in quently abnormal. Platelet aggregation patterns in a,5 storage
amounts of a-granule proteins such as platelet factor 4, beta pool deficiency are similar to ad storage pool deficiency as
(6)-thromboglobulin, and platelet-derived growth factor as the deficiency in 6 granules appears more severe than the
well as the dense granule proteins are noted (a6 storage pool deficiency of a granules in these individuals. Platelet counts
deficiency). Other patients have a decrease in @ granules only, are normal in the storage pool deficiencies. Morphology is
with normal dense granules and normal amounts of dense normal except in the gray platelet syndrome and Chediak—
granule constituents (ad storage pool deficiency). Deficiency Higashi syndrome.
of the « granules leads to an agranular appearance of platelets
on the Wright-stained peripheral blood smear and has been PRIMARY SECRETION DEFECTS (ENZYMATIC PATHWAY
termed gray platelet syndrome. The Quebec platelet disorder DEFECTS) These disorders resemble storage pool deficien-
is reported to have a deficiency of a granule proteins related cies but have no abnormalities of the platelet granules
to excessive proteolysis. Platelet factor 3 (PF3) activity is and have normal amounts of platelet granule contents. The
reduced in these disorders and appears related to the defects causes of release defects are deficiencies of enzymes and
in platelet aggregation. other “second messengers” that transmit the signal from
The inheritance pattern of these disorders has not been surface receptors to cause platelet release of granule con-
well defined but in some instances appears to be an autoso- tents. The bleeding tendency and laboratory findings in
mal dominant pattern. Patients with storage pool deficiency these disorders are similar to those found in the storage
have a mild to moderate mucosal bleeding tendency. Easy pool deficiencies.
bruising, epistaxis, menorrhagia, postpartum bleeding Cyclo-oxygenase deficiency results in deficient conver-
and bleeding after dental extractions and tonsillectomy are sion of membrane-associated arachidonic acid to thrombox-
often present. Gastrointestinal bleeding is uncommon, and ane A,. Thromboxane synthetase deficiency and other defects
hemarthrosis is not present. Storage pool deficiencies have of thromboxane A, generation and calcium mobilization have
been found in association with other congenital abnormali- been described.”
ties (see Table 25-5). The Hermansky—Pudlak syndrome Treatment of storage pool deficiencies and primary
describes patients with oculocutaneous albinism and dense secretion defects has primarily been with judicious use of
granule storage pool deficiency. Individuals with Chediak— platelet transfusions for prevention or treatment of bleeding.
Higashi syndrome also have oculocutaneous abnormalities, For procedures with low risk of bleeding or where bleeding is
including ocular albinism and characteristic silver-gray hair easily controlled by local measures, no additional treatment
with dense granule storage pool deficiency. Other congeni- may be needed. Prednisone has been reported to improve the
tal abnormalities, including Wiskott—Aldrich syndrome and bleeding time but not platelet aggregation defects, presum-
the syndrome of thrombocytopenia with absent radii (TAR ably owing to an effect to promote vascular integrity. Cryo-
syndrome), have been described to have an associated precipitate and DDAVP have improved the bleeding time in
platelet storage pool deficiency. patients with Hermansky-Pudlak syndrome. DDAVP has been
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 593

used in patients with gray platelet syndrome to shorten the THE FACTOR VIII/VWF COMPLEX IN PLASMA
prolonged bleeding time. However, efficacy in treating
bleeding has not been reported in those studies. Red blood Chromosome 12
X Chromosome
cell transfusion may also improve the platelet adhesion Endothelial cell
defect, possibly by supplying ADP. Avoidance of nons- Liver cell Megakaryocyte
teroidal anti-inflammatory agents or other drugs that induce
platelet dysfunction is important in the management of these
disorders. Factor VIII VWEF subunit

DEFECTS IN PLATELET COAGULANT ACTIVITY |

VWF multimers
Platelet disorders with failure to generate thrombin are con-
sidered to have defects in platelet anticoagulant activity. Very
few patients with this problem have been described. In one
(Factor VIII) (vsWF multimers)
individual extensively studied, there was a failure of platelet
microvesiculation in response to stimulation leading to Figure 25-8 ® Factor VIII/von Willebrand factor (VWF) complex in
decreased Va—Xa and VIla—IXa binding which slowed the plasma.
normal coagulant response.
The bleeding pattern in these disorders is different from
other platelet function defects since it involves an abnormal- The role vWF plays in hemostasis is quite important, as
ity of coagulant activity. Easy bruising and epistaxis are not seen from the bleeding manifestations in patients with von
predominant features. Bleeding after surgery, tooth extrac- Willebrand’s disease. vWF is an important adhesive protein
tions and childbirth occur. Spontaneous pelvic hematomas following vascular injury. It serves as a ligand between
and hematomas after trauma are also seen. This pattern of platelets, vascular endothelium, and other adhesive proteins
bleeding more closely resembles the bleeding in hemophilias. such as fibronectin (Fig. 25-9). VWF has distinct domains for
The bleeding time is normal in affected individuals but binding to platelet GPIb/IX, GPIIb/IIIa, and subendothelial
the prothrombin timeis abnormal. Specific assays for platelet components heparin and collagen. Platelet GPIb serves as the
factor 3 activity, which measures the platelet contribution to
major receptor for vWF. Platelet adhesion is dependent
on subendothelium, GPIb, and vWF. In von Willebrand
coagulation, should be abnormal.
Treatment for bleeding is usually with platelet transfu- disease, the platelets that are intrinsically normal exhibit
sions to provide platelet procoagulant activity. Concentrates abnormal adhesion because of the absence or dysfunction of
of prothrombin complex proteins have been used but have a
risk of thrombosis so should be used only for serious bleed-
ing problems.

von Willebrand Disease Platelet |

GP Ib GP Ilb-Illa
von Willebrand disease is an inherited deficiency of vWF that
may be confused with a primary platelet defect because of the
similarities in clinical presentations between von Willebrand
disease and platelet disorders. von Willebrand disease was orig-
inally termed parahemophilia, because it is a congenital bleed-
Plasma
ing disorder; however, it has a different inheritance pattern and
different bleeding pattern than hemophilia.
vWE is a large, multimeric glycoprotein coded for by a
gene located on chromosome 12 (Fig. 25-8). VWF is synthe-
sized by vascular endothelial cells and stored in Weibel-Palade
H
Palade bodies. A small amount of vWF is also synthesized in
Endothelium 4 arian aA
megakaryocytes and stored in the platelet « granules. Polymer-
ization of VWF occurs in endothelial cells with the production Subendothelium COLLAG
of high-molecular-weight multimers. vWF-cleaving protease VWF

(ADAMTS 13) reduces the size of the multimers at the time Figure 25-9 ™ Schematic representation of the interactions
vWE is secreted into the plasma. Circulating vWF forms a between vWF, platelets, and collagen of the subendothelium. vWF
complex with factor VIII, the protein that is deficient or defec- synthesized by endothelial cells is released in plasma and is stored
tive in hemophilia A. vWF acts as a carrier protein for factor in the a granules of platelets, and can be released after stimulation.
VWF mediates platelet adhesion through binding to collagen and to
VIII, serving to protect the factor VIII molecule from prote-
platelet glycoprotein Ib (GPlIb) in the presence of ristocetin, as well
olytic degradation and increase its concentration at the site of as the platelet GPIlb/Illa in the presence of physiologic agonists
tissue injury. In normal individuals, plasma levels of factor VIII (thrombin, collagen, ADP). vWF also binds to factor VIII (FVIII) and
closely correlate with plasma levels of vWF. heparin.
594 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

VWE. VWE promotes secondary hemostasis by functioning as binding of normal vWF to platelets caused by point mutations
a carrier for factor VIII. In von Willebrand disease, absence or in platelet glycoprotein Iba (GPIba).*°*’ The disorder also
dysfunction of vWF results in decreases in factor VII and appears tobe inherited in an autosomal dominant pattern. There
abnormal secondary hemostasis. is variable thrombocytopenia, possibly due to shortenedplatelet
VWF can be measured antigenically and functionally survival from this increased vWF binding to platelets.
using the antibiotic ristocetin. Ristocetin was removed from
use after clinical trials revealed it produced thrombocytope- LABORATORY EVALUATION
nia. Further evaluation revealed it produced agglutination of The routine laboratory evaluation of patients suspected of
platelets in the presence of vWF. This has led to its use as a having von Willebrand’s disease includes a bleeding time,
diagnostic agent in the evaluation of von Willebrand disease platelet count, factor VIII coagulant activity, quantitative
in vitro with the ristocetin-induced platelet aggregation measurements of plasma vWF antigen (vWF:Ag) by crossed
(RIPA) test. Factor VII is measured by a functional clotting immunoelectrophoresis or agarose gel electrophoresis, and
assay. Measurements of factor VII, vWF antigen, and vWF functional assays of plasma vWF by RIPA (vWF activity).**
activity are important in differentiating von Willebrand Subtypes of von Willebrand disease can be distinguished
disease from hemophilia A and to define the types of von according to the vWF:Ag, vWE activity, factor VII coagulant
Willebrand’s disease. activity, and multimeric pattern on gel electrophoresis. All
von Willebrand disease comprises both quantitative and types of von Willebrand disease will have reduced vWF
qualitative abnormalities of the large multimeric VWF glyco- activity but other laboratory findings will vary between the
protein. Affected individuals exhibit mucocutaneous bleeding types. Platelet-type von Willebrand disease also has reduced
typical of defects of primary hemostasis, in contrast to the plasma vWF levels, reduced low molecular weight vWF mul-
joint and deep muscle bleeding typical for hemophilia. The timers and enhanced RIPA, similar to type 2 von Willebrand
disorder has an autosomal pattern of inheritance but has vari- disease. Since the defect occurs on the platelet surface, VWF
able penetrance for expression in affected individuals. from affected individuals will bind normally to normal
Severe cases are characterized by recurrent, potentially life- platelets and vWF from normal individuals will bind abnor-
threatening bleeding, whereas mild cases may go undetected. mally to platelets of affected individuals.
Diagnosis of von Willebrand disease is often difficult
because of the variability of abnormal laboratory results TREATMENT
within affected individuals or family members. vWF and fac- For patients with type I von Willebrand disease, DDAVP is
tor VIH are both acute phase reactant proteins, and plasma considered the initial treatment of choice.” DDAVP stimu-
levels may be increased with stresses such as inflammation, lates the release of vWF from endothelial cell Weibel-Palade
surgery, and pregnancy or with estrogen therapy. Further- bodies. Baseline plasma vWF antigen and activity increase
more, the levels of VWF vary with ABO blood types, with the about three- to fourfold after DDAVP administration. vWF
lowest levels being seen in individuals of O blood type. stores within the endothelial cells are depleted after about 3 to
4 days of DDAVP treatment. Patients requiring longer treat-
CLASSIFICATION ment or those who do not achieve an adequate response to
von Willebrand disease can be classified into three general DDAVP may require the use of intermediate purity factor VIII
categories: products such as Humate P, which contain intact vWF, or
cryoprecipitate. In type 1 patients who receive cryoprecipi-
1. Type 1, an autosomal-dominant disorder accounting for
tate, there is evidence that the endothelial cells are also stim-
70% of all cases, which is characterized by a quantitative
ulated to increase de novo synthesis of VWF.
decrease in normal vWF and mild bleeding;
Patients with type 3 von Willebrand disease do not make
2. Type 2, which has variable inheritance with numerous sub-
vWE and do not respond to DDAVP. Therefore, they must
types, accounts for most of the remaining cases, and is
characterized by a qualitative abnormality in the structure be treated with intermediate purity factor VIII products or
cryoprecipitate.
of vWF; and
3. Type 3, a rare autosomal-recessive disorder characterized There are many subtypes of type 2 von Willebrand dis-
by absent levels of VWF multimers and a severe bleeding ease, some of which respond to DDAVP and some of which
do not. DDAVP has been reported to cause thrombocytopenia
diathesis very similar in presentation to hemophilia A.*
in the type 2b variant.** In many of the type 2 variants,
von Willebrand Normandy is an unusual type 2 variant response to therapy is difficult to quantitate and may require
of von Willebrand disease that is characterized by a defect in measurement of the bleeding time or PFA-100 with determi-
the vWF binding to factor VIII. This disorder is sometimes nation of correction of platelet function after administration
confused with mild hemophilia A because the factor VIII of appropriate therapy. Addition of DDAVP to cryoprecipitate
coagulant level is reduced but the vWF antigen and activity has been utilized as an effective combination in certain
levels are normal or elevated. Clinically, the bleeding diathe- patients.
Sis associated with von Willebrand Normandy is similar to Antifibrinolytic agents can be utilized as an adjunctive
other patients with type 2 von Willebrand’s disease.*5 therapy in all patients with von Willebrand disease. For
A platelet-type von Willebrand’s disease has also been dental procedures, local measures are used and replacement
described. It is a platelet disorder characterized by increased therapy may not be necessary.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 595

Treatment of platelet-type von Willebrand disease with correlation of the metabolic changes with the exact mechanism
measures to increase plasma vWF may actually cause throm- of the functional defect has not yet been firmly established.
bocytopenia by inducing clearance of platelets from the circu- There may also be a defect in platelet activation since platelets
lation. Infusion of smaller amounts of cryoprecipitate than in some uremic individuals demonstrate reduced fibrinogen
used to treat the other forms of von Willebrand disease and binding, secretion and aggregation responses.
platelet transfusions if the thrombocytopenia is severe may be Mild thrombocytopenia with platelet counts as low as
useful. There has been a report of successful use of recombi- 100 X 10°/L, decreased adhesion, abnormal aggregation, and
nant factor VIIa in this disorder. increased bleeding times are abnormal laboratory findings
often seen in patients with uremia. Platelet aggregation studies
in uremic patients typically show no characteristic patterns.
Acquired Qualitative Platelet Disorders The anemia of renal failure may contribute to the hemo-
Platelet dysfunction can also be caused by many systemic ill- static defects observed in uremic patients. Hematocrit values
nesses and medications (Table 25-11). In these diseases, the of less than 25% may prolong bleeding times. Red cells may
platelet dysfunction is a secondary manifestation and not typi- play a role in hemostasis by improving factor VIII coagulant
cally one of the primary presenting features of these disorders. function and by providing ADP, a platelet-aggregating agent.
Acquired causes of platelet dysfunction are more commonly Erythropoietin (r-HuEPO) therapy has proved to be effective
encountered than congenital platelet disorders; however, the in improving hemostasis and decreasing the bleeding tenden-
results of platelet function tests can appear similar to those seen cies in uremic patients on dialysis. It has been theorized that
with congenital disorders. To differentiate between acquired t-HuEPO may also increase platelet number and aid aggrega-
and congenital causes of platelet dysfunction, a careful history tion in addition to raising the haemoglobin and hematocrit.
and physical examination as well as family history must be Hemodialysis or peritoneal dialysis is the treatment of
obtained. choice to correct the hemostatic defect in uremia. However,
platelet function abnormalities may remain. Administration of
UREMIA
cryoprecipitate to patients unresponsive to dialysis has been
Bleeding has long been recognized as a common complication reported to shorten the prolonged bleeding time and decrease
of uremia. Patients with acute and chronic renal failure gener- bleeding. The use of DDAVP in uremic patients has also
ally exhibit bleeding from mucous membranes. Petechiae, pur- prevented clinical bleeding in patients following surgical pro-
pura, epistaxis, ecchymosis, and gastrointestinal bleeding are cedures and has shortened the prolonged bleeding time.°°
common. Severe hemorrhage within serous cavities and Conjugated estrogens have been reported to shorten the
muscles may also occur. bleeding time in uremia and limit bleeding in patients who
A number of different laboratory findings and clinical develop gastrointestinal telangiectasias. The mechanism of
symptoms suggest that platelet dysfunction with abnormal action is not clear.
platelet—vessel wall interaction is the major cause of platelet
LIVER DISEASE
dysfunction and hemorrhage. Studies have shown an abnormal-
Chronic liver disease is often associated with a significant
ity in the interaction of vWF and platelet GPIIb/IIIa complex in
hemorrhagic diathesis as a result of multiple alterations in
uremic patients; however, platelet membrane glycoproteins Ib,
hemostasis, including platelet dysfunction. Mild to moderate
IIb, and IIa are quantitatively normal. Other platelet abnormal-
thrombocytopenia is seen in approximately one-third of
ities seen in uremia include abnormal prostaglandin synthesis,
patients with chronic liver disease as a result of splenic
decreased membrane procoagulant activity, decreased platelet
sequestration secondary to congestive splenomegaly associ-
serotonin release, abnormal B-thromboglobulin levels, elevated
ated with portal hypertension. Abnormal platelet function
intracellular calcium, and decreased thromboxane synthesis.
tests found in patients with chronic liver disease include
Increased levels of “uremic toxins,” such as guanidosuccinic
reduced platelet adhesion; abnormal platelet aggregation to
acid and phenols, are rather consistent findings in patients with
ADP, epinephrine, and thrombin; and abnormal PF3 availability.
uremia. The acquired platelet defects associated with uremia
An acquired storage pool deficiency has also been suggested.
are thought to be primarily mediated by these products. A
The exact mechanism responsible for the platelet defects seen
in chronic liver disease is not known.
Treatment of bleeding in a patient with chronic liver dis-
ease may require several modalities simultaneously to correct
the multiple abnormalities present. Transfusion with platelet
concentrates may ameliorate the bleeding and thrombocy-
Renal disease (uremia)
Liver disease topenia associated with chronic liver disease; however, the
Paraproteinemias expected rise in platelet count following transfusion may be
Myeloproliferative disorders blunted if splenic sequestration or increased platelet con-
Acquired von Willebrand disease sumption occurs. DDAVP may improve the qualitative
Cardiopulmonary bypass
platelet defect associated with this disorder. The numerous
Acquired storage pool deficiencies
Drug therapy coagulation factor deficiencies common in chronic liver dis-
ease typically require the administration of fresh frozen
596 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

plasma to correct the coagulation abnormalities. Use of con- manifestations include ecchymosis, epistaxis, and mucocuta-
jugated estrogens may decrease the overall bleeding tendency neous bleeding from the gastrointestinal and genitourinary
after an acute episode. tracts. Thrombosis occurs in both the arterial and venous cir-
culation and includes deep vein thrombosis, pulmonary
PARAPROTEINEMIAS embolism, stroke, myocardial infarction, and thrombosis of
Patients with lymphoproliferative malignancies may exhibit the hepatic, portal, splenic, and mesenteric veins. Patients
both hemorrhagic diatheses and hypercoagulability. These may present with bleeding and thrombosis simultaneously, or
hemostatic alterations are complex and multifactorial. Clini- alternate with bleeding and thrombotic episodes as the disease
cally significant bleeding is a manifestation associated with progresses. The precise mechanisms that cause the problems
multiple myeloma, Waldenstr6m’s macroglobulinemia, and are unknown.°! Intrinsic platelet function defects are noted
other related malignant paraprotein disorders. The pathogen- frequently. In many cases, thrombocytosis is a contributing
esis of hemorrhage and other recognized hemostatic abnor- factor, especially for thrombotic problems in patients with an
malities has been proposed to be caused by the interaction of MPD. In patients with polycythemia rubra vera increased
the paraprotein with platelets and coagulation factors. Clini- whole blood viscosity may play a role.
cal bleeding and platelet dysfunction are seen in approxi- Aggregation patterns in these disorders are usually not
mately 60% of patients with IgM myeloma or Waldenstrém’s characteristic. The most consistently noted laboratory abnor-
macroglobulinemia as compared to approximately 40% of the malities include abnormal release and aggregation in response
patients with IgA myeloma and 15% of patients with IgG to epinephrine, collagen, and ADP, but these findings vary
myeloma. from patient to patient with the same type of MPD. The bleed-
The hemorrhagic diathesis of paraproteinemias is charac- ing time is prolonged in many cases but does not correlate
terized by a prolonged bleeding time and platelet dysfunction. with the risk for bleeding or thrombosis in these patients.
The occurrence of bleeding correlates with the impairment of Other reported abnormalities include acquired storage pool
platelet function. Thrombocytopenia and inhibition of coagula- defects, abnormal prostaglandin and arachidonic acid metab-
tion factors may also contribute to the bleeding tendency. olism, and platelet hyperactivity.
Patients may present clinically with spontaneous epistaxis and Thrombosis is a major complication of PV owing to an
ecchymosis, or unexplained postoperative bleeding in the face increased red cell mass and whole blood viscosity and throm-
of a normal platelet count and coagulation profile. Patients bocytosis. Thrombocytosis is generally moderate, in the range
with malignant paraprotein disorders should not ingest aspirin of 500 X 10°/L to 1 x 10!°/L, and is seen in approximately
or aspirin-containing drugs because this may exacerbate the 50% to 60% of cases. Platelet function defects occur in
platelet defect. Uremic platelet dysfunction associated with approximately 50% to 60% of cases. Platelet function defects
renal failure often seen in the malignant paraproteinemias may are thought to be responsible for many of the hemostatic
also contribute to the hemorrhagic episodes. problems seen. Abnormal platelet aggregation in response
Platelet abnormalities include decreased aggregation in to epinephrine, ADP, and collagen has been reported in
response to various aggregating agents, altered shape change, PV. Platelet activation and intravascular coagulation with
abnormal release reactions, and a prolonged bleeding time. increased plasma levels of B-thromboglobulin, low platelet
Acquired von Willebrand’s syndrome causing reduced levels of serotonin levels, and abnormal levels of fibrinogen and pro-
vWF/factor VIII complexes, other circulating anticoagulants, thrombin have also been reported in cases of PV.
amyloid-associated coagulopathies, impaired fibrin formation Patients with idiopathic myelofibrosis may experience
and polymerization, hyperviscosity syndrome, nephrosis, and bleeding, such as ecchymosis and urogenital hemorrhage.
DIC represent additional abnormalities that predispose patients Thromboembolic complications, with the exception of
with paraproteinemia to bleeding. hepatic and portal vein thrombosis, occur rather infrequently.
Thrombocytopenia, unrelated to the paraprotein effect, It has been suggested that of all the MPDs, idiopathic
may result from marrow replacement, chemotherapy, or hyper- myelofibrosis has the greatest degree of platelet function
splenism and is acommon finding contributing to the incidence defects. Decreased platelet adhesion and prolonged bleeding
of significant bleeding. times are relatively common findings in patients with idio-
Therapy for patients with qualitative defects resulting pathic myelofibrosis. Storage pool deficiencies and impaired
from multiple myeloma and related disorders includes platelet aggregation have also been reported.
plasmapheresis to reduce the circulating paraprotein concen- ET presents with thrombocytosis and is associated with
trations and chemotherapy to inhibit paraprotein production. both bleeding and thrombosis, although bleeding episodes
Blood product support and other therapies may be required occur more often. Evidence suggests that thrombocytosis
when additional hemostatic abnormalities are present. alone does not account for the bleeding and thrombotic
episodes seen in ET, but the combination of thrombocytosis
MYELOPROLIFERATIVE DISORDERS and abnormal platelet function determine the hemostatic
The MPDs include polycythemia vera (PV), idiopathic defects in ET. Decreased platelet aggregation in response to
myelofibrosis, CML, and essential thrombocythemia (ET) epinephrine as well as decreased platelet retention are noted
(see Chaps. 17 and 18). This group of disorders has many in patients with ET. Impaired PF3 availability and platelet
clinical and hematologic features in common, but the hemo- hyperactivity have been reported in patients with thrombosis
static and thrombotic problems are variable. The hemorrhagic in ET. Several platelet defects have been described in ET, but
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 597

what predisposes patients to bleeding or thrombotic manifes- CARDIOPULMONARY BYPASS

tations is still uncertain. In the United States, more than 200,000 patients per year
Bleeding and thrombosis occur least frequently in patients undergo cardiopulmonary bypass surgery. Alterations in hemo-
with CML. When present, bleeding includes mucocutaneous stasis and life-threatening hemorrhage have been documented.
hemorrhages, retinal hemorrhages, and hematuria. It is thought Various hemostatic abnormalities are implicated in the develop-
that acquired platelet defects may be responsible for this bleed- ment of the hemorrhagic manifestations of cardiopulmonary
ing as a result of dysplastic platelet production. Thrombocytope- bypass surgery. These abnormalities include decreases in
nia, as occurs with transformation to blast crisis, also results in platelet number and function, factor deficiencies resulting from
a bleeding tendency. Decreased or absent platelet aggregation in consumption and hemodilution, increased fibrinolytic activity,
response to ADP, epinephrine, and collagen have been reported DIC, and inadequate or excess neutralization of heparin with
in cases of CML. A prolonged bleeding time is quite frequently protamine. Postbypass diffuse microvascular bleeding occurs
seen in patients with CML in remission as well as in a blast with greater frequency in patients undergoing repeat surgery or
crisis. A factor V deficiency has been reported in some cases of complicated cardiac procedures as compared with patients
CML. Increases in platelet vWF have also been noted. undergoing surgery for the first time. During cardiopulmonary
Management of patients with MPDs aims at reducing the bypass, a prolonged bleeding time can be seen even though the
risk of hemorrhage and thrombotic complications.°? Cytoreduc- platelet count is only mildly low, in the range of 100 x 10°/L.
tive chemotherapy is often used. In patients with marked Platelet activation and platelet dysfunction account for the
thrombocytosis and cerebral ischemia, plateletpheresis in addi- majority of bleeding complications seen during this procedure.*°
tion to chemotherapy may be helpful. The use of antiplatelet Proposed mechanisms include platelet activation with a granule
drugs such as aspirin is controversial. Antiplatelet therapy may release during exposure to the extracorporeal unit, and plasmin
be useful to prevent recurrences of thrombosis or to treat a degradation of platelet membrane receptors.
specific complication of pain and erythema of the hands termed Platelet concentrates are administered to stop bleeding.
erythromelalgia but may worsen a preexisting bleeding ten- Fresh frozen plasma and cryoprecipitate should be given only
dency. Hemorrhage may require administration of platelets to to treat bleeding associated with coagulation factor deficien-
replace endogenous dysfunctional platelets. cies. DDAVP is often given prophylactically for high-risk
patients or to stop bleeding after it occurs. Aprotinin is also
ACQUIRED VON WILLEBRAND DISEASE used to decrease postoperative bleeding, particularly in those
Acquired von Willebrand disease is a rare bleeding disorder patients undergoing repeat or complicated procedures. It is
that has been found in patients with myeloproliferative disor- thought that aprotinin maintains platelet function by inhibit-
ders, lymphoproliferative diseases, monoclonal gammopathies, ing plasmin degradation of platelet membrane receptors.
and collagen vascular diseases, and after certain infectious Anti-fibrinolytic agents may also help to minimize post-
processes.’ Most patients are 40 vears of age or older, with no operative blood loss.
previous history of bleeding. Bleeding presents with an insidi-
ous onset and manifests as mucocutaneous or posttraumatic ACQUIRED STORAGE POOL DEFICIENCIES
hemorrhage. Acquired storage pool deficiencies have been reported in
Pathologically, this disorder is caused by production of patients with SLE, ITP, TTP, DIC, HUS, MPDs, hairy-cell
an antibody that specifically interacts with vWF. Acquired leukemia, acute nonlymphocytic leukemia, chronic lympho-
von Willebrand disease is caused by a diverse group of disor- cytic leukemia, and in cardiopulmonary bypass. The acquired
ders, and the associated antibodies are a heterogeneous group platelet defect in each disorder may be variable and can be
that interact differently with the vWF protein from patient to caused by either production of dysfunctional platelets (i.e.,
patient. Because these antibodies are so varied, the laboratory MPDs) or depletion of storage pool constituents resulting
findings seen in acquired von Willebrand disease are also from injury or activation (DIC).
variable from patient to patient but may include a prolonged
bleeding time, decreased plasma levels of factor VIII coagu- DRUG THERAPY
lant activity, VWF:Ag, and vWF activity, and abnormalities in A large variety of pharmacologic drugs may affect platelet
vWE multimeric patterns. Platelet vWF is generally normal. function (Table 25—12). Some drugs may induce thrombocy-
The typical rise in factor VIII coagulant activity and vWF topenia. Other drugs alter platelet responses. Drug-induced
activity after infusion of cryoprecipitate seen in congenital alterations of hemostasis that cause activation of platelet
von Willebrand disease is not seen in the acquired form function clinically manifest as thrombosis whereas drugs that
because of neutralization of these activities by antibody. inhibit platelet function clinically manifest as hemorrhage.
Spontaneous remission or remission after therapy for Individual susceptibility to the effects of these drugs varies
the underlying disease is typical of this disorder. Therapeutic greatly. Most often, the acquired drug-induced hemostatic
use of DDAVP has been observed to correct the bleeding abnormalities observed are transitory and disappear when the
time and increase the level of vWF. A response to the use of drug is discontinued. However, other effects, such as those
corticosteroid therapy has been documented. Plasmapheresis seen in heparin-induced thrombocytopenia and thrombosis,
to remove antibody has been reported to be effective in a may be irreversible or disappear more slowly.
few patients who are bleeding and do not respond to other Drugs that inhibit platelet function do so by a variety of
measures. mechanisms that include altering prostaglandin synthesis,
S98 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

ibit at : Other drugs suppress platelet function by inhibiting

Table 25-12 Drugs that can Inh cAMP production. cAMP plays a major role in mediating
G
Platelet Function Beoa: ny platelet activity. Platelet response is inhibited when intracellu-
lar levels of cAMP are raised. Drugs such as dipyridamole
Aspirin inhibit phosphodiesterase, an enzyme capable of inactivating
Sulfinpyrazone
cAMP, resulting in increased cAMP levels in platelets. Dipyri-
Dipyridamole
Ticlopidine damole has a mild effect on platelet function by itself and does
Clopidogrel not prolong the bleeding time; however, when administered
Antimicrobials with aspirin, it exerts a greater antiplatelet effect.
Penicillins Ticlopidine is utilized in patients undergoing cardiac
Cephalosporins
catheterization and coronary stent placement and in patients
Dextran
GP Ub/IIla inhibitors
with transient ischemic attacks or completed strokes to inhibit
Abciximab platelet function. The mechanism of action of ticlopidine is
Tirofiban inhibition of ADP-induced platelet-fibrinogen binding. The effect
Eptifibatide of ticlopidine is irreversible and lasts the life of the affected
platelet. Ticlopidine is occasionally used in conjunction with
aspirin therapy to induce two separate mechanisms of platelet
inhibition. When drugs such as these with differing mechanisms
phosphodiesterase activity, platelet membrane or membrane are utilized together, the hemorrhagic complications may
receptors, and cyclic adenosine monophosphate (cAMP) increase. Clopidogrel has a similar mechanism of action.
levels. However, the mechanism of platelet inhibition for Antimicrobial drugs such as penicillin, ampicillin, and
many drugs is still uncertain. carbenicillin may inhibit platelet function through interference
Aspirin has been well documented to inhibit platelet with membrane or membrane receptors. When administered in
aggregation and platelet secretion in response to ADP, epineph- high doses, bleeding may occur. Cephalosporin antibiotics
rine, and low concentrations of collagen. Prolonged bleeding may also inhibit platelet function. These drugs affect platelet
times may be associated with the use of aspirin. The effect of aggregation, platelet secretion, and platelet adhesion.
aspirin on the bleeding time is generally dose dependent, but Dextran is also known to prolong the bleeding time.
extremely prolonged bleeding times at lower dosages have been Platelet function tests such as aggregation, PF3 availability,
reported in susceptible individuals (syndrome of intermediate and platelet retention have all been shown to be abnormal with
platelet dysfunction) or those with underlying platelet function the use of dextran. It is believed that dextran interferes with the
defects. Aspirin inhibits prostaglandin synthesis by irreversible platelet surface membrane to cause platelet dysfunction.
acetylation and inactivation of cyclo-oxygenase (COX-1), Several GPIIb/IIIa receptor antagonists have been devel-
thereby inhibiting endoperoxide and thromboxane A, synthesis, oped and approved for intravenous human use in acute coronary
which are two important mediators of platelet release. The syndromes or for percutaneous coronary interventions, such
inhibitory effect of aspirin on thromboxane A, synthesis remains as angioplasty or stent placement, to prevent reocclusion.
for the life span of the platelet. Aspirin also inhibits endothelial Abciximab (Reopro) is a mouse-human chimeric Fab fragment
cell synthesis of prostacyclin; however, inhibitory action is pre- that exhibits irreversible, noncompetitive binding to platelets.
dominantly against platelet cyclo-oxygenase. Low-dose aspirin Recovery of platelet function is noted by 48 hours after admin-
treatment takes advantage of this differential effect on vascular istration, but platelet-bound antibody is still detectable for
and platelet cyclo-oxygenase. Patients with hemophilia, von 14 days after administration. Eptifibatide (Integrelin) is a cyclic
Willebrand disease, or major underlying hemostatic defects may heptapeptide that contains a lysine-glycine-aspartic acid (KGD)
develop significant or spontaneous bleeding after aspirin inges- sequence similar to that found on adhesive proteins such as
tion and should avoid aspirin or aspirin-containing compounds. fibrinogen or vWF. This peptide has reversible, competitive
Salicylate and choline magnesium trisalicylate do not contain an binding to platelets, with recovery of platelet function 2 to
acetyl group and do not inhibit cyclo-oxygenase. These drugs 4 hours after administration. Tirofiban (Aggrastat) is a nonpep-
are preferable in patients with underlying hemostatic disorders. tide GPIHb/Ia inhibitor that also exhibits reversible, competi-
Certain other nonsteroidal anti-inflammatory agents tive binding to platelets with a similar recovery of platelet
(NSAIDs), such as indomethacin, ibuprofen, phenylbutazone, function in 2 to 4 hours.
and naproxen, can induce significant platelet dysfunction by Bleeding is a potential complication of all of these drug
inhibiting prostaglandin synthesis. These drugs inhibit cyclo- treatments and is usually noted when excessive doses of
oxygenase, but the effect is reversible. Newer NSAIDs, such heparin are used concomitantly with these drugs. Heparin
as COX-2 inhibitors, do not affect platelet function but have dosing based on body weight has reduced the risk of serious
been reported to cause gastrointestinal bleeding. The cause of and fatal bleeding complications. Thrombocytopenia is a less
the bleeding remains uncertain. frequent complication but may be severe (less than 10 X
Sulfinpyrazone is a competitive inhibitor of platelet 10°/L). Onset of thrombocytopenia is rapid, usually within
cyclooxygenase. Bleeding times are not prolonged, and platelet 12 to 24 hours of treatment, and increases the hemorrhagic
aggregation is generally normal. Its complete mechanism of risk in patients treated with these agents. Thrombocytopenia
action is not well understood as an antiplatelet agent. caused by these agents is often mistaken for heparin-induced
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 599

thrombocytopenia because patients typically are given both Primary Purpura

agents concomitantly. The precipitous onset of thrombocytope-
nia after initiation of therapy may be helpful in differentiating Primary purpura comprises disorders that result in bruising
GPIIb/Ila inhibitor-induced thrombocytopenia from heparin- but are not associated with any specific disease. Simple pur-
induced thrombocytopenia. Thrombocytopenia appears to pura or “devil pinches” occur as a result of skin fragility.
occur more frequently with abciximab than with eptifibatide or The bruising is usually mild and occurs with minimal
tirofiban. The mechanism responsible for thrombocytopenia is trauma. Often, there may be a family history of easy bruis-
not known but may be a result of accelerated immune clearance ing. Mechanical purpura occurs as a result of sudden
of platelets from the circulation. increases in capillary pressure and usually manifests as
Numerous other conditions have been associated with an petechiae. Sneezing, coughing, Valsalva maneuvers, or
acquired platelet defect. These include paroxysmal nocturnal seizures may cause this problem. Senile purpura, which is
hemoglobinuria, infectious mononucleosis, severe B,, defi- seen in older individuals, is similar to that occurring in ind1-
ciency, diabetes, hyperbetalipoproteinemia, congenital heart viduals who are receiving corticosteroid therapy. In these
defects, and hypothyroidism. The pathogenesis in these vari- disorders, the purpuric lesions usually occur on the hands
ous disorders in uncertain. and arms and result from loss of subcutaneous tissue and
support of blood vessels in affected skin. Factitious purpura
is caused by self-induced trauma and usually is found on
Vascular Disorders areas of the body that are easily accessible. The purpura may
be caused by pinching, suction applied to the skin, or a blow
Purpura can be caused by abnormalities of skin, connective
to the skin. An entity termed psychogenic purpura has been
tissue, or blood vessels, or may be a result of inflammatory
described which is seen in individuals with emotional prob-
processes that are not associated with quantitative or qualita-
lems, often after severe trauma or extensive surgery, which
tive platelet disorders. These may be congenital or acquired
may be a hypersensitivity reaction to red blood cell mem-
vascular disorders and are listed in Table 25-13.
brane components or DNA hypersensitivity. Various skin
disorders such as Schamberg’s purpura (progressive pig-
mentary dermatosis) and related conditions result in pur-
puric lesions. These lesions are usually seen bilaterally on
both lower legs and appear as a result of a non-inflammatory
Primary Purpura disorder of capillaries of the skin. If the disorder is long-
Simple purpura standing, hemosiderin pigmentation is noted in previously
Mechanical purpura
involved areas.
Senile purpura
Factitious purpura
Schamberg’s purpura
Secondary Purpura Secondary Purpura
Infectious purpura
Waterhouse—Friderichsen syndrome Secondary purpura results from another disease process and
Purpura fulminans is just one of the manifestations of the disease process.
Septic emboli These disorders are acquired and have no associated family
Allergic purpura history.
Schonlein—Henoch purpura
Drug sensitivity
INFECTIOUS PURPURA
Metabolic purpura
Scurvy
Purpura may result from various infectious diseases. Bacter-
Cushing’s syndrome ial infections such as meningococcemia, streptococcal or
Diabetes mellitus staphylococcal bacteremia, or diphtheria; rickettsial infec-
Protein C deficiency tion with Rocky Mountain spotted fever; and parasitic infec-
Psychogenic purpura tion by malaria may cause purpura. The purpura may be
Gardner—Diamond syndrome
DNA hypersensitivity
caused by direct damage to blood vessels by the particular
Purpura secondary to dysproteinemia organism (vasculitis) or result from the effects of endotoxin
Waldenstr6m’s purpura on blood vessels. Septic emboli to the skin (ecthyma gan-
Cryoglobulinemia grenosum) may be seen in endocarditis. In the Waterhouse-
Amzyloidosis Friderichsen syndrome, meningococcemia results in adrenal
Hyperviscosity syndrome
Vascular and connective tissue disorders
hemorrhage with adrenal insufficiency and shock. Purpura
Hereditary hemorrhagic telangiectasia fulminans is an acute purpuric syndrome that usually occurs
Angiodysplasia during septic shock, with development of ischemia followed
Giant hemangiomas (Kasabach—Merritt syndrome) by thrombosis and necrosis of affected areas, often the fin-
Ehlers—Danlos syndrome gers and toes.°° Meningococcemia, streptococcal infections,
Marfan syndrome
and viral infections may provoke this life-threatening prob-
Pseudoxanthoma elasticum
Osteogenesis imperfecta lem.°’ If the individual survives, necrosis of the fingers
and toes may require amputation. DIC is often seen with
600 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

Waterhouse—Friderichsen syndrome or purpura fulminans. defect in the synthesis of collagen in the walls of small
Activated Protein C (Drotrecogin alpha) concentrate and blood vessels. Vitamin C is a cofactor for hydroxylation of
hyperbaric oxygen have been used to treat this disorder. proline and lysine in the production of collagen and keratin.
A platelet function defect may also be present, with a
ALLERGIC PURPURA decrease in the platelet adhesion reaction. Scurvy may pre-
Allergic purpura is the result of an allergic vasculitis. sent with leg swelling, pain, or discoloration. Subperiosteal
Schénlein-Henoch purpura is a vasculitis involving the bone bleeding is characteristic of scurvy and may be noted
skin, gastrointestinal tract, kidneys, heart, and central ner- radiologically. Gingival bleeding and hemarthrosis may also
vous system.°** This syndrome is considered an immune be present. Perifollicular hemorrhages with hyperkeratosis
complex disease, and is characterized by involvement of the hair follicles and “corkscrew” hairs are characteristic
of capillaries with a diffuse perivascular infiltration by neu- of this problem. Vitamin C administration orally cures the
trophils, lymphocytes, and macrophages. Some patients disorder.
have IgA deposition in the kidney and elevated serum IgA Cushing’s syndrome caused by corticosteroid excess
levels. This syndrome may be related to IgA nephropathy results in purpura (Fig. 25-11). The pathogenesis is identical
whereby similar IgA deposition in the kidney is noted. In to that seen with steroid or senile purpura, including atrophy
Schonlein—Henoch purpura, the onset is abrupt, with a mac- of the subcutaneous tissue and increased blood vessel
ular rash (usually symmetric) involving the lower extremi- fragility. Other cutaneous stigmata of Cushing’s syndrome
ties that becomes purpuric. In some individuals, the joint include a “moon” face, pigmented abdominal striae, a “buf-
and gastrointestinal symptoms predominate and the cuta- falo hump” on the lower neck and upper back, with wasting
neous expression is minimal. This disorder is seen most of the extremities and an obese abdomen.
commonly in children. Renal dysfunction is common and Diabetes mellitus may be associated with retinal hem-
typically reversible in children. Overt renal failure is most orrhages. This is a result of vascular proliferation caused
common in affected adults. Schonlein—Henoch purpura may by retinal hypoxia. These fragile vessels may leak fluid or
last several weeks and then recur (Fig. 25-10). hemorrhage because of their increased permeability. Laser
Drug hypersensitivity may also result in allergic pur- photocoagulation and optimal control of hyperglycemia are
pura. This must be differentiated from drug-induced thrombo- treatments used for this problem. No endogenous platelet or
cytopenias that result in purpura. The mechanism by which coagulation disorder is present in this situation.
these drugs cause purpura is not understood. Aspirin, Protein C deficiency may occur on a congenital or
atropine, iodides, penicillin, quinine, and sulfonamides have acquired basis and is a risk factor for thrombosis. An
been described to cause allergic hypersensitivity purpura. unusual syndrome that resembles purpura fulminans can be
present in neonates, resulting from inherited homozygous
METABOLIC PURPURA protein C deficiency.’ This disorder presents hours after
Metabolic purpura are caused by hormonal or biochemical birth and is usually lethal. Protein C deficiency results from
abnormalities. Scurvy is caused by a deficiency of vitamin oral anticoagulant therapy, because protein C is a vitamin
C. This was a common problem several centuries ago in K-dependent protein. Rapid depletion of protein C by war-
sailors who did not have access to vitamin C on long voy- farin appears to cause a unique thrombotic disorder termed
ages. In modern times, this disorder is found in refugees the warfarin skin necrosis syndrome. This unusual disorder
from war or famine, adults with inadequate or fad diets, and presents as painful, erythematous areas on the buttocks,
in alcoholics. Deficiency of vitamin C appears to cause a breasts, legs, or penis, which rapidly turn to hemorrhagic

Figure 25-10 ® Anaphylactoid (Schénlein-Henoch) purpura.

Figure 25-11 @ Steroid purpura (skin manifestations).
Purpuric lesions of the foot.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 60]

bullae and then become necrotic. Cessation of warfarin and

administration of plasma that contains protein C are appro-
priate treatments. Vitamin K administration may be helpful
but will usually not reverse the disorder soon enough to
prevent necrosis.

PURPURA SECONDARY TO DYSPROTEINEMIA

Purpura caused by dysproteinemias encompasses several disor-
ders. Benign hypergammaglobulinemic purpura (Waldenstrém’s
purpura) is a disorder of women that presents with recurrent pur-
pura on the lower extremities and resultant hemosiderin staining
of the skin similar to Schamberg’s purpura.” Unlike Scham-
berg’s purpura, polyclonal hypergammaglobulinemia and an
elevated erythrocyte sedimentation rate are noted. Mild anemia
may be present. Purpura are made worse by leg dependency or
by wearing constrictive garments and improved with leg eleva- Figure 25-13 @ Amyloid purpura. Note characteristic periorbital
tion and support stockings (Fig. 25-12). Skin biopsy reveals distribution.
necrotizing vasculitis. Patients may develop Sjégren’s syndrome
or keratoconjunctivitis sicca, suggesting this may be a vasculitis
amyloidosis is placed in a head-down position for procto-
that results from a connective tissue disorder.
scopic examination, with characteristic purpura around the
Cryoglobulinemia is caused by production of cryopre-
eyelids (Fig. 25-13). Bleeding is caused by deposition of
cipitable serum proteins or protein complexes. This may
amyloid protein around small blood vessels, resulting in
result from primary plasma cell dyscrasias or from chronic
vessel fragility. Low factor X levels from binding of factor X
viral infections such as hepatitis C. Exposure to cold may
to amyloid fibrils, hyperfibrinolysis related to excessive
cause purpura on the skin of the extremities or face. The pur-
urokinase activity, and platelet function alterations may
pura may blister or ulcerate. The proposed pathogenesis for
enhance the bleeding tendency. Treatment for bleeding
the purpura in the cryoglobulinemic disorders is vascular
caused by amyloidosis involves treatment of the underlying
damage owing to precipitation of cryoglobulins.
disease with chemotherapy. Splenectomy has also been
Amyloidosis may cause purpura as a result of several
used. Administration of plasma and platelet transfusions are
mechanisms. Easy bruising is common in amyloidosis, with
not usually effective. Anti-fibrinolytic therapy, especially if
“pinch” purpura and ecchymosis from skin pressure during
hyperfibrinolysis is present, and recombinant Factor VIla may
shaving. Postproctoscopic purpura occurs when a patient with
be useful in controlling bleeding.
Hyperviscosity syndrome results from hypergamma-
globulinemia owing to an increase in plasma viscosity.
Blood flow is restricted because of hyperviscosity involving
development of ischemia and vascular injury, with resultant
hemorrhage. Purpura, gingival hemorrhages, and retinal
hemorrhages are common. Central nervous system bleeding
may be lethal. This disorder is usually seen in IgM syn-
dromes such as Waldenstrém’s macroglobulinemia, and
IgA or IgG3 multiple myeloma. It is rarely seen in
multiple myeloma with other IgG subtypes. Treatment is
with plasmapheresis to remove excessive IgM or IgG, and
chemotherapeutic treatment of the underlying plasma cell
dyscrasia to reduce paraprotein production.

Vascular and Connective Tissue Disorders

These disorders result from abnormal blood vessel structure,

with purpura and bleeding caused by increased permeability
to blood. Most of these disorders are congenital.

HEREDITARY HEMORRHAGIC TELANGIECTASIA

Hereditary hemorrhagic telangiectasia (Osler-Weber—Rendu
syndrome) is caused by the development of telangiectases
of mucous membranes and skin (Fig. 25-14). This is an
Figure 25-12 @ Typical lower extremity vascular lesions seen in
patients with paraproteinemia. autosomal-dominant disorder that usually becomes obvious
602 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

Treatment is usually surgical removal or radiation, if

surgery is not possible. Interferon has been used in a single
case report.

CONGENITAL CONNECTIVE TISSUE DISORDERS

Several rare congenital disorders of connective tissue
proteins have been noted to cause purpura and bleeding.
Ehlers-Danlos syndrome is an autosomal-dominant disorder
characterized by hyperdistensible joints and fragile skin. Six
different subtypes of the disorder are described related to
abnormal collagen synthesis.°? Hematomas, mucosal bleed-
ing after dental procedures or from the gastrointestinal tract
can occur. Bleeding is believed to be due to abnormalities of
collagen in blood vessel walls leading to vascular fragility.
These patients may be mistaken for individuals with hemo-
Figure 25-14 @ Tongue of a patient with hereditary hemorrhagic philia because of the bleeding tendency and joint abnormal-
telangiectasia. Note the multiple vascular telangiectases, which can ities. Laboratory tests of hemostasis are usually normal in
occur in the nares, the oral mucous membranes, and throughout
affected individuals. Surgery, trauma and use of aspirin
the gastrointestinal tract. Recurrent bleeding requiring transfusions
is a common manifestation. should be avoided as possible because of bleeding and poor
wound healing. Individuals with type IV Ehlers—Danlos
syndrome are prone to arterial aneurysms and rupture of
in adolescence or early adulthood. Lesions develop on the arterial blood vessels.
tongue, lips, palate, face, and hands. Similar lesions are pre- Marfan syndrome is an autosomal-dominant genetic dis-
sent in nasal mucosa and throughout the gastrointestinal tract. order due to mutations of the gene for fibrillin resulting in
The lesions blanch when pressure is applied, in contrast to abnormalities of connective tissues and risk for bleeding and
petechiae, because these are vascular structures. Chronic bruising. Individuals with this disorder are usually tall and
epistaxis and gastrointestinal bleeding occur and usually have long limbs. Dislocation of the lens of the eye, aortic
worsen as the patient grows older. Iron deficiency anemia is valvular regurgitation, and aortic dissection may occur. Easy
usual. The bleeding is difficult to control but may be treated bruising or postpartum or postsurgical bleeding may occur
with nasal packing, cauterization, or laser photocoagulation. but are usually not prominent features of this disorder. Labo-
Topical use of tranexamic acid may control epistaxis acutely. ratory studies of hemostasis are normal.
Administration of parenteral iron or red blood cell transfu- Pseudoxanthoma elasticum is an autosomal recessive
sions is usually necessary. Estrogens, the estrogen receptor disorder affecting elastic fibers of connective tissue of the
antagonist tamoxifen, and danazol have been reported to help skin and arteries. Several types of the disorder have been
in individual patients.°' described with mutations of a transmembrane transporter pro-
tein. The skin of affected individuals has a grooved, thickened
appearance. Characteristic hyaline (angioid) streaks are noted
ANGIODYSPLASIA
in the retina. Easy bruising, gastrointestinal, uterine and uri-
Angiodysplasia is a disorder involving blood vessels of the
nary bladder bleeding is common. Bleeding is thought to
gastrointestinal tract. The cutaneous lesions of hereditary
occur as a result of rupture of calcified blood vessels. Athero-
hemorrhagic telangiectasia are not present. Vascular perme-
sclerosis due to calcification of medium-sized and large blood
ability leads to chronic gastrointestinal bleeding, anemia, and
vessels usually occurs, and intermittent claudication is fre-
iron deficiency. This disorder has been associated with von
quent. Tests of hemostasis are normal and there is no effective
Willebrand disease and may be caused by the effect of VWF
therapy.
on vascular structures. An acquired form of angiodysplasia
Osteogenesis imperfecta is another rare autosomal
has been reported in patients with aortic stenosis. In these
dominant disorder caused by mutations of genes which code
patients, aortic valve surgery has improved the bleeding.
for peptides of type | collagen. Skin is mostly type | colla-
Hemicolectomy has been used to treat the gastrointestinal
gen and defects in supporting structures in skin are believed
bleeding.
to be the cause for bruising in this disorder. Four different
clinical types of the disorder are reported which are caused
GIANT HEMANGIOMAS (KASABACH-MERRITT by numerous mutations of the genes for these collagen pep-
SYNDROME) tides. Affected individuals may demonstrate easy bruising,
Giant hemangiomas may occur congenitally soon after epistaxis, hemoptysis, and, infrequently, intracranial bleed-
birth and affect the skin, liver, and spleen. Unless these ing. Postoperative wound bleeding can also occur. Reports of
lesions spontaneously regress, DIC may develop. These benefit with the use of recombinant factor VIla and desmo-
hemangiomas have numerous thin-walled blood vessels pressin have been published. Affected individuals have char-
that produce thromboplastic substances to incite DIC. acteristic blue-colored sclera.
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 603

~ Case Study 1_ Case Study 2

A 23-year-old African-American woman in the 14th week of An obese 36-year-old white female emergency medical tech-
her first pregnancy presents to the hospital with mild confu- nician complains of easy bruising of the arms and legs for sev-
sion, headaches, nausea, easy bruising, and petechiae. Physical eral months. This bruising is episodic and seems unrelated to
examination reveals a temperature of 37.7°C, blood pressure trauma during work. She reports no stinging or burning sensa-
of 150/100 mm Hg, pulse of 80 beats per minute, and respira- tions before the appearance of bruises. No bleeding from the
tions of 14 per minute. Mild confusion and decrease in light nose, mouth, or other mucous membranes is noted. No bleed-
touch sensation of the right arm are noted. Scattered petechiae ing into joints or muscles has occurred. Family history is neg-
cover the inner thighs, and ecchymoses on the forearms are ative for bleeding or bruising. Physical examination reveals
present. No other bleeding is observed. The uterus is enlarged, normal skin turgor with a few discrete fresh ecchymoses on
and a fetal heartbeat is detected. The spleen tip is not palpable. the thighs and forearms but no other obvious bleeding. Platelet
Admission laboratory findings show a hematocrit of 21%, count is 380 x 10°/L; bleeding time is 5 minutes, 30 seconds;
white blood count of 10 x 10°/L, platelet count of 18.0 x APTT, PT, and fibrinogen studies are normal.
10°/L, and reticulocyte count of 7%. PT, APTT, and fibrinogen
are normal. FDP is positive, with a titer of 32, and D-dimer is QUESTIONS
positive at 0.5 to 1.0 ug/mL. The Direct Antiglobulin Test
(DAT) is negative. The peripheral blood smear shows moder- 1. What additional information will be helpful in defining a
ate poikilocytosis with schistocytes, nucleated red blood cells, cause for this bruising?
and polychromasia. Serum creatinine is 2.5 mg/dL, blood urea 2. What conditions should be considered in the differential
nitrogen is 50 mg/dL, calcium is 7.6 mg/dL, and albumin is diagnosis? Why?
2,4 mg/dL. Urinalysis is remarkable with 3+ proteinuria and 3. What additional testing can be performed to confirm a
numerous red blood cells. Bone marrow aspiration and biopsy diagnosis?
reveal erythroid and megakaryocytic hyperplasia. Hyaline
thrombi are seen in blood vessels of the biopsy. Electrocardio- ANSWERS
gram and chest radiograph are normal.
1. Psychiatric history or history of abuse? Access to antico-
«
agulants such as heparin or warfarin?
QUESTIONS 2. Simple or mechanical purpuras, psychogenic purpura, or
1. What is the most likely diagnosis? How should the fictitious purpura.
3. Thrombin clotting time assay to detect low levels of
patient be treated?
heparin, von Willebrand factor assays and Factor VIII
2. What other conditions should also be considered in the
clotting activity to investigate for mild von Willebrand
diagnosis? What additional testing could be performed
disease, platelet function studies (PFA assay or platelet
to confirm or reject these diagnoses?
aggregation studies) as there could be another platelet
function defect not evident with the bleeding time assay.
ANSWERS Erythrocyte sedimentation rate, cryoglobulin testing and
1. TTP is the most likely diagnosis. Primary treatment is serum protein electrophoresis may uncover a vasculitis,
with plasma exchange. Addition of corticosteroids, or hypergammaglobuinemic purpura.
cyclosporine A, splenectomy, chemotherapy or mono-
clonal antibody immunotherapy (Rituximab) may be
necessary for refractory or chronic relapsing disease.

Case Study3
2. Hemolytic-uremic syndrome, pre-eclampsia/eclampsia
syndromes, and disseminated intravascular coagulation
are other considerations for a pregnant female with
microangiopathic hemolytic anemia. Idiopathic throm- A 67-year-old white man with a history of cigarette smoking
bocytopenic purpura and gestational thrombocytopenia developed sudden onset of tingling and weakness of the left
do not cause microangiopathy. ADAMTS 13 testing arm that lasted 10 minutes before resolving, consistent with
often reveals the presence of autoantibodies to the a transient ischemic attack. He was examined soon after-
ADAMTS 13 metalloprotease and ADAMTS 13 levels ward in the hospital emergency department and no residual
are less than 5%, not in the other disorders. neurologic abnormalities were found. The spleen was not
palpable and no bleeding or bruising were present. Labora-
tory findings included a hematocrit of 43%, white blood
count of 11.2 X 10°/L, with a normal white blood cell dif-
ferential, and platelet count of 760 x 10°/L. Bone marrow
aspirate and biopsy revealed megakaryocytic hyperplasia
with clustering of megakaryocytes but no granulocytic or
erythrocytic hyperplasia, no fibrosis, and normal storage
iron. Bone marrow culture exhibited growth in the absence
of added growth factors, consistent with a diagnosis of a
continued
604 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders

Case Study 3 Case Study 4

myeloproliferative disorder. Smoking cessation and aspirin A 17-year-old Jewish woman complains of recurrent episodes
were advised to prevent recurrent thrombotic episodes. The of bleeding from the nose and mouth since birth and heavy
patient refused hydroxyurea treatment. menstrual bleeding since menarche. She has never had bleed-
ing into joints or muscles. Her brother also has a history of
QUESTIONS nasal bleeding. Physical examination does not reveal any
telangiectases of the nose or mouth. Skin and joints appear
1. What conditions are considered in the differential diag- normal. Gynecologic examination is normal. Platelet count
nosis of thrombocytosis in this patient? is 250 X 10°/L. Bleeding time is prolonged at 18 minutes.
2. Which characteristics present in this case differentiate Appearance of platelets on the peripheral blood smear is
myeloproliferative disorders from reactive causes of normal.
thrombocytosis?
QUESTIONS
ANSWERS
1. What conditions should be considered in the differential
1. Most likely diagnosis is a myeloproliferative disorder. diagnosis for this bleeding? Why?
Reactive thrombocytosis is still possible. The clinical 2. What additional testing can be performed to evaluate
features are most indicative of essential thrombo- this bleeding tendency?
cythemia. Other myeloproliferative disorders to consider 3. How would each of the possible conditions in the differ-
are polycythemia rubra vera, idiopathic myelofibrosis ential diagnosis be treated to minimize further bleeding
and chronic myelogenous leukemia but these disorders episodes?
exhibit elevations in other cell lines not seen in this
patient.
ANSWERS
2. The clustering of megakaryocytes in the bone marrow
specimens and spontaneous growth of marrow cultures 1. The symptom of mucocutaneous bleeding, normal
(without added growth factors) suggest a myeloprolifer- platelet count and prolonged bleeding time suggest a
ative disorder. The increase in white blood cell count platelet function disorder. Coagulation screening test
could be seen with a reactive marrow. Absence of results are not known. von Willebrand disease is the
splenomegaly does not differentiate between essential most likely possibility as it is the commonest platelet
thrombocythemia and reactive process. Arterial throm- function disorder. Glanzmann’s thrombasthenia is
boses are common in essential thrombocythemia but the uncommon but occurs in individuals of Askenazi Jewish
patient also is at risk due to the history of cigarette extraction. Bernard-Soulier syndrome patients usually
smoking. JAK2 mutation analysis, if positive, would exhibit large platelets.
confirm a myeloproliferative disorder as it is positive in 2. Factor VII clotting activity assay and von Willebrand
95% of cases of polycythemia rubra vera and 50% of factor assays to evaluate for von Willebrand disease,
cases of essential thrombocythemia and idiopathic platelet function testing to evaluate for other platelet
myelofibrosis. function abnormalities.
3. von Willebrand disease is treated with DDAVP if the
disorder is mild and with intermediate purity factor VIII
concentrates which contain functional von Willebrand
factor when severe or when prolonged therapy is neces-
sary. Other platelet function disorders are treated with
platelet transfusions or activated factor VII. Anti-
fibrinolytic therapy and estrogen therapy to reduce
menstrual bleeding may be helpful.

Woo ont

1. A patient presents with a platelet count of 212 X 10°/L a. Mucosal bleeding

and a bleeding time of 12 minutes. These results most b. Hemarthrosis
probably suggest: c. Retroperitoneal hemorrhage
a. Decreased platelet production d. Deep muscle hematomas
b. Defective platelet function . Which of the following is not characteristic of
to

c. Increased platelet production Bernard- Soulier syndrome?

d. Increased platelet destruction a. Prolonged bleeding time
2. Which of the following clinical manifestations is most b. Absent platelet aggregation in response to bovine
characteristic of a platelet disorder: VWE or human vWE plus ristocetin
 Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders 605

c. Abnormal platelet aggregation in response to ADP, 7. What disorders are classically associated with
collagen, and epinephrine thrombocytosis?
d. Abnormality in platelet membrane GPIb/IX a. Myeloproliferative syndromes
4. Which of the following is not characteristic of b. Autoimmune disorders
Glanzmann’s thrombasthenia? c. Immunodeficiency syndrome (HIV)
a. Prolonged bleeding time d. Renal disease
b. Giant platelets with thrombocytopenia 8. Which of the following is not an inherited vascular defect?
c. Absent platelet aggregation in response to ADP, a. Ehlers—Danlos syndrome
thrombin, collagen, and epinephrine b. Marfan syndrome
d. Normal platelet aggregation to ristocetin and c. Amyloidosis
bovine vWF d. Giant hemangiomas
5. Which of the following statements is not true regarding 9. Which condition is classified as nonimmunologic throm-
acute idiopathic thrombocytopenia (ITP)? bocytopenia?
a. Thrombocytopenia occurs following a viral infection. a. DIC
b. Spontaneous remissions are common. bs TTk
c. It is found primarily in young children. c. Storage pool defects
d. Platelet counts are generally higher than in d. Post-transfusion purpura
chronic ITP. a
10. Which condition is classified as immunologic thrombo-
6. Thrombocytopenia, fever, renal disease, microangio- cytopenia?
pathic hemolytic anemia, and neurologic complications a. DIC
are hallmark characteristics of: barEe.
a. Hemolytic uremic syndrome (HUS) c. Storage pool defects
b. Disseminated intravascular coagulation (DIC) d. Bernard—Soulier syndrome
c. Thrombotic thrombocytopenic purpura (TTP)
d. Idiopathic thrombocytopenic purpura (ITP) See answers at the back of this book.

# Qualitative platelet disorders may be caused by abnormal-

--) SUMMARY CH ities of the platelet surface glycoproteins (Bernard—
Soulier syndrome and Glanzmann’s thrombasthenia),
= Quantitative and qualitative platelet disorders and vascu- deficiencies of platelet granules (storage pool defects), or
lar disorders are abnormalities of primary hemostasis that abnormalities of the platelet release mechanism (release
result in bleeding and purpura. defects).
g Thrombocytopenia is the most common primary hemosta- gw von Willebrand disease is one of the most common con-
tic disorder. genital defects of primary hemostasis.
m The mechanisms responsible for thrombocytopenia include mvon Willebrand factor results in a lack of binding of
deficient marrow production of platelets, sequestration of platelets at the site of injury, with bleeding characteristic
platelets in the spleen or hemangiomas, or accelerated loss of platelet defects.
or destruction of platelets from the circulation. m Vascular purpura is caused by abnormalities of blood ves-
a Thrombotic thrombocytopenic purpura (TTP) is a sels without associated platelet or plasma protein defects.
microangiopathic process that leads to thrombocytopenia
with thrombosis and bleeding.

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 Chapter |

Disorders of Plasma
Clotting Factors
Sharon L. Schwartz, MS, MT(ASCP)SH, CLS(NCA)

Introduction OBJECTIVES
The Plasma Clotting Factors, At the end of this chapter, the reader should be able to:
Associated Disorders, and
Laboratory Evaluation 1. List various defects that impair the coagulation system.
Factor | (Fibrinogen) . List the clotting factors and their synonymous names.
Factor Il (Prothrombin)
Factor V (Proaccelerin; . List the vitamin K-dependent factors.
Labile Factor) . Describe the genetic mutations known to affect factor II and factor V, and the conse-
Factor VII (Proconvertin; quences thereof.
Stable Factor; Plasma
Thromboplastin . Describe the differences between hemophilia A and von Willebrand disease.
Component) . Name the respective factor deficiency responsible for causing hemophilias A, B, and C.
Factor VIII (Antihemophilic
Factor) and von . Describe various etiologies and treatment modalities for various factor deficiencies.
Willebrand Factor . Describe laboratory methods used to identify factor deficiencies.
Factor IX (Christmas Factor;
Plasma Thromboplastin . Describe circulating anticoagulants/inhibitors.
cn
ONG
Nec
1)
COn
NOR

Component [PTC]) 10. Describe laboratory methods used to identify circulating anticoagulants/inhibitors.
Factor X (Stuart-Prower
Factor)
Factor XI (Plasma
Thromboplastin
Antecedent [PTA])
Factor XII (Hageman Factor)
Factor XIII (Fibrin-Stabilizing
Factor)
Prekallikrein (Fletcher Factor)
High-Molecular-Weight
Kininogen (Fitzgerald
Factor; Williams—
Fitzgerald—Flaujeac Factor)
Circulating
Anticoagulants/Acquired
Inhibitors
Specific Inhibitors
Nonspecific Inhibitors:
Lupus Anticoagulants
Case Study 1
Case Study 2
Case Study 3
Case Study 4
Case Study 5
607
608 Chapter 26 Disorders of Plasma Clotting Factors

Case Study 6
Case Study 7
Case Study 8
Case Study 9
Case Study 10

INTRODUCTION have the amino acid serine in their active sites. The activation
of the zymogens that participate in the coagulation pathways
This chapter covers disorders of secondary hemostasis: the leading to the formation of an insoluble fibrin clot were dis-
plasma clotting factors, their defects, and how these defects cussed in Chapter 24.
directly affect hemostasis. Information is provided in detail on Plasma clotting factor defects can impair hemostasis.
each of the clotting factors, the effects of their deficiencies, and This can occur as a result of:
associated disorders (Tables 26-1 and 26-2). Recommended
1. Decreased synthesis of one or more factors
laboratory tests and their results are discussed. In addition, dis-
2. Production of abnormal molecules that interfere with the
cussions include information on specific and nonspecific
coagulation pathways
inhibitors. The recommended therapy for each plasma clotting
3. Loss or consumption of the coagulation factors
factor defect is discussed. A summary of the materials pro-
4. Inactivation of one or more factors by inhibitors or
vided, and several case studies are presented at the end of the
antibodies. '
chapter.
The transformation of liquid blood into a semisolid clot Bleeding disorders related to clotting factor deficiencies
is a very complex process. The end result is the cessation of or dysfunction (secondary hemostasis) have a clinical presen-
bleeding, or hemostasis. Clotting factors circulate as inactive tation that differs from platelet-related (primary hemostasis)
zymogens, Or precursors of enzymes, known as serine pro- disorders. Most of the clotting factor deficiencies involve
teases: proteolytic enzymes that cleave peptide bonds that delayed and/or inadequate fibrin formation (except in cases of

Minimum for Half-life Laboratory Clinical

Factor Deficiency Hemostasis (hours) Findings Findings

I Afibrinogenemia 50-100 mg/dL No clot formation Umbilical stump bleeding,

Abnormal PT, aPTT, easy bruising, ecchymosis,
TT; absent fibrinogen gingival oozing,
hematuria, poor
wound healing
Hypofibrinogenemia Abnormal PT, aPTT, TT; Mild bleeding episodes
low fibrinogen
Dysfibrinogenemia Fibrinogen: antigen— Possible hemorrhage/
normal; clottable thrombosis; possibly
activity—abnormal asymptomatic
Hypoprothrombinemia 30-40% Abnormal PT, aPTT Postoperative bleeding,
epistaxis, menorrhagia,
easy bruising
Parahemophilia 10% Abnormal PT, aPTT, BT Epistaxis, menorrhagia, easy
bruising
Hypoproconvertinemia 10% Abnormal PT, normal Epistaxis, menorrhagia,
aPTT cerebral hemorrhage
Hemophilia A 30% Abnormal aPTT; May be severe, moderate,
normal PT, BT mild—spontaneous
hemorrhage, hemarthroses,
crippling, muscle
hemorrhage, post-
traumatic/postoperative
bleeding
 Chapter 26 Disorders of Plasma Clotting Factors 609

Minimum for Half-life Laboratory Clinical

F ne _ Deficiency _Hemosta 5 one) Bindings LESS

von Willebrand nenI 30-40% 16-24 Variable ety rear Mucosal scans
and variants: variable studies, BT, aPTT, superficial wound
inheritance FVII:C, vWF:Ag, RCA, bleeding—variable
multimers depending on level of F
VIII:C, vWF
Ix Hemophilia B 30-40% 20 Abnormal aPTT; May be severe, moderate,
(Christmas disease) normal PT mild—spontaneous
_ hemorrhage, hemarthroses,
crippling, muscle
hemorrhage, post-
traumatic/postoperative
bleeding
xX Stuart-Prower 10% 65 Abnormal PT, aPTT Menorrhagia, ecchymoses,
deficiency CNS bleeding, excessive
bleeding postpartum
XI Hemophilia C 20-30% 65 Abnormal aPTT; Mild bleeding, bruising,
normal PT epistaxis, retinal
hemorrhage, menorrhagia
XII Hageman trait Unknown 60 Abnormal aPTT; Asymptomatic: thrombotic
normal PT tendency
XIll Factor XIII deficiency 1% 150 Normal PT, aPTT; clot Umbilical cord stump
lysis in 5 Molar urea bleeding, poor wound
solution healing, minor injuries
causing delayed bleeding,
fetal wastage, excessive
fibrinolysis, male sterility,
intracranial hemorrhage
PK Prekallikrein Unknown 35 Abnormal aPTT: shortening Asymptomatic
(Fletcher factor) with interval incubation
with activator
HMWK Fitzgerald factor Ua pown 156 _ Abnormal aPTT _ Asymptomatic

PT == aries time; aPTT= activated Gi ieee time; BT =Fert time; HMWK = =ee molecular-weight eee
Ie = thrombin time; CNS= central nervous system.
Source: From Pittiglio, DH, et al: Treating hemostatic disorders. A problem oriented approach. In Pittiglio DH: Hemostasis Overview. American Asso-
ciation of Blood Banks, Arlington, VA, 1984, p 28, with permission.

") table 26-2 Factor Deficiencies

and Test Results =
Factor Fibrinogen 5 M Urea Solubility

Fibrinogen ABN N/A

XI ABN
XI
Prekallikrein ABN*
HMWK ABN |
: Pa
BOG
far
dr
MAAN| l\Zz2AzZAZAZZZZZZzZ
ZZ
Ze
Ae
Wee
*The aPTT willihe after aa contact
a activation.
N = normal; ABN = abnormal; BT = bleeding time; PT = prothrombin time; aPTT = activated partial thromboplastin time;
(bh thrombin time; N/A = not applicable.
Source: From Pittiglio, DH, et al: Treating hemostatic disorders. A problem-oriented approach. In Pittiglio, DH: Hemostasis Overview. American
Association of Blood Banks, Arlington, VA, 1984, p 31, with permission.
610 Chapter 26 Disorders of Plasma Clotting Factors

factor XIII deficiency and most cases of von Willebrand dis- aggregate. In the final stages of coagulation, these soluble
ease); whereas platelet function is normal. Bleeding or hemor- fibrin monomers spontaneously polymerize to form soluble,
rhage can be more profuse because larger vessels are usually but unstable, fibrin strands. The production of an insoluble,
involved (during surgery, childbirth, or after trauma); the stable clot requires the formation of covalent cross-linkages
___location of the hemorrhage is within muscle, deep-tissue, or of the alpha and gamma chains between the soluble fibrin
joint cavities; or it can be delayed, (i.e., a stabilized injury strands, catalyzed by thrombin-activated factor XIII (see
begins oozing or actively bleeding again). Secondary hemo- Factor XIII). This insoluble, stable clot is resistant to enzy-
static disorders may be inherited as sex-linked or autosomal matic digestion or chemical degradation. Normal serum does
genetic traits; they may also be acquired due to dietary not contain detectable fibrinogen; it is consumed in the clot-
vitamin K deficiency, liver disease, hemorrhage, or a consump- ting process.
tive coagulopathy (e.g., disseminated intravascular coagulation
[DIC]); induction of anticoagulant therapy, inflammatory dis- AFIBRINOGENEMIA
orders, or treatment with various drugs. Afibrinogenemia refers to the absence of any chemically, anti-
Laboratory evaluation for clotting factor defects always genically, or functionally detectable fibrinogen in the plasma. It
begins with screening tests: prothrombin time (PT) and acti- is a rare (estimated prevalence 1:1,000,000), homozygous, con-
vated partial thromboplastin time (aPTT). Abnormal results genital disorder inherited as an autosomal recessive trait. Fib-
obtained from one or both screening tests must be followed- rinogen levels may be less than 5 mg/dL. There can be profuse
up with differential tests which include repeating the abnor- bleeding after slight trauma and delayed wound healing. Initial
mal screening tests on mixtures of patient plasma with pooled symptoms include bleeding from the umbilical cord stump and
normal plasma (known as mixing studies), to further charac- mucosal tissues (e.g., epistaxis, gastrointestinal bleeding),
terize the abnormal screening test results. The addition of which can be fatal. Other symptoms can include intracranial
pooled normal plasma to the patient’s plasma sample will nor- bleeding, menorrhagia, and hemarthrosis. Mild to moderate
malize, or “correct” the results of the abnormal screening test if thrombocytopenia can occur, but platelet counts are rarely less
there is a deficiency of one or more factors. The inability to cor- than 100,000/uL.*° Platelet adhesion and aggregation are
rect the screening test result, or a correction followed by pro- abnormal in patients with this disorder; fibrinogen is required
longation after a 2-hour incubation at 37°C, would suggest for normal primary platelet aggregation because it binds to stim-
the presence of an inhibitory substance. The inhibitor sub- ulated platelets at the glycoprotein IIb/Ila (GPIIb/IIIa) receptor,
stance can be the result of an immune response (antibody) forming a linkage with adjacent platelets and reinforcing
produced by the patient, or an anticoagulant such as heparin the platelet plug at the site of vascular injury (see Chap. 24). *
or other thrombin inhibitor which has been administered to Afibrinogenemia, therefore, produces a prolonged bleeding time
the patient or contaminated the specimen. (see Table 26-1).
The laboratory evaluation for this disorder should
The Plasma Clotting Factors, Associated include: PT, aPTT, fibrinogen assay (clottable activity and
antigen), and thrombin time (TT) (Table 26-3). The results of
Disorders, and Laboratory Evaluation these tests will show marked abnormalities: terminal prolon-
Factor | (Fibrinogen) gation of the PT, aPTT, and TT (no clot forms; no detectable
endpoint), and the absence of measurable fibrinogen. Mix-
Factor I, more commonly known as fibrinogen, is a large gly- ture of the patient’s plasma with pooled normal plasma will
coprotein (molecular weight 340 kD) produced solely by the correct these defects.
liver, and is critical in the final stages of coagulation. It is the Treatment for afibrinogenemia requires the administra-
most abundant of the plasma coagulation proteins; up to tion of cryoprecipitate; this preparation contains appreciable
5 grams/day are produced by the liver, with a normal plasma amounts of fibrinogen. Fresh frozen plasma (FFP) may be
concentration of approximately 200 to 400 mg/dL. A minimum used; however, volume overload is a major concern due to the
of approximately 100 mg/dL is required to maintain normal number of units required to sufficiently elevate the fibrinogen
hemostasis. The normal plasma half-life is 3 to 5 days.?* While level. Whole blood transfusions may be required if significant
the majority of fibrinogen is present in the plasma (75%), it can bleeding has occurred.
also be found in interstitial fluid, and is present in platelet alpha
granules by absorption from the plasma. The fibrinogen mole- HY POFIBRINOGENEMIA
cule consists of three pairs of different polypeptide chains, des- Hypofibrinogenemia may be inherited as a heterozygous,
ignated as A-alpha (Aq), B-beta (BB), and gamma (vy), joined autosomal recessive disorder. The level of fibrinogen is found
by disulfide bonds. The interaction of these chains, combined to be in the range of 20 to 100 mg/dL. Bleeding episodes are
with the activity of the enzyme thrombin, ultimately yields a usually mild and not spontaneous. Bleeding may occur after
network of insoluble strands called fibrin. Fibrin monomers are trauma or surgery. Acquired hypofibrinogenemia can be
produced by thrombin’s enzymatic cleavage of fibrinopeptides caused by liver disease or DIC.
A and B from the terminal ends of the Aw and Bf chains of The degree of prolongation of the PT, aPTT, and TT is
the fibrinogen molecule. These fragments contain negatively dependent on the level of fibrinogen present in the plasma.
charged amino acids; once removed, the intermolecular Treatment for this condition may include the use of cry-
repulsive force is reduced, allowing the fibrin monomers to oprecipitate and/or FFP.
 Chapter 26 Disorders of Plasma Clotting Factors 611

Afibrinogenemia Hypofibrinogenemia Heparin

ABN N N
ABN ABN N/ABN
ABN ABN ABN
Thrombin time ABN ABN ABN
Reptilase time ABN ABN
Fibrinogen (clottable) Undetectable ABN
Fibrinogen (antigen) Absent ABN
Platelet aggregation ABN N

N = normal; BT = bleeding time; ABN = abnormal; aPTT = activated partial thromboplastin time; PT = prothrombin time.
Source: From Girolami, A, et al: Rare and quantitative and qualitative: Abnormalities of coagulation. Changes Hematol 14:388, 1985,
with permission.

DYSFIBRINOGENEMIA To evaluate the cause of an abnormal TT, a reptilase time (RT)

Alteration of the structure of the fibrinogen molecule, either should be determined.*’ Reptilase, a thrombin-like enzyme
in its amino acid sequence or carbohydrate composition, leads extracted from the snake venom of Bothrops atrox, only
to the formation of an abnormal glycoprotein. The alterations cleaves fibrinopeptide A from the fibrinogen molecule and
are caused by mutations in the genes that direct the synthesis forms a fibrin clot. It can be used to differentiate between the
of the peptide chains. These changes can affect the normal presence of heparin, antithrombin anticoagulants, or dysfib-
interactions of fibrinogen with its enzymes and cofactors, rinogenemia.'” Reptilase is unaffected by heparin, hirudin,
thereby causing defective clot formation, as well as impaired and antithrombins; therefore, the clotting time will be normal
fibrinolysis. in their presence, provided an adequate amount of normal fib-
Dysfibrinogenemia is inherited as an autosomal domi- rinogen is present (see Table 26-3). Hypofibrinogenemia and
nant trait. More than 300 different structural variants have afibrinogenemia will also have prolonged RTs.
been identified. These molecules may exhibit abnormal fib- Clinically, patients with dysfibrinogenemia are usually
rinopeptide release by thrombin, abnormal polymerization of asymptomatic; the condition is discovered incidentally when
the monomers, defective cross-linking, or a combination of laboratory tests are performed for unrelated reasons. Occasion-
these defects. It may also be acquired as a result of liver ally, it is associated with mild bleeding occurring only after
disease, cancer, fibrinolysis, and DIC. These disorders may trauma. However, there are recent reports indicating an associ-
induce relatively minor structural changes in the molecule, ation with thrombosis.'! This may be a result of abnormalities
producing a functional dysfibrinogenemia with similarities to in the interaction of the fibrinolytic enzyme: plasmin, with the
the congenital forms. abnormal fibrin clot, causing a resistance to lysis.
Patients with dysfibrinogenemia usually have a normal Treatment for dysfibrinogenemia may involve FFP or
PT and aPTT. Bleeding time and platelet function are unaf- cryoprecipitate, if bleeding occurs. If recurrent thrombosis
fected. Specific testing of fibrinogen will usually be abnor- exists, anticoagulant therapy is indicated.'*
mal. Polymerization of fibrin monomers induced by treating
the abnormal molecules with a high concentration of thrombin HY PERFIBRINOGENEMIA
(i.e., the Clauss clot-based fibrinogen assay; see Chap. 33), has Fibrinogen is also an acute-phase reactant. Physiologic
been found to be normal in several cases.° However, with stresses such as trauma, pregnancy, and tissue inflammation
lower thrombin concentrations (i.e., the thrombin time cause fibrinogen concentrations to increase, which is mani-
test), polymerization is delayed because of the slower cleav- fested in an elevated erythrocyte sedimentation rate. High fib-
age rate of the abnormal peptide.’ A common laboratory rinogen concentrations may be seen in acute hepatitis and
finding is a normal quantitative level of functional (clottable) may also be associated with atherosclerosis, arterial thrombo-
fibrinogen with a mild to markedly prolonged TT. The level sis, or both."*
obtained by an antigenic assay of fibrinogen is frequently
higher than the level determined by functional assay. Antico- Factor Il (Prothrombin)
agulants such as heparin, hirudin (a direct thrombin inhibitor
derived from leeches), and synthetic direct thrombin Factor H (F Il), synonymously known as prothrombin (mole-
inhibitors, will prolong the TT. Heparin is an unbranched, cular weight 71.6 kD), is a single-chain glycoprotein synthe-
heavily sulfated polysaccharide derived from bovine lungs or sized in the liver. It is the most abundant factor, has the
porcine intestines. It acts as a cofactor to a naturally occurring longest half-life of the vitamin K—dependent clotting proteins,
anticoagulant: Antithrombin III (AT), accelerating the rate and circulates as a zymogen (precursor) to a serine protease,
at which AT normally inhibits thrombin 1000-fold (see thrombin.'* Prothrombin is converted to thrombin by the pro-
Chap. 28). It requires normal levels of AT to exert its effect. teolytic action of activated factor X (F Xa) in complex with
O12 Chapter 26 Disorders of Plasma Clotting Factors

activated factor V (F Va), platelet phospholipids (PF3), and disease, or the presence of antibodies to prothrombin must be
calcium ions. This is known as the prothrombinase complex, differentiated from dysprothrombinemia and hypoprothrom-
which is assembled on the platelet surface and activates binemia in order to determine the proper course of therapy.
prothrombin (F II) to thrombin (F Ia). The generally accepted Treatment may include administration of FFP or PCC.
normal adult reference range is 50% to 150% (0.5 to 1.5 U/mL),
PROTHROMBIN G20210A MUTATION
and may vary by institution or published reference. Defi-
A single-point mutation in the prothrombin gene, located on
ciency of prothrombin delays the generation of thrombin, thus
chromosome 11, was discovered in 1996 that was associated
contributing to hemorrhagic symptoms. The prothrombin
with a mildly elevated prothrombin concentration and an
time (PT) is an in vitro analysis of the time required to con-
increased risk of venous thrombosis.'’ The mutation, known as
vert plasma prothrombin into endogenous thrombin, using an
prothrombin G20210A, consists of a single base change
exogenous source of phospholipid, tissue thromboplastin
resulting from a G (guanine) to A (adenine) transition at
(tissue factor), and calcium ions; thereby cleaving fibrinogen
nucleotide 20210, in the 3’-untranslated region of the pro-
to a fibrin endpoint. This assay is affected by defects or defi-
thrombin gene.'* It is the second most common cause of inher-
ciencies in any of the components of the prothrombinase
ited thrombophilia (predisposition to thrombosis). Patients
complex, as well as the concentrations of fibrinogen, pro-
who are heterozygous for this mutation carry a two- to fivefold
thrombin, and factor VII (F VII).
increased risk of venous thromboembolism as compared with
normal individuals. This genetic defect is not commonly
HYPOPROTHROMBINEMIA
found in patients with arterial thromboembolic disease, but it
It has been suggested that the mode of inheritance of
has been identified as a risk factor for myocardial infarction in
hypoprothrombinemia is autosomal recessive.'> Patients who
young women! and cerebrovascular ischemic disease in
are either doubly heterozygous or homozygous with this rare
young patients.”? Prothrombin G20210A is predominantly
condition may have hemorrhagic symptoms with prothrom-
(although not exclusively) restricted to the Caucasian popula-
bin levels from 2% to 25% of normal activity. Deficiency
tion. In the presence of other acquired risk factors such as
may also be acquired by dietary vitamin K deficiency or oral
pregnancy, oral contraceptives, malignancy, antiphospholipid
anticoagulant (warfarin, Coumadin®) therapy. However, the
antibody (lupus anticoagulant), recent surgery or trauma, the
type and severity of symptoms may vary with the level of
incidence of thrombosis is increased.*!”?
functional prothrombin available. Epistaxis, menorrhagia,
The elevation of F II activity, which may only be slightly
postpartum hemorrhage, hemorrhage following surgery or
greater than levels in patients without the mutation (approxi-
trauma, and broad-spectrum antibiotic use (which destroy the
mately 115% to 130%; 1.15 to 1.30 U/mL), may still be
normal flora of the gastrointestinal tract responsible for vita-
within normal limits, making this an insensitive method of
min K synthesis) are exhibited with prothrombin levels rang-
detection. Therefore, the mutation can be detected only by
ing from less than 2% to less than 50%. Ingestion of aspirin
direct analysis of genomic DNA, since the polymerase chain
increases the bleeding tendency by affecting platelet function.
reaction (PCR) technology is only one (initial) part of analyz-
Prothrombin levels approaching 50% activity generally do
ing such single nucleotide polymorphisms.
not cause hemorrhagic symptoms. Variable prolongation of
both the PT and aPTT, and a normal TT, are observed in indi-
viduals with hypoprothrombinemia. These screening assays Factor V (Proaccelerin; Labile Factor)
are not specific for hypoprothrombinemia. Definitive diagno-
sis is dependent on specific assays for functional activity or Factor V (F V) ( molecular weight 350 kD) is a single-chain
antigenic concentration of prothrombin, or both. In hypopro- glycoprotein, synthesized by the liver and also present in the
thrombinemia, both the functional activity and antigenic con- alpha granules of platelets, which is secreted during platelet
centration of prothrombin are decreased. Mixing studies with activation.**** It has a short half-life in vivo, and is also
pooled normal plasma will show full correction of the PT or referred to as the “labile factor” because of rapid deterioration
aPTT (see Tables 26—1 and 26-2). of its activity in plasma at room temperature. F V, also known
Treatment of hypoprothrombinemia may include the as proaccelerin, is the catalyst in the conversion of prothrom-
administration of fresh frozen plasma or prothrombin com- bin (F II) to thrombin (F Ia), functioning as a cofactor within
plex concentrate (PCC), which contains factors II, VII, IX, the prothrombinase complex. It is converted to its active
and X. Vitamin K supplementation will restore only low form, F Va, by the actions of thrombin (F Ia) or F Xa, in the
factor levels associated with dietary deficiency or oral antico- presence of calcium ions and phospholipids. The normal adult
agulant therapy. reference range is 50% to 150% (0.5 to 1.5 U/mL), and may
vary by institution or published reference.
DYSPROTHROMBINEMIA In 1947, Owren discovered F V deficiency in a 21-year-
The mode of inheritance in dysprothrombinemia is the same old woman with a lifelong bleeding history.*>?° Studies demon-
as in hypoprothrombinemia.'® A structural defect causes strated that this clotting factor was not vitamin K dependent.
impaired functional activity, although the antigenic concen- Congenital F V deficiency, also known as parahemophilia, is
tration is normal. Bleeding manifestations may occur that are inherited as an autosomal recessive trait.” It is an extremely
similar to those described for hypoprothrombinemia. Vitamin rare condition that occurs with a probability of 1 individual per
K deficiency, induction and therapeutic warfarin therapy, liver 1 million population (see Table 26-1).
 Chapter 26 Disorders of Plasma Clotting Factors 613

Ecchymoses, epistaxis, menorrhagia, and gingival, gas- 90% of the cases positive for APCR; approximately 20% to
trointestinal tract, umbilical, and central nervous system 60% of patients with recurrent venous thrombosis were found
bleeding are associated with deficiencies of F V. Hemorrhagic to be positive for the mutation.'® The clottable activity of F V
manifestations are usually noted in individuals with less than is unaffected by this mutation. Detection of the R506Q defect
10% F V activity. Hemarthrosis seldom occurs even in can be accomplished only by genomic DNA analysis. In
severely deficient patients. Combined deficiencies of factors V addition, APCR has been found to be present in patients with-
and VIII have been reported in several families. The com- out the factor V Leiden mutation; other mutations have been
bined factor V and VIII activities and antigen levels are characterized, however, they are still undergoing research.
decreased, ranging from 5% to 30% of normal.” This syn- There are also cases of acquired APCR associated with malig-
drome has been found to be the result of a gene mutation nancies, lupus anticoagulants, and third trimester pregnancy.
which produces a protein that affects the intracellular trans- This illustrates the importance of detecting APCR in patients
port of these factors from the endoplasmic reticulum to the who may be at an increased risk of thromboembolic disease.
Golgi apparatus. Its defective transport leads to reduced Screening for the presence of APCR is easily performed by
secretion of these coagulation factors.’® clottable assays utilizing modified aPTT or Russell’s viper
Acquired F V deficiency is associated with a variety of venom time methods. By either method, the plasma clotting
disorders such as liver disease, carcinoma, tuberculosis, and time with the addition of activated protein C (APC) is com-
DIC.*° It can also result from the presence of F V-specific pared to the clotting time of the plasma without added APC.
antibodies acquired after childbirth, due to autoimmune dis- Normal patients produce prolonged clotting times in the pres-
eases, Or exposure to certain types of fibrin glue used in sur- ence of APC, because of its ability to inactivate F Va and F
gical procedures.*! Villa; the clotting time without APC will therefore be signif-
In the laboratory evaluation, the PT and aPTT are icantly shortened. Patients with APCR will produce shortened
prolonged, and both are corrected on mixing with pooled nor- clotting times for both tests, indicating the inability of APC
mal plasma. The TT is normal. Approximately one-third of to exert its effect on F Va or F VUIa, hence the description
patients may present with an abnormal bleeding time (BT), “resistance to APC” (refer to Chap. 28 for a further discussion
possibly related to platelet alpha granule-associated F V defi- of the technique.)
ciency.*” Prior to analysis, the centrifuged plasma must be
platelet-poor (less than 10,000/uL) to avoid contamination of
the plasma F V level by platelets’ endogenous F V. Plasma Factor VII (Proconvertin; Stable Factor;
factor activity by clotting factor assay is decreased or absent. Plasma Thromboplastin Component)
Correction with the addition of pooled normal plasma will not
occur in the presence of an inhibitor. A predominant plasma protein of the extrinsic coagulation
F V-deficient patients are often treated with fresh plasma pathway is F VII (molecular weight 50 kD), a heat- and
or FFP. Cryoprecipitate does not contain adequate amounts of storage-stable component for which it is also known as “‘sta-
F V to be used therapeutically. ble factor.” It is also known as proconvertin, for its pivotal
role in coagulation. It is produced by the liver requiring vita-
FACTOR V LEIDEN MUTATION (R506Q) min K for its synthesis (vitamin K—dependent), and circulates
Under normal conditions, F Va (and, as will be discussed, as an inactive zymogen (F VII), as well as in low levels of its
F VIIa) must be inactivated to prevent ongoing thrombin gen- activated form, F VIla.*° The normal adult reference range is
eration. This is accomplished by the naturally occurring antico- generally 50% to 150% (0.5 to 1.5 U/mL).
agulant, protein C; and its cofactor, protein S. Protein C is The division of the coagulation cascade into the intrinsic
activated by thrombin (which is bound to thrombomodulin on and extrinsic pathways was created as a useful tool for labo-
the endothelial cell surface; see Chap. 28). Activated protein C ratory diagnoses. It is now recognized that this division does
(APC) is responsible for inactivation of F Va and F VIIa . not exist in vivo. Initiation of coagulation in vivo begins with
In 1993, a new and very common inherited throm- the extrinsic pathway, involving components of the vascula-
bophilic syndrome was described: activated protein C resis- ture and cellular elements of the blood. A major component is
tance (APCR). A molecular defect (single point mutation) was tissue factor (TF), also known as tissue thromboplastin, which
identified involving the transition of a guanine (G) to adenine functions as a cofactor. TF, a transmembrane lipoprotein nor-
(A) at nucleotide 1691 of the F V gene. This results in the sub- mally found exclusively extravascular, is synthesized by
stitution of arginine (R) with glutamine (Q) at amino acid monocyte/macrophages and endothelial cells. Its surface
number 506; this is one of the cleavage sites where APC expression can be induced by inflammatory cytokines; this
must bind to the factor V molecule.** This mutation, named seems to be one of the most important pathophysiological
factor V Leiden (R506Q), obliterates this site and leads to mechanisms of DIC in sepsis. TF is also present in many
impaired degradation (inactivation) of F Va by APC, resulting other tissues, such as brain, lung, and placenta. In the event of
in continued thrombin generation, promoting thrombosis vessel injury, TF and F VII/F Vila form a complex (TF/F
(Fig. 26-1). Factor V Leiden is present in 3 to 7% of Vila) in the presence of calcium ions. This complex, known
Caucasians of Northern European descent,” but it is at least as an extrinsic tenase complex, activates F X to F Xa. The
10 times more prevalent than other known genetic throm- TF/F Vila complex is also capable of activating F IX, bypass-
bophilic disorders. This mutation is responsible for more than ing the need for contact activation of factors XI and XII.
614 Chapter 26 Disorders of Plasma Clotting Factors

APC

Factor VIII(a) Factor V(a) Factor VIII(i) Factor V(i)

© i

Factor VIII(a) Factor V(a) Factor VIII(i) Factor V(a)

Leiden Leiden

i = inactivated
a = activated
Thrombin

Figure 26-1 ™ Activated protein C (APC) resistance of factor V Leiden. (Top) Normal inactivation of factors V and VIII:C by APC. (Bottom)
The binding site for APC on the factor V Leiden molecule is altered, thereby permitting activated factor V (factor Va Leiden) to continue
thrombin generation and subsequent fibrin formation.

Positive feedback mechanisms exist that further activate more Factor VII deficiency can be acquired with liver disease,
F VII. Another name for F VII is “plasma thromboplastin warfarin therapy, broad-spectrum antibiotic use, or dietary
component (PTC)” for its involvement in this mechanism. vitamin K deficiency. Treatment can include FFP, PCC, and/or
Homozygous congenital F VII deficiency is a rare, vitamin K supplementation. In addition, a genetically engi-
autosomal-recessive trait.*°°’ Clinically, these patients can neered preparation structurally similar to human plasma-derived
present with deep muscle hematomas, hemarthroses, epistaxis, F VIIa: recombinant activated factor VII (rF VIIa), can be used
and menorrhagia. Patients having less than 1% F VII activity in congenitally deficient patients with a hemorrhagic diathesis.
can have severe hemorrhagic manifestations. Heterozygotes rFVIla was also approved by the FDA in 1999 as a treatment
are usually asymptomatic, having factor activities between option in hemophiliacs with inhibitors (see Hemophilia A; see
40% and 60% of normal. also Circulating Anticoagulants/Acquired Inhibitors).
In the laboratory, the PT is prolonged, with a normal
aPTT (F VII is not measured by the aPTT test system). The
PT can be fully corrected by mixing with pooled normal Factor VIII (Antihemophilic Factor) and von
plasma. Testing with a Russell’s viper venom reagent, Willebrand Factor
known as the Stypven time, will be normal because it
Factor VIII (F VIII) is a large glycoprotein ( molecular weight
directly activates F X (Table 26-4). Documentation of F VII
330 kD), essential in the middle phase of coagulation.'* It is
deficiency requires the performance of a clotting factor
not completely known where the procoagulant F VIII is
activity assay.
synthesized, although studies have tended to support the liver
as the source.**“? However, severe hepatic failure does not
cause F VIII deficiency. Some evidence suggests that a cell
type present in several organs may be the major source, such
as fibroblasts, lymphocytes, monocytes or macrophages, and
vascular endothelial cells.*°
Results Normal Values F VIII is secreted into the plasma, where it circulates as a
macromolecular complex with von Willebrand factor (vWF),
>14 sec 10.0-14.0 sec
<36 sec designated as FVIII:vWF (Table 26-5).41 vWF is synthesized
Factor VII assay <50% within megakaryocytes and endothelial cells as a high-molecu-
Stypven time << SEG lar-weight (HMW) monomeric glycoprotein (molecular weight
Other factor activities >50% 250 kD), which dimerizes and polymerizes into even higher
PT = prothrombin time; aPTT = activated partial thromboplastin molecular weight molecules known as multimers. These multi-
time; sec = seconds. mers are secreted and circulate in the plasma as a heterogeneous
mixture, with molecular weights ranging from 600 to 20,000
 Chapter 26 Disorders of Plasma Clotting Factors 615

receptors on the platelets’ membranes, forming a “molecular

bridge” that results in platelet adhesion to the subendothelium.
A conformational change in vWF after binding to collagen pro-
motes binding to the platelet receptor. This reaction, coupled
with the fibrin-forming action of F VIII:C at the same site, is the
_ Sbbreviation | critical process of hemostasis: the formation of the hemostatic
eee VI Eante (AHF) lei WANES plug. Selected properties of FVII:C and von Willebrand factor
Antigenic portion F VIIL:Ag are summarized in Table 26-6. FVIII:C is also thermolabile and
von Willebrand factor vWE rapidly loses activity at room temperature, unless the plasma is
Antigenic portion VWF:Ag frozen at or below -70°C.
Functional {adhesion)_ VWF. RCo; RCA
The generally accepted normal adult reference range for
*In sisi i two proteins ae a aerbuileai doinniee abe both F VII:C and vWF is 50% to 150% (0.5 to 1.5 U/mL), with
viated as FVII:vWF. 40% considered as a minimum to maintain hemostasis. Defects
AHF = antihemophilic factor.
Source: From Marder, VJ, et al: Standard nomenclature for factor and/or deficiencies of F VIII:C and vWF cause hemorrhagic
VIII and von Willebrand factor: A recommendation by the Interna- disorders with variable severities, known as hemophilia A and
tional Committee on Thrombosis and Haemostasis. Thromb von Willebrand disease (vWD), respectively. Each is discussed
Haemost 54:871, 1985, with permission.
separately in detail. F VIII:C and vWF are also acute-phase
reactants; increased levels are seen with inflammation, stress,
or pregnancy. There is a link between elevated levels of
kD.* vWF is stored and secreted from granules within endothe- FVII:C and a hypercoagulable state.*° Inactivation of FVIIa
lial cells, known as Weibel-Palade bodies; and from the alpha by APC is required to prevent ongoing thrombin generation and
granules in platelets.** Its plasma half-life is approximately 24 thromboembolic complications. During some inflammatory
hours. Only 1% to 2% of the complex functions as the clotting processes, the elevated factor level, in combination with other
factor, or coagulant, designated as F VIII:C, and also known as vascular and activated platelet-derived substances, may pro-
the antihemophilic factor (AHF), that is measurable by clotting mote the formation of thrombi. Oral contraceptives and estro-
assays and is critical to hemostasis. FVII:C has a short half-life gen replacement therapy can also elevate FVII:C and vWF
of approximately 8 to 12 hours. It functions as a cofactor, hav- levels. If there are predisposing factors such as surgery, trauma,
ing no enzymatic activity of its own. Proteolytic cleavage by systemic infection, immobilization, or an unrecognized abnor-
thrombin activates F VIII:C to become F VIII:Ca; it then disso- mality of the anticoagulant-fibrinolytic mechanism, the risk
ciates from vWF, allowing it to react with platelet phospho- of pulmonary embolism, deep vein thrombosis, or stroke
lipids, calcium ions, and activated F IX (F [Xa) to form the increases dramatically.
intrinsic tenase complex, that accelerates the conversion of F X The laboratory assessment of F VIII:C and vWF
to F Xa (see Chap. 24). The remaining portion of the FVII:vWF must include assays for functional activity and antigenic
complex mediates platelet adhesion and also stabilizes F VII:C, concentration, as well as multimeric structure. In addition,
protecting it from enzymatic degradation or inhibition. The platelets must be included in some functional assays in
FVIIL:vWF complex serves as a carrier to concentrate FVII:C order to effectively evaluate the platelet-vWF interactions.
at the site of vessel damage. After injury, vWF interacts with Each of the assay types is addressed in the sections that
exposed subendothelial collagen™ and glycoprotein Ib (GP Ib) follow.

Hactor oak sc vonn Willebrand Becton)

Ee e Gronvintieeis erences: Sateen cells,

endothelial cells megakaryocytes
Plasma concentration 50-150% 50-50%
Molecular weight 330 kD 600—20,000 kD
Principal biologic activity Procoagulant; cofactor Platelet adhesion to vessel wall
Functional assay aPTT intrinsic tenase formation BT, PFA-100°, RCA, RIPA
Antigenic assay IRMA, ELISA ELISA; IEP (Laurell); automated latex
Agglutination
Inheritance Sex-linked recessive Autosomal
Clinical disorder caused by Hemophilia A von Willebrand disease
_ deficiency _

“An plasma, the two proteins areApress as a rndcromolealtayEe onbiek FVILL:vWFBeomplen

BT= bleeding time; RCA= ristocetin cofactor assay; RIPA= ristocetin-induced platelet agglutination; IRMA = immunoradiometric assay;
ELISA = enzyme-linked immunosorbent assay; IEP = immunoelectrophoresis.
Source: From Marder VJ, et al: Standard nomenclature for factor VII and von Willebrand factor: A recommendation by the International Committee
on Thrombosis and Hemostasis. Thromb Haemost 54:871, 1985 with permission.
616 Chapter 26 Disorders of Plasma Clotting Factors

F VILI:C (i.e., known to have a coefficient of variation [CV] as high as

F VIII:C levels can be indirectly assessed with the aPTT, 15%).** There are at present newer methods using enzyme-
which will be prolonged if levels are decreased or absent. The linked immunosorbent assay (ELISA) or latex immunotur-
aPTT will normalize when the patient’s plasma is mixed with bidimetry, that provide a more sensitive, more precise, faster,
pooled normal plasma. Quantitative analysis requires the per- and automated measurement of vWF-dependent function.
formance of an aPTT-based factor activity assay. In the RCA method, the formalin-fixed platelets are not
metabolically active; therefore, the response to ristocetin is
VON WILLEBRAND FACTOR agglutination. In the RIPA method, metabolically active
The function of vWF cannot be determined by standard clot- (viable) platelets are exposed to ristocetin; agglutination ini-
ting assays because it does not directly cause fibrin formation. tially occurs, but subsequent physiologic changes result in
A method to qualitatively assess the platelet and vWF func- the release of endogenous ADP and other platelet-derived
tional interaction, known as the ristocetin-induced platelet agonists. This response is considered aggregation, because
agglutination (RIPA) assay, uses an aggregometer: a device once vWF binds to its receptor (GP Ib), it induces the subse-
that records the change in optical density of a platelet suspen- quent GPIlIb/IIla receptor-dependent aggregation response*!
sion as they aggregate/agglutinate in response to stimulating (Fig. 26-2). GPIIb/IIa is normally the physiologic binding
agents (agonists). RIPA assays evaluate the patient’s site for fibrinogen during in vivo aggregation, but in the
vWF-platelet interaction, through binding with GP Ib. This absence of fibrinogen or when vWF is at high concentrations,
action can be simulated in-vitro using platelet-rich plasma such as at the site of vascular injury, vWF may also induce
and a substance known as ristocetin: a glycopeptide antibiotic this receptor-mediated aggregation process.*' The RIPA assay
first used in the late 1950s. It is structurally similar to van- will produce an aggregation and secretion, or biphasic
comycin, and is isolated from the actinomycete Norcardia response in normal patients.
lurida. Very soon after its introduction, it was removed from The quantity of plasma vWF antigen can be measured by
clinical.use when it was shown to cause thrombocytopenia. It several methods: ELISA; Laurell rocket immunoelectrophore-
was not until 10 years later, that it was shown to agglutinate sis (IEP), using an anti-vWF:Ag-impregnated agarose gel
normal platelets in vitro, but cause little to no agglutination in (Fig. 26-3); and an automated method, which utilizes anti-
patients with vWD.* A snake venom from the Bothrops vWF-coated latex particles. These methods do not assess the
species, known as botrocetin, can also cause vWF-dependent functional ability of the protein.
agglutination; but its action differs from ristocetin in that it
first binds to vWF, then the complex: vWF/botrocetin, binds VON WILLEBRAND FACTOR MULTIMERIC
to the receptor, causing agglutination. Ristocetin only reacts STRUCTURE
with the high molecular weight (HMW) multimer fraction. vWF multimer analysis of plasma or platelet lysates is per-
Botrocetin is useful to determine the reactivity of vWF with formed using sodium dodecyl] sulfate (SDS)-agarose-gel elec-
GP Ib regardless of the multimeric structure.*° Normal or trophoresis, followed by Western blotting with enzyme-linked
hemophilic plasma added to the vWD plasma would induce antibodies, and then staining to visualize the banding pattern
agglutination due to the presence of vWF. The RIPA assay (Fig. 26-4). The multimers are separated on the basis of their
uses ristocetin at final concentrations of 1.2 to 1.5 mg/mL. molecular size, with the lower molecular weight multimers
Studies suggest that ristocetin reduces the negative charge at migrating faster than the HMW multimers. The normal distri-
the platelet membrane’s surface, thereby reducing the repul- bution pattern shows a progression from the smallest to the
sive forces between platelets and/or between platelets and largest multimers.*” Defects in the banding pattern are visu-
vWF, promoting platelet agglutination.*’ It also induces a mor- ally apparent. There can be a decrease in staining intensity or
phologic change in vWF to promote binding to GP Ib. Altering a complete absence of banding with quantitative deficiencies
the concentration of ristocetin (e.g., 0.2 to 0.6 mg/mL), useful of vWF; with qualitative defects, an absence of HMW and/or
in detecting variant forms of vWD, is known as the low-dose intermediate molecular weight multimer bands can be seen.
RIPA method (LD-RIPA). RIPA is advantageous in detecting
both vWD and disorders related to membrane GP Ib defects BLEEDING TIME
(1.e., Bernard-Soulier syndrome; see Chap. 25). One of the earliest and most common screening tests of over-
The RIPA assay is not sufficiently sensitive to mild all hemostasis, and still in use, is the bleeding time
reductions of vWF; therefore, it only serves as a qualitative (BT) method. This assessment of platelet-vWF interaction is
indicator. A method that quantitatively measures plasma a medical procedure performed on the patient. The BT test is
VWE activity, known as the ristocetin cofactor activity assay dependent on adequate numbers of platelets in circulation
(vWF-R:CoF, or RCA), uses aggregometry to record the rate with normal function. In addition, capillary contractility and
of agglutination of a standardized suspension of formalin- secondary hemostasis function in a minor capacity. The first
fixed normal platelets, ristocetin (1.0 mg/mL), and the method, described by Duke (1912), involved puncturing the
patient’s plasma. The rate of agglutination (slope of the reac- earlobe. This puncture was extremely variable in depth. An
tion tracing) of the patient’s plasma is compared to a calibra- improved method by Ivy (1941) involved the application of a
tion curve, constructed by plotting the slopes of dilutions of an pressure cuff inflated to 40 mm Hg, which controlled the
assayed reference plasma versus the vWF activity. This assay, intracapillary pressure to maximize sensitivity and repro-
however, is time-consuming, labor intensive, and imprecise ducibility. An incision was made on the forearm with a lancet;
 Chapter 26 Disorders of Plasma Clotting Factors 617

A 1 Minute

0% sdb

10%
20%
30%

Figure 26-5 @ Quantitative immunoelectrophoresis of vWF:Ag;

Laurell rockets in agarose gel. The first four peaks are the standard
curve dilutions, followed by various vWF:Ag levels. Peak height is
proportional to concentration.

Type 2B von Willebrand’s

institution or published reference. This test is not specific for

any particular disorder and is often unreliable; it is a poor
predictor of bleeding risk and does not correlate with the pres-
ence of bleeding at other internal anatomic sites. The results
B 1 Minute can be affected by various factors, such as: thrombocytopenia
(less than 50,000/uL), low hematocrit (less than 30%),
Figure 26-2 @ Ristocetin-induced platelet aggregation. A. (1) Nor- hypofibrinogenemia/afibrinogenemia, uremia, ingestion of
mal response to ristocetin 1.2 U/mL. (2) Normal response to risto-
aspirin or non-steroidal anti-inflammatory agents, other med-
cetin 0.6 U/mL (low-dose). B. Abnormal response to ristocetin 0.6
U/mL characteristic of type 2B von Willebrand’s disease. Each mark ications, or vascular defects. In addition, the technical skill of
on the x axis represents 1-minute intervals. the performing personnel, skin temperature, patient age, as well
as the ___location and direction of the incision, can affect the test
outcome. Scarring is also a possibility. Considering these
this was also variable in depth. In a method described by limitations, it is virtually always prolonged in patients with
Mielke (ca. 1969), the Ivy method was improved with the use severe vWD or other platelet defects, such as Glanzmann’s
of a template device; this limited the incision to a consistent thrombasthenia or Bernard-Soulier syndrome (see Chap. 25).
length (9 mm) and depth (1 mm). This “template BT” pro- Milder forms of vWD, however, may have normal or only
vided better standardization. Today, the method is performed slightly prolonged BTs.°°
using a disposable, spring-loaded surgical blade device. Lab- A recently developed method, which assesses the platelets’
oratory or medical personnel apply the device to the patient’s ability to adhere and form a hemostatic plug, is known as
forearm, creating a small incision of a standard length (5 mm) the PFA-100® or Platelet Function Analyzer® (Dade-Behring,
and depth (1 mm), while maintaining 40 mm Hg with a blood Miami, FL). This device utilizes a two-cartridge system, each
pressure cuff. As the blood is wicked away with absorbent containing a membrane coated with collagen, and combined
paper at 10- to 15- second intervals, the time to the cessation with either epinephrine or ADP. In the center of the membrane
of bleeding is recorded (see Chap. 33). A normal template is an aperture (150 tum) over a capillary tube, through which
BT is generally 2 to 9 minutes, with slight variations by blood must pass. Vacuum pressure is applied to the cartridge
618 Chapter 26 Disorders of Plasma Clotting Factors

B, and
common of the hemophilic syndromes (hemophilias A,
ve bleedin g disorde r
C). Hemophilia A is a sex-linked recessi
es. Approx imatel y | in
that has been documented for centuri
5.000 to 10,000 affected males are born annually. The fifth-
d
century Talmud described a bleeding episode that occurre
after circumcision. Modern rabbinic comma nd forbids the cir-
cumcision of any child in whom the diagnosis of hemophilia
has been made.°! The disorder was found in the Royal House
of Stuart in Europe and Russia. Queen Victoria, a carrier, was
the source of hemophilia in four subsequent generations.*””*
The defect in hemophilia A is not an absence of the
F VIII complex but rather a molecular defect or absence of its
coagulant portion, F VIII:C,** caused by either a point muta-
tion involving a single nucleotide, deletion of all or part of the
gene, or a mutation affecting gene regulation. The vWF anti-
genic component (vWF: Ag) of the F VIII complex is found to
be normal in patients with hemophilia A. The gene for F
VIII:C resides on the X chromosome. Males inheriting an
affected X chromosome will manifest the disease, whereas
females with one affected X chromosome will be silent
(obligatory) carriers of the trait (asymptomatic). They do not
exhibit clinical bleeding because one allele is capable of pro-
ducing sufficient, functional F VIII:C to maintain hemostasis.
The ratio of F VIII:C to the vWF antigen (F VIII:C/vWF:Ag)
should be approximately 1:2 in the carrier.’ In approximately
one-third of newly diagnosed cases of hemophilia A, there
may be no previous family history of bleeding. This suggests
that a spontaneous mutation may have occurred, or that
there could be several generations of female silent carriers of
thetrait:2

1 2 3
Patients with a F VIII:C activity of less than 1% (less
than 0.01 U/mL) are classified as severe hemophiliacs. They
have a profound hemorrhagic disease, evidenced by intracra-
Figure 26-4 = vWF multimers. Samples of (7) normal plasma,
nial and intramuscular bleeding, hematuria, and spontaneous
(2) von Willebrand’s plasma, and (3) cryoprecipitate, underwent
electrophoresis in sodium dodecyl! sulfate (SDS)-agarose gel. hemorrhage, requiring daily factor replacement therapy.
A Western blotting technique was performed. The gel on nitrocellu- Frequent, spontaneous hemarthroses are the primary symp-
lose paper was incubated first with a rabbit anti-human vWF: tom, involving the knees, elbows, ankles, shoulders, hips, and
Ag antibody and then with goat anti-rabbit IgG, after which it was wrists, that causes crippling deformities. Moderately severe
stained.
hemophilia demonstrates levels of F VII:C activity between
2% and 5% (0.02 to 0.05 U/mL). These patients have less fre-
after a citrated whole blood sample is added. This subjects the quent spontaneous bleeds; however, profuse bleeding still
platelets to the shear forces that may be encountered in vivo, as occurs with circumcision, surgery, trauma, or even minor
they are drawn up through the capillary tube. When they injuries. Patients with activities greater than 5% to 30%
encounter the coated membrane aperture, they are stimulated to (greater than 0.05 to 0.30 U/mL) are classified as having mild
adhere and aggregate, ultimately occluding the aperture. The hemophilia. These patients may go undiagnosed until a bleed-
time required for this occlusion to occur is known as the ing episode occurs, such as with surgery or trauma. Approxi-
“closure time” (CT). Normal values for the CT are established mately 10% to 15% of patients with hemophilia A develop
by each laboratory. Patients with vWD or other platelet dysfunc- antibodies, or inhibitors, to F VIII:C°° that are usually time
tion disorders have abnormal CTs. This test parallels results of and temperature dependent in vitro, and are immunoglobulin
the BT test, but is superior to the BT in standardization, preci- (IgG) in nature. They are capable of destroying F VIII:C
sion, and causing less patient distress. Its limitations, however, at 37°C, neutralizing the coagulant present in therapeutic
are also related to platelet count, as well as hematocrit, erythro- infusions, and complicating treatment.°’ Administration of
cyte sedimentation rate, and specimen age. F VIII:C concentrates may lead to a rise in antibody titers in
patients who have developed antibodies (an anamnestic
HEMOPHILIA A response) to F VIIN:C (see Circulating Anticoagulants/
Hemophilia A (classic hemophilia) is a hereditary coagulopa- Inhibitors).>!
thy, second in overall frequency of the inherited bleeding dis- There are many forms of treatment available for hemo-
orders after von Willebrand disease. It is, however, the most philia A. Concentrates of human plasma F VIII:C were widely
 Chapter 26 Disorders of Plasma Clotting Factors 619

used prior to 1984. Since then, many patients have been the aPTT method, known as the “Bethesda Assay,” and
treated with highly purified, heat- or solvent-detergent quantifying the inhibitor titer in Bethesda units (see Spe-
processed F VIII concentrates, to inactivate hepatitis B, C, cific Inhibitors).°° Patients with inhibitors may show dilu-
and human immunodeficiency viruses (HIV). Although tional increases in factor activity during factor assays.
cryoprecipitate is a rich source of F VIII:C, it is not a product These inhibitors must be differentiated from a nonspecific
of choice due to the high incidence of parenteral viral trans- inhibitor (see Circulating Anticoagulants/Inhibitors) by
mission, because antiviral processing is not possible. New additional testing and clinical presentation.
technologies that produce synthetic DNA-recombinant F VIII
(rF VII:C) have significantly lowered the incidence of viral VON WILLEBRAND DISEASE
transmission. Patients in whom antibodies to human F VIII:C In 1926, Dr. Erich von Willebrand evaluated a large family
have developed, may often respond to treatment with porcine from the Aland Islands for a severe bleeding disorder. Both
F VIII; however, they may form antibodies to this product as sexes were affected, and presented with prolonged bleeding
well. Factor IX concentrate, activated PCC, or Factor Eight times despite normal platelet counts and clot retraction tests.°!
Inhibitor Bypassing Activity™ (FEIBA™), an anti-inhibitor Dr. von Willebrand concluded that this was a previously
coagulant complex agent, has been used in severe cases. undescribed bleeding disorder.
These products contain factors II, IX, X, activated F VII, and vWD differs from classic hemophilia A in three cardinal
a small amount of F VIII:C antigen, which can restore manifestations: (1) autosomal inheritance rather than sex-linked,
hemostasis by providing factors beyond F VIIE:C in the coag- (2) consistently prolonged BTs, and (3) mucocutaneous bleed-
ulation mechanism to form thrombin. As discussed under the ing rather than hemarthroses and deep muscle hemorrhage. It
topic of F VII, rFVIa was approved by the FDA as a treat- was not until the 1970s that vWF and F VIII:C were found to
ment option in hemophiliacs who have inhibitors to control be different proteins produced by different cells under different
life-threatening hemorrhage. Thirty percent (0.30 U/mL) genetic control.
FP VUI:C activity is the minimum level required to maintain von Willebrand’s disease is the most common inherited
normal hemostasis. bleeding disorder, affecting both males and females equally. It
The laboratory findings for patients with hemophilia A results from mutations within the vWE gene, which causes
would be: prolonged aPTT, normal PT, and a normal BT either quantitative or qualitative defects of plasma vWF.
(Table 26-7). Mixing studies with pooled normal plasma These differences arise due to the characteristics of the muta-
corrects the prolongation of the aPTT. The deficiency 1s tion(s) and the autosomal inheritance pattern, which may be
further characterized by low to absent levels of F VIII:C, either dominant or recessive. The gene for vWF resides on
normal levels of vWF:Ag and RCA, and normal platelet chromosome 12; therefore, the disorder may occur in either a
function assays. The presence of an inhibitor can be estab- heterozygous or homozygous mode. These variations in
lished when mixing studies with pooled normal plasma do genotype produce various phenotypes of the disease. vWD
not correct the prolonged aPTT. The inhibitor activity is can be divided into three main types, designated as types 1, 2,
determined using a modified one-stage factor assay utilizing and 3. Type 2 vWD is further subdivided into four subtypes,

vonm Willebrand Disease (Type 1)

See FVIC WE
Inheritance Sex-linked recessive Autosomal, dominant
Clinical presentation Hemarthroses; muscle, soft tissue bleeding Mucosal bleeding: gingival, gastrointestinal,
menorrhagia
Bleeding tendency Moderate to severe Mild to moderate
Laboratory Tests
Bleeding time N N/ABN (variable)
PFA-100° N ABN
Platelet count N N
N N/ABN (variable)
N N
A BN N/ABN (variable)
A BN N/ABN (variable)
N ABN
Caray 2k
N ABN
WE Multimer ‘Pattern N ma Pattern with decreased total concentr, ation

owe= er. ABN= ANS FF VIL C = factor VIII aera activity; PT = Ain Rewari time; aPTT= activated pane tirombontestin ti
time;
Ag = von Willebrand factor antigen; RCA= ristocetin cofactor assay; PFA-100°= platelet function analyzer; RIPA= ristocetin-induced
vWF:
platelet agglutination.
620 Chapter 26 Disorders of Plasma Clotting Factors

designated as 2A, 2B, 2M, and 2N. There is an additional dis- prophylactically with a synthetic analog of vasopressin (the
ease category related to the platelet VWF receptor, known as antidiuretic hormone): desmopressin, also known as DDAVP
platelet-type/pseudo-vWD, which has a similar presentation (1-deamino-8-b-arginine-vasopressin), before dental work,
as vWD, but does not involve a mutation of the vWF gene. surgery, and after bleeding episodes, thereby avoiding expo-
There are also acquired forms of vWD. Each is discussed sure to blood products. This medication causes the vWF and
separately. F VIII:C activity to transiently increase, approximately two-
Since vWF circulates as a complex with F VIII:C, to-fivefold, by stimulating the release from endothelial stores.
patients having reduced or absent levels of VWF:Ag may also This medication can also be useful in patients with mild
have reduced or absent levels of F VIII:C. However, levels of hemophilia A. Some patients do not respond to DDAVP; a
F VUI:C are usually slightly greater than the level of trial course may be administered in advance of elective pro-
vWF:Ag, presumably because there is a normal gene present cedures to determine the degree of response by the patient.
for F VHI:C, and the potential binding sites are saturated Patients who fail to respond to DDAVP must receive cryopre-
when levels of vWF:Ag are reduced.*! The ABO blood type cipitate infusions for prophylaxis or treatment.
has also been found to have a significant effect on the amount
of vWF:Ag produced; individuals who are type A, B, or AB TYPE 2 vWD The type 2 vWD subtypes (15% to 20% of
have much greater mean plasma vWF:Ag concentrations than cases) exhibit qualitative defects of the VWF molecule, with
type O individuals, affecting the severity of symptoms." unequal reductions in the components of the FVIII/vWF
Patients with vWD present with mucocutaneous bleeding: complex, and abnormalities in the multimeric distribution.
frequent instances of epistaxis, ecchymosis, easy bruisability, They are associated with missense mutations of the vWF
gastrointestinal bleeding, menorrhagia, and hemorrhage gene, and inherited as autosomal dominant traits unless
following surgery, childbirth, or dental extractions. These stated otherwise.
manifestations are caused by the inability of platelets to adhere
to subendothelial collagen following injury to the blood Type 2A
vessel.°° vWE and F VII:C are acute-phase reactants, known Type 2A vWD is the most common of the type 2 disorders. It
to increase during stress, inflammation, pregnancy, with oral exhibits a dysfunctional defect of vWE, suggested by a
contraceptive use, estrogen replacement, or after surgery. This disproportionately lower vWF activity than antigenic concen-
variability creates difficulties in the evaluation and diagnosis of tration. There is an absence of the intermediate and HMW
patients suspected of having vWD. Therefore, it may be neces- multimers in the plasma and platelets. Bleeding symptoms are
sary to study these patients on multiple occasions to determine moderately severe; BTs, CTs, RIPA, and vWF activity are
if vWD is the cause of their bleeding symptoms. markedly abnormal; platelet count, PT, and LD-RIPA are nor-
Due to the various types of vWD that have been described, mal. Levels of F VIII:C and vWF:Ag may be normal or
the clinical presentation may vary. Patients who have severe decreased. The aPTT may be affected, dependent on F VIII:C
forms of vWD generally present with a normal PT, a prolonged levels. Treatment involves the use of vWF concentrates or
aPTT that corrects on mixing with pooled normal plasma, nor- commercial F VII concentrates with a near- normal comple-
mal platelet counts, and an abnormal BT test (see Table 26-7). ment of multimers.*! DDAVP is ineffective as a treatment
Laboratory assessment should include assays for F VIII:C modality.
activity, VWF:Ag, RCA, RIPA, LD-RIPA, and vWF multi-
meric analysis. Multimeric analysis will further confirm the Type 2B
specific variant type; identification is essential for proper Type 2B vWD exhibits a qualitative defect of the vWF in
treatment (Table 26-8). which there is an increased affinity for platelet GP Ib. In this
disorder, the plasma shows an absence of only the HMW mul-
VON WILLEBRAND DISEASE TYPES AND SUBTYPES timers, whereas the platelet-associated multimers are nor-
TYPE 1 vWD Type 1, or classic vWD, is the most common mal.°’ The HMW multimers are most important for normal
type (70% to 80% of cases), inherited as a heterozygous auto- platelet function; each subunit contains numerous binding
somal dominant trait. It is associated with partial deletions of sites for the GP Ib receptor on resting platelets and the
the vWF gene that produces a partial quantitative defect, GP IIb/IIIa receptor on activated platelets. This high number
causing only mild symptoms. Patients have reduced levels of of platelet-binding sites correlates with the highly adhesive
a normally functioning vWF, with parallel decreases in properties of this fraction, thereby facilitating aggregation.
vWF:Ag concentration. This may produce either normal A differential laboratory finding in this variant is an abnormal
or abnormal BTs, or CTs according to PFA-100® analysis. LD-RIPA response, i.e., enhanced aggregation is seen when
F VUEC activity can be normal or decreased; this may cause testing with very low concentrations of ristocetin (e.g., 0.2 to
the aPTT to be normal or increased dependent on the F VII:C 0.6 mg/mL). This increased affinity for the GP Ib receptor is
level. The PT and platelet counts are normal. RIPA may be not an intrinsic abnormality of the platelets. The production of
normal or decreased, with a normally absent response to low- the abnormal vWF protein spontaneously binds to normal
dose RIPA, as well. The multimeric pattern is normal but platelets, increasing their clearance from the circulation and
decreased, i.e., the distribution of the various molecular thereby removing the HMW multimers.*! This may be seen
weight multimers on agarose gel electrophoresis is complete in vitro as spontaneous self-aggregation: an aggregation
but equally reduced. Patients with type 1 vWD can be treated response without the addition of any agonist. This subtype
621
Disorders of Plasma Clotting Factors

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622 Chapter 26 Disorders of Plasma Clotting Factors

must be differentiated from another form of vWD with simi- variant requires the use of VWF concentrates or cryoprecipi-
lar findings (see platelet-type/pseudo-vWD). There is no evi- tate. DDAVP is ineffective owing to the vWF defect.
dence of an increased incidence of thromboembolic disease.*!
A mild transient or persistent thrombocytopenia may be seen Type 3 vWD
in some patients. Bleeding symptoms are moderate; BTs and Type 3 vWD is the most severe form (1% to 3% of cases),
CTs are increased. The levels of vWF activity, antigen, and F inherited as a homozygous or double heterozygous autosomal
VIII:C activity may be either normal or decreased; the aPTT recessive trait. Total deletion of the vWF gene causes a com-
will be increased relative to the F VIII:C level. The PT is plete deficiency of vWF in the plasma, plateiets, and endothe-
normal. The RIPA will show a normal response. Definitive lial cells. VWF antigen, activity, and multimers are nearly
diagnosis requires the presence of an increased LD-RIPA undetectable. F VII:C activity is usually 2% to 5% (0.02 to
response, an absence of HMW multimers in the plasma, and 0.05 U/mL), due to the lack of its carrier protein.”” BTs, CTs,
a normal platelet-associated multimer distribution. Treatment RIPA, and aPTTs are severely abnormal. The PT, platelet
of this subtype requires the use of vWF concentrates. DDAVP counts, and LD-RIPA are normal. This form may initially be
administration is contraindicated; the increased release of the confused with hemophilia A by its clinical presentation (e.g.,
abnormal vWE molecules with increased binding to normal hemarthroses, intramuscular bleeding). Treatment of this
platelets causes accelerated clearance of normal platelets, form of vWD requires the replacement of vWF and F VIII:C
resulting in a worsening of the thrombocytopenia.*! Subtyp- with concentrates that contain a normal complement of mul-
ing is essential in determining the appropriate treatment. timers, and which has been treated to inactivate any viruses.
Development of anti-vWF alloantibodies is possible if
Type 2M patients are infused with plasma products, which reduces the
Type 2M (‘“multimer”) vWD is a very rare form in which the effectiveness of therapy.’* Allo-antibody formation has been
qualitative defect of vWF causes decreased or absent binding reported in 10% to 15% of type 3 (severe) vWD patients as a
of vWF to GP Ib, as in type 2A; however, the multimer distri- result of transfusion of vWF-containing products, similar to
bution is normal. There are two known mutations in the GP Ib patients with hemophilia A who produce anti-F VHI:C anti-
binding site that selectively impair ristocetin-induced binding bodies (see Specific Inhibitors).”*
of vWF to the platelet, but do not affect botrocetin-induced
binding.” Occasionally, larger than normal multimers may be Platelet-Type/Pseudo-vWD
seen (Vicenza variant).”? vWF activity is disproportionately Platelet-type/pseudo-vWD (Pt/p-vWD) is an abnormality
decreased as compared to levels of the antigen and F VIII:C, intrinsic to the platelet, although its initial laboratory findings
which may be normal or slightly decreased. The binding of are identical to those of type 2B vWD. It is classified as a
vWF to F VUI:C is normal.“ BTs and CTs are increased with pseudo-vWD because there is no genetic mutation in the VWF
normal platelet counts. The PT is normal, and the aPTT gene. It is inherited as an autosomal recessive trait. A molec-
will be normal if the levels of F VIII:C and vWF antigen are ular abnormality in the GP Ib receptor causes spontaneous
normal. The RIPA response is decreased with a normal binding of the HMW multimers, clearing them from the plasma
LD-RIPA. Treatment requires the use of vWF concentrates or and causing increased platelet turnover. A mild thrombocytope-
cryoprecipitate. DDAVP is ineffective owing to the vWF nia develops and larger-than-normal platelets are present on a
defect. peripheral smear.*' The presence of a positive LD-RIPA
response, as in type 2B, may cause misdiagnosis. Classification
Type 2N is crucial, because different treatments are required for these
Type 2N (“Normandy”) vWD is inherited as an autosomal two disorders. Differentiation can be assisted by performing a
recessive trait, and is defined by a markedly decreased affinity test known as the vWF binding assay. In this method, the
of vWF to F VIII:C. Genetic (missense) mutations alter the F patient’s plasma vWF is tagged with a vWF-specific mono-
VUl:C-binding region of vWF. The majority of these clonal antibody, and reacted with normal formalin-fixed
patients are either homozygous or compound heterozygous for platelets, at various concentrations of ristocetin. This assay
these mutations; another group of patients with this variant cannot rule out Pt/p-vWD; however, a patient with type 2B
appears to be heterozygous for one of the mutations that pro- vWD would show increased vWF binding at lower concentra-
duces the F VIII:C binding defect as well as co-inheriting an tions of ristocetin than normal vWF or vWF in Pt/p-vWD.7
allele associated with type 1 vWD.°’’!” The levels of VWF An abnormal vWF binding assay is diagnostic of type 2B
antigen and activity are normal or decreased, the multimer vWD. Diagnosis of Pt/p-vWD requires a normal vWF bind-
distribution is normal, and the level of F VIII:C is dispropor- ing assay result, in conjunction with other test results typical
tionately lower than levels of VWF, which may have caused of this type (as mentioned earlier). Platelet concentrates are
some patients to be misdiagnosed in the past as hemophiliacs. used to treat patients with this defect.
The disorder may be considered an autosomal form of hemo-
philia A. The platelet-dependent function of vWF is normal, ACQUIRED VON WILLEBRAND DISEASE SYNDROME A
therefore the RIPA and LD-RIPA will be normal. Platelet rare bleeding disorder, with symptoms identical to vWD but
counts, BTs, and CTs are normal. The F VIHI:C is structurally without a genetic defect, can develop in association with a
normal, but has a shortened half-life in the plasma due to variety of conditions or without any apparent cause. This dis-
its inability to bind to its carrier protein. Treatment of this order is known as acquired von Willebrand Disease (AvWD)
 Chapter 26 Disorders of Plasma Clotting Factors 623

syndrome because it does not involve mutations of the vWF normal hemostasis, the age at presentation, a negative family
gene. It can occur at any age, but usually affects the elderly; history, and the presence of an underlying disorder can pro-
either sex can be equally affected. Hemorrhagic symptoms vide a presumptive diagnosis. Detection of vWF-specific
vary considerably among patients and there is a negative fam- antibodies provides a definitive diagnosis. In addition, analy-
ily history. While the incidence of AvWD syndrome is sis of the vWF propeptide (vWF:Ag II): a fragment cleaved
extremely rare, it is most often associated with monoclonal from the mature vWF antigen within the endothelial cell prior
gammopathies, such as multiple myeloma and Waldenstr6m to its release, can assist in distinguishing congenital vWD
macroglobulinemia; and with lymphoproliferative disorders, from AvWD syndrome. vWF:Ag II and mature vWF antigen
such as the lymphomas or chronic lymphocytic leukemia. occur in equimolar amounts in congenital WWD. However, in
Other disorders can include: Wilms tumor; congenital cardiac AvWD syndrome, due to immune complex clearance of the
defects or mitral valve prolapse; angiodysplasia; other solid mature vWF antigen, vWF:Ag II is in excess.”
tumors; autoimmune disorders; and collagen vascular diseases, Some cases of AVWD syndrome can be eradicated by
such as systemic lupus erythematosus (SLE).”? Moderate to treating the underlying disorder. This may involve surgery,
severe bleeding from mucosal surfaces (e.g., epistaxis, gastroin- chemotherapy, radiation, or immunosuppression. Treatment
testinal and gingival bleeding) are the primary symptoms, also must be tailored to the specific cause; the presence of an
occurring following invasive procedures or surgery.’° autoantibody will affect the survival of any vWF-containing
AvWD syndrome can arise from either a reduced rate product administered to the patient. High-dose intravenous
of synthesis of vWF or rapid clearance from the circulation. immunoglobulin (IVIG) therapy is effective in these patients.
Reduced synthesis can occur in hypothyroidism; laboratory The circulating autoantibody is neutralized by the high con-
results resemble those found in type | vWD. Treatment with tent of anti-idiotypic antibodies (i.e., antibodies that interact
thyroid hormone replacement causes a return to normal with the antigen-combining region of other antibodies) in the
hemostasis. Rapid clearance can be attributed to: IVIG preparation.” This allows a spontaneous rise in F VII:C
and vWE levels to occur which can last 2 to 3 weeks, as pro-
* Development of vWF-specific antibodies which form immune
phylaxis for surgical procedures or to maintain hemostasis.
complexes that are cleared by the reticuloendothelial system;
However, most cases are managed with supportive measures
* Selective adsorption of HMW multimers onto the surface of
such as controlling acute hemorrhage or preventing complica-
malignant cells due t6 aberrant expression of the GP Ib recep-
tions from hemorrhage. These measures may include the use
tor, or to activated platelets in essential thrombocythemia; or
of DDAVP, F VIII:C/vWE concentrates, high-dose [VIG ther-
¢ Proteolytic or mechanical degradation: e.g., induced by certain
apy, corticosteroids, plasma exchange, immunoadsorption, or
drugs, or associated with congenital cardiac defects, such as
recombinant activated F VII concentrates.*°*!
ventricular septal defects or mitral valve prolapse; these create
turbulent blood flow disturbances around the defect leading to
the increased activity of a catabolic enzyme of vWF.
Factor IX (Christmas Factor; Plasma
Surgical correction of the defect or cessation of drug Thromboplastin Component [PTC])
usage can resolve the coagulopathy.
AvWD syndrome may also occur without the presence Factor IX (F IX) is a single-chain glycoprotein (Mr approx-
of an underlying disorder. An antibody spontaneously devel- imately 60 kD) that is synthesized by the liver and is
ops that is directed against the vWF:F VIII complex, resulting vitamin K dependent. The gene coding for its production
in a concomitant reduction of F VHI:C, vWF:Ag, and RCA; also resides on the X chromosome. In the coagulation
or it binds vWF alone, resulting in normal F VHI:C activity sequence, F IX participates in the intrinsic pathway, where
without any measurable vWF activity.”” Autoantibodies that it is activated by F Xla in the presence of calcium ions to
interfere with vWF function or cause rapid clearance from the become a serine protease, F [Xa. A second mechanism of
circulation are usually of the IgG type, but occasionally can activation, which bypasses the intrinsic contact system
be of the IgM or IgA type.°' (F XII, high-molecular-weight kininogen, prekallikrein),
Laboratory findings for AVWD syndrome are those typi- occurs through TF and F VIla (see Factor VI).** F [Xa, in
cally seen in vWD: variable aPTT, decreased vWF activity the presence of F VIIIa, calcium ions, and platelet phospho-
discordant with the level of vWF antigen, variable levels of F lipids (PF3), forms the intrinsic tenase complex, that
VIII:C and vWF antigen, and prolonged BTs. Multimer activates F X.° Although F [Xa alone is capable of slowly
analysis most commonly shows a loss of the HMW multi- activating F X in the presence of phospholipid and calcium
mers, as seen in type 2A vWD. The PT, platelet count, and ions, neither F VIII alone nor F VUI plus thrombin activates
other factor activity levels are normal. These results are indis- F X in the absence of F [Xa.® Factor IX is as critical to
tinguishable from the inherited form. It may be possible to hemostasis as F VHI:C. The generally accepted normal adult
demonstrate inhibitory activity by performing an RCA assay reference range is 50% to 150% (0.5 to 1.5 U/mL).
on a mixture of patient plasma with pooled normal plasma,
which would remain abnormal; in addition, adding patient HEMOPHILIA B
plasma to pooled normal plasma may cause a reduction of Factor [X deficiency, also known as hemophilia B, is also a
assayed vWF:Ag. One or both of these assays may be posi- sex-linked bleeding disorder, as in hemophilia A. Females
tive. Clinical history of the patient reflecting previously with the deficiency are quite rare; they are obligate carriers
624 Chapter 26 Disorders of Plasma Clotting Factors

of the disorder. However, spontaneous mutations do occur. chain. This reaction, in the intrinsic pathway, is catalyzed by
The clinical symptoms of hemophilia A and B are identical; the intrinsic tenase complex (F [Xa, F VIIa, calcium ions,
it was originally believed that these were the same disorder. and platelet phospholipid). This same bond is cleaved by the
In 1947, it was observed that mixing plasmas from two extrinsic tenase complex (F VIla, TF, and calcium ions) from
unrelated hemophilic patients would normalize the clotting the extrinsic pathway.’° The normal adult reference range is
time in the mixed plasma.***°Hemophilia B became known 50% to 150% (0.5 to 1.5 U/mL).
as “Christmas disease” by Biggs and colleagues,*’ named Inherited F X deficiency is extremely rare. F X, also
after the family of the first patient they had seen with the known as Stuart-Prower factor, gets its name from two cases
disorder. Since then, F IX has also been known as Christ- found to have this abnormality. At the time of their diagnoses,
mas factor, or synonymously as plasma thromboplastin Mr. Stuart was the 36-year-old son of a consanguineous rela-
component (PTC). tionship between an aunt and nephew within a large, interre-
The incidence of inherited F [X deficiency in the normal lated family living in the Blue Ridge Mountain region of
population is approximately | per 100,000.** The level ofF IX North Carolina®’; and Miss Prower was a 22-year-old English
activity determines the severity of the disorder, with the most woman with a history of two significant hemorrhagic
severe deficiencies having less than one percent activity, and episodes following dental extraction and _ tonsillectomy,
milder deficiencies having between 5% to 25% activity. In who also had a brother that died of postoperative hemorrhage
severe hemophilia B, there is a total absence of F [X, demon- following tonsillectomy at the age of 5.*' Its transmission is
strated by the absence of activity by factor assay as well as autosomal recessive, with an occurrence of less than 1 per
antigenic assay. Some hemophilia B patients synthesize a 500,000; however, the heterozygous state can occur in
nonfunctional variant of the F LX molecule, whereas others do approximately | per 500.
not have identifiable F IX antigen in the plasma and, there- Deficiency of F X may occur at any age, but the most
fore, have a true absence of synthesis of the molecule.*’ severe hemorrhagic symptoms occur in the very young.
Measurement of F [X antigen and clottable activity levels Bleeding sites vary according to the severity of the deficiency.
greatly increases the accuracy of determining carrier status.”° Clinical symptoms range from easy bruising; epistaxis; hema-
Acquired deficiency states can be seen in patients with liver turia; gastrointestinal bleeding; or menorrhagia in mildly
disease, nutritional vitamin K deficiency, and in patients on affected patients, to hemarthrosis; central nervous system
oral anticoagulant therapy. hemorrhage; and severe postoperative hemorrhage in the
Treatment of patients with severe to moderate F IX most severely affected patients.
deficiency consists of infusions of FFP, PCC, or purified F Usually, the diagnosis of inherited F X deficiency can be
IX concentrates. Patients with mild deficiencies usually made by an appropriate family history and laboratory data.
receive prophylactic treatment in association with both However, differentiating between inherited and acquired defi-
minor and major surgery. The most serious complication ciencies should include the consideration of liver disease and
in patients with hemophilia B, as in hemophilia A, is the vitamin K deficiency. Acquired F X deficiencies usually coin-
formation of antibodies/inhibitors to F IX. These are cide with other vitamin K—dependent factor disorders.
extremely difficult to handle, especially in life-threatening F X deficiencies have been reported in patients with
situations, and they occur in approximately 10% of patients amyloidosis.’* Rare, variant forms of F X deficiencies exist
with F IX deficiency.?!” due to amino acid substitutions or deletions, which produce
The laboratory findings in F IX deficiency include: a different patterns of results with PT, aPTT, Stypven time and
normal PT, TT, and BT. There is a prolongation of the aPTT F X antigen levels.”
that corrects when mixed with pooled normal plasma. Laboratory testing results typically seen in this defi-
Patients with inhibitors would demonstrate non-correction ciency include a prolonged PT and aPTT, that corrects when
of the aPTT in mixing studies. Factor assays for F IX mixed with pooled normal plasma. The TT and BT are nor-
activity produce decreased to absent levels. Patients with mal. The Russell’s viper venom time (Stypven time test) is
inhibitors may show dilutional increases in factor activity prolonged, due to its dependence on normal levels of factors
during factor assays; these must be differentiated from II, V, X, fibrinogen, and phospholipid. Factor activity assay,
a non-specific inhibitor (see Circulating Anticoagulants/ typically using the PT methodology, is decreased or absent
Inhibitors, later in this chapter) by additional testing and (Table 26-9). Factor assays based on either the PT or aPTT
clinical presentation. method may be used to determine factor activity (F X is part
of the common pathway, which is measured by both tests);
Factor X (Stuart-Prower Factor) however, there are variant forms of F X deficiency in which
discrepancies in the results can occur, depending on which
Factor X (F X) (M, 58.8 kD) is a vitamin K—-dependent glyco- methodology is used.
protein. It is composed of a light chain and a heavy chain, Treatment consists of transfusions with FFP or PCC.
held together by a single disulfide bond.”*°* The heavy chain However, the need for such measures should be guided by the
contains the catalytic ___domain of the protein.” It is synthesized severity of the hemorrhagic episode. Levels of 10% are con-
by the liver and released into the plasma as a precursor to a sidered adequate for hemostasis. If the deficiency is acquired
serine protease. The conversion of F X to its proteolytic form, by poor diet or oral anticoagulant therapy vitamin K supple-
F Xa, involves the cleavage of a peptide bond in the heavy mentation may be therapeutic.
 Chapter 26 Disorders of Plasma Clotting Factors 625

Replacement therapy is not required for patients with F

XI deficiency unless they require surgery. Preoperative infu-
sion of fresh plasma or FFP, or the supernatant plasma after
the removal of cryoprecipitate, may be required to avoid
Result Ref. Values severe hemorrhage. An increase in factor activity to 20% to
>14.0 sec 10.0-14.0 sec 30% of normal is adequate for hemostasis.'°' Circulating
E-367sec 23-36 sec alloantibodies (inhibitors) can arise in patients with severe
F X activity <50% 50-150% F XI deficiency after exposure to plasma products. Further
Stypven time >25 sec <5 SeC plasma infusions will not control bleeding, but there may be
Other factor activities >50% 50-150%
a response to activated PCC (FEIBA™), such as those used to
PT = prothrombin time; aPTT = activated partial thromboplastin treat patients with F VII or IX inhibitors.'°
time; sec = seconds. The laboratory features associated with a F XI deficiency
include: a normal PT, and a prolonged aPTT that corrects
when mixed with pooled normal plasma. The BT and TT are
both normal. F XI activity assays produce decreased or absent
Factor XI (Plasma Thromboplastin levels. Freezing and thawing the plasma can cause cold acti-
Antecedent [PTA]) vation of the contact system and significantly shorten the
aPTT in F XI-deficient patients. Therefore, it is advisable to
Factor XI (F XI) (M, 143 kD) is a plasma glycoprotein that perform the factor activity assay on a fresh plasma sample
participates in the intrinsic coagulation pathway. It is consid- when there is a high suspicion of deficiency.'°? Normal F XI
ered to be one of the factors in the contact activation pathway activity 1s considered to be 70% to 130% (0.70 to 1.30 U/mL).
involved in early coagulation. Synthesized by the liver and Heterozygous individuals have levels that range between 20%
secreted into the plasma as a zymogen to a serine protease, it and 70% (0.20 to 0.70 U/mL), whereas patients homozygous
circulates as a complex with high-molecular-weight kinino- for F XI deficiency have levels of less than 15% (less than
gen (HMWK).'” Structurally, it is composed of two identical 0.15 U/mL). Because of the variability of factor activity in
polypeptide chains linked by a single disulfide bond.'°!!” some individuals, repeat testing is warranted in questionable
After contact activation by a negatively charged surface, cases.
F XIla cleaves the same peptide bond on each chain, forming The “cascade” theory of blood coagulation, which
F Xla. The resulting serine protease is composed of two light included the intrinsic, extrinsic, and common pathways of
chains containing the active sites and two heavy chains hemostasis, was discussed in Chap. 24. Activation of the
bridged by disulfide bonds.'°*'* The heavy chains of F Xla surface-mediated pathway (contact activation system) can be
are necessary for binding to HMWK and its substrate, F [X.'° achieved with F XII, PK, F XI, and HMWK. With the excep-
tion of F XI, which is associated with hemorrhagic tenden-
HEMOPHILIA C cies, the remaining proteins participate in the inflammatory
F XI deficiency, or hemophilia C (also once known as Rosenthal response, complement activation, fibrinolysis, and kinin
syndrome), is seen predominately in the Ashkenazi Jewish formation.''°
population. It is inherited as an autosomal recessive trait. The Recent studies have found that a high level of F XI
incidence in the general population is approximately | in activity, such as greater than 120% (greater than 1.2 U/mL),
100,000.'°° The heterozygous frequency within the Ashkenazi is a risk factor for venous thrombosis.''' F XI participates in
Jewish population is nearly | in 8.'°” the intrinsic coagulation pathway leading to the formation of
F XI is the only factor among the contact activation thrombin; however, F XI can also be activated by thrombin,
pathway components (consisting of F XII, HMWK, and both in the presence and in the absence of negatively charged
prekallikrein [PK]), in which a deficiency may lead to a bleed- surfaces.''* This feedback mechanism leads to further throm-
ing diathesis. However, plasma levels do not always predict the bin generation, necessary for the formation of fibrin, and for
occurrence of postoperative or posttraumatic bleeding. Symp- protection against fibrinolysis.''* Thrombin mediates this pro-
toms can be mild, ranging from bruising, epistaxis, menorrha- tective mechanism by activating thrombin-activatable fibri-
gia, hematuria, prolonged or delayed postpartum bleeding, and nolysis inhibitor (TAFI),''* which removes the sites necessary
bleeding following dental extractions, to severe hemorrhage for binding and activating plasminogen. Therefore, the risk
requiring massive replacement therapy. Levels of F XI may of thrombosis associated with high levels of F XI may be
fluctuate with time, and bleeding episodes vary in response to a explained by the role of F XI in the inhibition of fibrinolysis.
variety of surgical procedures.'** There is, however, some
degree of correlation between the severity of hemostatic chal- Factor XII (Hageman Factor)
lenge and severe bleeding.'"' Homozygous F XI-deficient
patients may have factor levels ranging from less than 1% activ- Factor XII (F XII) (M, 76 kD), also known as Hageman
ity up to 10%. Antigenic levels parallel the clottable factor factor, is a single-chain beta-globulin.''? The gene has been
activity, demonstrating the decreased level of production of the identified on chromosome 5. It is synthesized by the liver, and
protein.*? Levels of less than 15% factor activity are considered is one of the factors of the contact activation pathway, circu-
severely deficient and result in postoperative bleeding. lating as an inactive zymogen. Contact with negatively
626 Chapter 26 Disorders of Plasma Clotting Factors

charged surfaces in-vitro (solid-phase activation) such as such as placenta, platelets, macrophages, and prostate; and
glass, celite, or kaolin; or chemical activation with ellagic contain the active catalytic site for transglutaminase activity.
acid, causes the auto-activation of F XII and its conversion to The B chains are synthesized in the liver and circulates as
a serine protease.''® This process initiates the intrinsic path- a free dimer or as a complex with the A chains. It is postu-
way of coagulation. In-vivo (fluid-phase) activation occurs by lated that the B chain contributes to the stabilization of
contact with the contents of cell membranes or negatively the A chain; however, the function of the B chain remains
charged subendothelial structures, such as collagen. In this unknown.
process, F XII undergoes a conformational change exposing Congenital F XIII deficiency is inherited as an autosomal
its active site, which then converts PK to kallikrein and sub- recessive trait, with a high frequency of consanguinity in
sequently activates F XI.''’ Additional F XIla is formed due families with this disorder.'*! Clinically, the homozygous
to cleavage by enzymes such as trypsin, plasmin, or deficiency presents with moderate to severe hemorrhagic
kallikrein. The proteolytic product is a two-chain molecule diatheses characterized by initial hemostasis followed by the
composed of a heavy chain and a light chain held by a disul- recurrence of bleeding 12 to 36 hours or more after the initial
fide bond.'°! The presence of small amounts of F XIla leads traumatic event. This results from the dissolution of the solu-
to the activation of its substrates: PK, F XI, and HMWK. ble, unstabilized fibrin clot. These soluble fibrin clots are
These in turn activate more F XII, which serves to amplify highly susceptible to degradation by plasmin. This disorder
the reactions. F XIla, F Xa, and kallikrein stimulate the con- can be diagnosed at birth because the most common clinical
version of plasminogen to plasmin, the enzyme critical to symptom is bleeding from the umbilical cord stump. There is
fibrinolysis. also poor wound healing and abnormal scar formation. An
Deficiency of F XII, also known as Hageman trait, acquired partial deficiency has been reported with several dis-
after the first patient identified with this deficiency in 1955, eases, including leukemias, DIC, and severe liver disease.
is a very rare disorder (1:1 million) inherited in an autoso- The development of [gG autoantibodies to F XIII have been
mal recessive fashion. Homozygotes are severely deficient reported, which have resulted in severe bleeding diatheses.
(less than 1% activity; 0.01 U/mL), while heterozygotes Treatments had once consisted of transfusion with FFP or cry-
may have 40% to 60% activity (0.40 to 0.60 U/mL). oprecipitate, which could cause circulatory overload and
Normal F XII activity levels fall in the range of 70% to virus transmission; there now are viral-inactivated F XIII con-
140% (0.70 to 1.40 U/mL). This disorder is asymptomatic; centrates available.
it is not associated with clinical bleeding or surgical hemor- All routine laboratory screening tests for hemostasis will
rhagic risk. Paradoxically, there may be an increased risk of indicate normal results; PT, aPTT, fibrinogen, BT, and platelet
thrombosis due to inadequate activation of the fibrinolytic count. The screening test for F XIII deficiency is based on the
pathway; it is notable that Mr. Hageman’s death was due to solubility of a recalcified plasma clot in a 5 molar (M) urea or
a pulmonary embolism. The deficiency is usually an inci- 1! M monochloroacetic acid solutions. If the plasma is defi-
dental discovery during presurgical screening. Laboratory cient in F XIII, the clot will dissolve within 24 hours. This is
analysis indicates a normal PT and a prolonged aPTT: a qualitative assay and does not reflect the level of F XIII
greater than 200 seconds in homozygotes that corrects on present, although activity of approximately 1% of normal is
mixing with pooled normal plasma. Specific factor analysis sufficient to prevent both an abnormal test result and symp-
yields decreased or absent F XII activity, which is required toms in-vivo.'** Actual F XIII activity can be assayed by a
for definitive diagnosis. chromogenic enzymatic method. The normal reference range
is 60% to 150% (0.6 to 1.5 U/mL).
Factor XIll (Fibrin-Stabilizing Factor)
Prekallikrein (Fletcher Factor)
In the final stages of the plasma coagulation process, the gen-
eration of thrombin, polymerization of fibrin, and activation Prekallikrein (PK) (M, of approximately 100 kD), is a single-
of factor XIII (F XIIa) is responsible for the formation of a chain protein synthesized by the liver.'?? Conversion to its
stable fibrin clot. F XIII is a proenzyme for a plasma transg- active form, kallikrein, is catalyzed by F XIla and HMWK.
lutaminase; in the presence of fibrin, thrombin and calcium Approximately 75% circulates bound to HMWK,'4 and 25%
ions convert F XIII to the enzymatic F XIIIa."'*"!? It is the circulates as free PK. Kallikrein, a serine protease, is impor-
only enzyme in the coagulation system that is not a serine tant in the activation of three other biologic systems: the fib-
protease. F XIIa functions as a catalyst, forming covalent rinolytic, complement, and kinin systems. Kallikrein cleaves
bonds between various protein substrates such as fibrin HMWKkK into bradykinin: an important mediator in the inflam-
monomers, alpha-2-antiplasmin, fibronectin, and collagen.!?° matory response; it leads to the conversion of plasminogen to
The action of this cross-linking of various plasma and extra- plasmin: a potent enzyme that degrades fibrin, fibrinogen, and
cellular matrix proteins contributes to hemostasis, wound factors V and VIII:C; and, through plasmin, it also activates
healing, and the maintenance of pregnancy. the complement components C3 and CS. Neutrophils and
F XII (M, 320 kD) circulates with fibrinogen. It has a monocytes are also attracted to the site of injury by the pres-
very long half-life of approximately one week. Extracellular ence of kallikrein.
or plasma F XIII consists of two subunits: two A chains and Deficiency of PK, known as Fletcher trait, is not associ-
two B chains. The A chains exist in various tissues and cells ated with clinical bleeding, even during surgery or after
 Chapter 26 Disorders of Plasma Clotting Factors 627

severe trauma. It has been reported to be inherited in both an Circulating Anticoagulants/Acquired

autosomal dominant and recessive trait.'*> There appears to be
no ethnic or racial predilection for this disorder; however,
Inhibitors
functional and antigenic PK, both absent in Fletcher trait Circulating anticoagulants, or acquired inhibitors of coagula-
plasma, have been most often reported in black Americans.'”° tion, can develop in patients with certain underlying disorders;
Patients with this deficiency have experienced thrombotic or spontaneously, without any apparent cause. They can have a
events (i.e., myocardial infarction, thromboembolism, and significant impact on hemostasis, or interfere with various in
multiple cerebral thromboses),'”’ and vascular permeability is vitro coagulation tests. They are composed of immunoglobulins
also defective.'?* (IgG, IgM, or IgA) and possess either auto- or alloantibody
A marked prolongation of the aPTT is characteristic of characteristics. There are two types of acquired inhibitors that
this disorder. Mixing studies of the patient plasma with are of major clinical importance: (1) specific inhibitors: their
pooled normal plasma will produce a normal aPTT. In addi- specificity is directed toward a single factor and (2) nonspecific
tion, this particular deficiency can be presumptively identified inhibitors, or the lupus anticoagulant (LAC): these interfere
by extending the contact activation time of the patient’s with the phospholipid components of the screening reagents,
plasma with kaolin-like aPTT reagents, beyond the usual 3 to which causes a prolongation of the clotting time, and therefore
5 minutes (e.g., 10 minutes, and 15 minutes), which will pro- suggests a hemorrhagic tendency. When inhibitors are detected
duce a progressively shorter aPTT. This phenomenon will not it is extremely important to determine their specificity: one type
occur with other types of factor deficiencies. can have serious, life-threatening hemorrhagic consequences,
Therapy is not indicated for this disorder because there whereas the other may cause thrombotic complications that
is no bleeding risk. Thromboembolic episodes may require range from mild, to severe and life-threatening, or they may be
management with anticoagulant therapy. transient and asymptomatic.

Specific Inhibitors
High-Molecular-Weight Kininogen
(Fitzgerald Factor; Williams—Fitzgerald- The specific inhibitors are characterized as antibodies. They
Flaujeac Factor). either directly inhibit (neutralize) the factor’s activity or cause
its increased clearance by forming immune complexes. They
High-molecular-weight kininogen (HMWK) (M, 200 kD) is a may occur secondary to transfusion, replacement therapy, or
single-chain glycoprotein produced by the liver. HMWK is both. They can also arise spontaneously, in a person without
known as the “‘contact activation cofactor” because it is required any underlying disorders. Inhibitors could potentially arise
for contact activation.'’? Studies indicate that plasma HMWK against any factor.
exists as a pro-cofactor that can be activated through cleavage F VIII:C inhibitors, the most frequently encountered, as
by kallikrein.'°° The kinin system is important in chemotaxis, well as rare instances of F IX inhibitors, can develop in
vascular permeability, and inflammation. Kallikrein cleaves patients with severe hemophilia as a result of transfusion and
HMWK into a two-chain molecule held by a disulfide bond. exposure to the respective deficient factor. These would be
This reaction releases bradykinin, a small peptide with actions classified as alloantibodies: antibodies produced against
related to pain and inflammation. The binding of HMWK to “nonself’ antigens. Their presence, however, does not
endothelial cells is necessary for the binding of F XI (with increase the frequency of bleeding episodes.'*? Hemarthroses,
which it is in complex) and its activation to F XIa, as well as for muscle and soft tissue hemorrhage continue to be the clinical
the activation of F [IX to F [Xa by F XIa."”! symptoms. F VIII:C inhibitors are predominately IgG anti-
Deficiency of HMWK, known as Fitzgerald trait or bodies with a specificity to the coagulant’s antigenic portion
Williams trait, has been described as an autosomal recessive of the F VIH/vWE complex, but do not interfere with the
disorder, and like PK, shows no predilection to race. HMWK function of vWF; there is no prolongation of the BT. An
deficiency is rare and not associated with any bleeding diathe- inhibitor should be suspected in any hemophiliac if transfused
sis. It presents with a markedly prolonged aPTT that corrects factor replacement products appear to have reduced effective-
in mixing studies with pooled normal plasma. Mild to moder- ness, hemostasis is difficult to achieve, or both.
ate acquired deficiencies can occur with liver disease or DIC. More frequently, however, inhibitors to F VIII:C may oc-
While there is no associated clinical bleeding disorder, cur as an autoantibody: an antibody produced against “self”
patients with HMWK deficiency have been observed with antigens; most often seen in patients with rheumatoid arthritis,
deep vein thrombosis and pulmonary embolus.'** systemic lupus erythematosus (SLE) and other autoimmune
Replacement therapy is not indicated with F XII, PK, or diseases; drug reactions, and lymphoproliferative diseases or
HMWK deficiencies. Patients requiring major surgery have multiple myeloma. They also occasionally occur in women
not had any incidence of bleeding. This is presumably be- during pregnancy or postpartum, and in the elderly with no
cause the extrinsic pathway of coagulation, through F VII and apparent underlying disease. This disorder is known as
tissue factor, remains intact, and F XI can be activated by acquired hemophilia. Its etiology is unknown and is apparently
thrombin generated from the extrinsic pathway.'**'** There- unrelated to any previous exposure to blood or blood
fore, F XI can be activated without the need for HMWK, PK, products.'* The clinical symptoms of this disorder may be
or F XI. severe and life-threatening, including large hematomas, gross
628 Chapter 26 Disorders of Plasma Clotting Factors

hematuria, retropharyngeal or retroperitoneal hematomas, or a low titer inhibitor (less than 5 BU) with no rise in titer after
cerebral hemorrhage.'* Hemarthroses are less common. The exposure to additional F VIII:C (i.e., “low responders”) can
bleeding cannot be controlled with factor replacement, and usually be treated with high concentrations of F VIII:C, in an
could further exacerbate the situation. Acquired hemophilia effort to overwhelm the antibody by saturating all its binding
should be suspected in anyone with no prior history who pre- sites. Other options include: porcine F VIII:C, recombinant
sents with massive bruising or hematoma.'*° The mortality rate F VIII concentrates, F IX concentrates, [VIG, and rFVIla.'*”
associated with acquired hemophilia is approximately 20%. Patients with high titer inhibitors (greater than 5 BU) with
A normal PT and prolonged aPTT, with non-correction a marked increase in titer after exposure to F VIII:C (ie.,
in a mixing study with pooled normal plasma, is a classical “high responders) present a treatment dilemma: options can
laboratory finding. However, some patients with mild to mod- include porcine F VIII:C concentrates; steroids, alone or in
erate inhibitors may still show correction on the initial mixing combination; immunosuppressive therapy; cytotoxic agents;
study. These antibodies also demonstrate a characteristic PCCs; high-dose IVIG, extracorporeal immunoadsorption or
known as “‘time-and-temperature dependency”: a 2-hour incu- plasmapheresis for patients with very high inhibitor titers.'°°
bation at 37°C, of the patient plasma/pooled normal plasma rFVIla is the newest therapeutic option available, used pri-
mixture from the initial mixing study, will show a further pro- marily to bring hemostasis under control while other modali-
longation of the aPTT when repeated after the incubation ties focus on suppressing the immune response. Inhibitors can
period. An “incubation control,” consisting of a new mixture persist for weeks or months; some titers may decline to near
prepared from separate, individually incubated patient and zero, only to recur as an anamnestic (memory) response in
pooled normal plasma samples, will not demonstrate the fur- some patients. Spontaneous remissions have been reported in
ther prolongation, indicating the effect of the inhibitor on the approximately 38% of cases, most often occurring in women
additionally provided F VIII:C from the pooled normal postpartum or in drug-induced reactions.!**'°°
plasma during the 2-hour period. This step is extremely Spontaneously acquired inhibitors to factors II, VII,
important in order to properly characterize the disorder, and and X are very rare. F II inhibitors are described in associa-
should be routinely performed for all abnormal screening tion with the LAC (see Nonspecific Inhibitors, following).
aPTTs with an order for follow-up testing. There have been several reports of F XI and F XII inhibitors;
Inhibitors to specific clotting factors are demonstrated by several cases were reported as F XI deficiency with acquired
performing factor assays for all suspected factors indicated by inhibitors posttransfusion and other cases occurring in
the results of the PT and/or aPTT. In the case of a F VIII:C patients with SLE.'**:'%° Acquired F V inhibitor is also rare;
inhibitor, only this factor will be reduced or absent. It is pos- approximately 100 cases are reported in the literature, with
sible for an inhibitor effect (dilutional increase in activity) to only one reported case in a F V-deficient patient; the remain-
occur in other factor assays, since the inhibitor in the patient’s ing cases occurred in elderly patients who previously had
plasma can react against the F VIII:C that is present in the normal levels.'*°
single factor deficient plasma used in the assay; e.g., F IX Antibodies to vWF can occur in patients congenitally
deficient plasma used in assays for F IX activity contains a deficient of vWF (severe vWD-type 3) as alloantibodies, or
normal amount of F VIII:C activity with which it can react. as autoantibodies in normal patients or in those with underly-
The inhibitor titer can be quantitated in a method ing disorders, causing AVWD syndrome (see acquired von
known as the Bethesda Inhibitor Assay. In this procedure, Willebrand disease syndrome, earlier in this chapter).
the patient’s plasma and various dilutions are incubated in
mixture with an equal volume of pooled normal plasma for
2 hours at 37°C; pooled normal plasma diluted with an Nonspecific Inhibitors: Lupus
equal volume of buffer serves as the control. The FVIII:C Anticoagulants
activity of the patient plasma mixture, each dilution, and
the control, is subsequently determined by factor assay. The Nonspecific inhibitors are not directed toward specific factors
percentage of residual F VIII:C in each mixture is then and are not usually associated with a hemorrhagic risk.'*°!*!
calculated, by comparison to the F VIII:C activity of the They are frequently discovered as an unexpectedly prolonged
control. A residual F VIII:C activity of 50%, or the amount aPTT on a routine preoperative evaluation. These results
of inhibitor that will inactivate 50% of the F VIII:C origi- would give the impression that the patient may have a factor
nally present, is defined as one Bethesda unit (BU) of deficiency. However, mixing studies with pooled normal
inhibitor. The inhibitor titer is determined by the dilution/ plasma does not produce a correction of the aPTT. This phe-
mixture with a residual FVIII:C activity of, or closest to nomenon was first recognized in patients with SLE,'? and
50%: e.g., 50% residual F VII:C at a 1:20 dilution is inter- consequently became known as the lupus anticoagulant
preted as a titer of 20 BU. This method can be applied to (LAC). This designation is a misnomer, however, because
any of the clotting factors. these inhibitors often occur in patients without SLE, who are
Treatment of patients with specific inhibitors is first otherwise healthy and have no underlying medical conditions.
aimed at stabilization of hemostasis; the overall goal is to Therefore, it would be more appropriate that they be termed
eradicate the antibody. The choice of therapeutic modality is lupus-like anticoagulants. LACs are immunoglobulins, usually
dependent on the titer (low versus high) and the patient’s IgG and/or IgM, that interfere with one or more phospholipid-
response on exposure to additional F VIII:C.'*° Patients with dependent coagulation tests, e.g., PT and aPTT. This laboratory
 Chapter 26 Disorders of Plasma Clotting Factors 629

phenomenon is a direct reaction against the negatively APLS'**; and thromboses associated with aCL syndrome are
charged (anionic) phospholipids present in the reagents used far more common than with LAC syndrome alone.'*
for coagulation screening assays. LACs do not inactivate the Venous thrombosis presents as deep vein thrombosis (DVT)
clotting factors in vitro: they inhibit the formation of the pro- with or without pulmonary embolism (PE); there may also be
thrombinase complex (F Xa, F Va, calcium ions, F II, and thromboses in unusual sites. Arterial thromboses present as
phospholipids) by binding to the phospholipids, causing a stroke, transient ischemic attacks, or myocardial infarction.'*°
prolongation of the clotting time.'**'°°'4? This in vitro aPL antibodies are actually directed against negatively
effect of “anticoagulation” is paradoxically associated with a charged phospholipid-protein complexes; they require the
hypercoagulable (thrombotic) state in-vivo. presence of specific proteins to facilitate phospholipid binding,
It was also noted that there was a high incidence of bio- such as prothrombin, or a natural anticoagulant protein: beta-
logical false-positive results on serologic tests for syphilis 2-glycoprotein-1 (B,GP-1), also known as apolipoprotein H.
(VDRL) in SLE patients. This method utilizes a phospholipid There have been some cases of patients with LAC that have
known as cardiolipin (diphosphatidylglycerol) extracted from had clinically significant bleeding, which in nearly all cases
bovine heart tissue: it is an anionic phospholipid found in the could be attributed to an abnormality other than the LAC.'*° In
inner mitochondrial membranes of cardiac and skeletal mus- some cases, the PT is prolonged and may or may not correct
cle cells, and some bacteria. These results were found to be with mixing studies. This could be attributed to an acquired
significantly associated with LACs and the risk of thrombo- hypoprothrombinemia, caused by an anti-prothrombin anti-
sis. It was determined that the sera of these patients contained body that binds prothrombin but does not neutralize its coagu-
IgG, IgM, and/or IgA anticardiolipin (aCL) antibodies. aCL lant activity in-vitro; the prothrombin activity and antigen
antibodies and LACs both complex with negatively charged are decreased to the same extent, due to immune complex
phospholipids bound to protein. These antiphospholipid anti- formation and rapid clearance.'**:'°° This has been named the
bodies (aPL) each comprise two related but clinically distinct hypoprothrombinemia-LAC syndrome. If the antibody was of
syndromes: the anticardiolipin antibody (aCL) syndrome and a neutralizing type, the prothrombin antigenic concentration
the LAC syndrome. Both are frequently associated with would have been normal. Serum anti-prothrombin antibodies
thrombosis, fetal loss, or thrombocytopenia, with or without can be detected by ELISA assays. Other causes of bleeding
autoimmune disorders.'*° These syndromes belong to a fam- can be attributed to thrombocytopenia, alone or in combina-
ily of disorders known as the Antiphospholipid antibody syn- tion with a moderate prothrombin deficiency.'*'*!
dromes (APLS). They are one of the most common causes of Antibodies to 8,GP-1 are frequently detected in patients
acquired coagulation defects associated with venous and/or with clinical symptoms of APLS. Approximately 20% of
arterial thrombosis.'** The APLS can occur spontaneously, patients testing negative for aCL antibodies will test positive
without any underlying autoimmune disorder, known as for anti-B,GP-1.'°* The presence of anti-8,GP-1 tends to sup-
primary APLS. A serious and often fatal manifestation of port a diagnosis of APLS in patients with symptoms even
APLS, known as catastrophic antiphospholipid syndrome though aCL or LAC testing may be negative.'** If patients test
(CAPS), is characterized by the development of multiple positive for aCL antibodies, but their clinical presentation is
organ thromboses (infarctions) over a very short period of not consistent with APLS, a negative result for anti-8,GP-1
time (days to weeks). The mortality rate is approximately could rule out the presence of APLS. aCL antibodies associ-
50%. APLS occurring in association with SLE and other ated with infections do not tend to be 8,GP-1-dependent.
autoimmune diseases, acquired immune deficiency syndrome They can often be observed during the convalescent phase of
(AIDS), infections of bacterial, viral, or protozoal origin; acute bacterial or viral infections and in individuals with
drugs such as procainamide or chlorpromazine, inflammation, syphilis. These infection-induced antibodies are usually tran-
malignancies, and lymphoproliferative disorders is known as sient and are not associated with an increased risk of clinical
secondary APLS.'*° Other antiphospholipid antibodies can be complications. Therefore, testing for anti-8,GP-1 can be more
detected using phosphatidylinositol, phosphatidic acid, phos- specific for APLS than aCL antibody testing.'* In general, all
phatidylserine, or phosphatidylethanolamine. patients that test positive for aCL antibodies should be
The LAC syndrome is much more frequently associated retested after 6 to 8 weeks to rule out transient antibodies that
with venous than with arterial thrombosis. The presence of the are usually of no clinical significance.'* Definitive diagnosis
LAC is more specific for APLS than the aCL antibody, how- of APLS requires a positive result for LAC and/or aCL anti-
ever, as the titer of aCL antibody increases its specificity for bodies that persist for at least 8 to 10 weeks and accompany
APLS also increases, especially if it is of the IgG isotype.'* the presence of arterial or venous thrombosis or complica-
Approximately 90% of patients with LACs have also been tions of pregnancy.
found to have at least one isotype of aCL antibody pre- The detection of aCL antibodies requires the perfor-
sent!4°-!47- however, there are clinical situations in which aCL mance of serological tests via ELISA methodology, using
antibody is present without the LAC.'* There is also a high microtiter plates coated with purified cardiolipin antigen,
incidence of thrombotic disease in patients with elevated aCL because they do not prolong clot-based assays. The ability to
antibodies; as the level of antibody increases so does the inci- detect LACs, however, is dependent on the sensitivity of the
dence of thrombosis, fetal loss, and thrombocytopenia.'** screening reagents and the preparation of the plasma prior to
Generally, the presence of aCL antibodies is considered to analysis. Platelets, that are rich in phospholipids, must be
be a more sensitive indicator than LAC in the detection of depleted from the test plasma by centrifugation or filtration to
630 Chapter 26 Disorders of Plasma Clotting Factors

less than 10,000 platelets/uL, to avoid neutralization of the phospholipid for prothrombinase production, and a shorter
inhibitor, especially if the sample is to be frozen for further clotting time is produced. A ratio, calculated from the two
testing at a later time (freezing and thawing will lyse the clotting times and their respective mean normal clotting
platelets). Hemolysis also poses an interference due to cell times, is used to determine the presence of an LAC. If the
lysis, which would also include platelets, as well as red and ratio is greater than 1.2, an LAC is present (see Table 26-10):
white blood cells; those specimens must be recollected.
Patient plasmas containing LACs usually demonstrate a pro- Example:
longed aPTT without correction on mixing study, and a nor- RVVT screening: 50 seconds (NL = less than
mal PT, mostly because PT reagents have a much higher 40 sec; mean =
phospholipid content which masks the effect. However, it has B8Isec)
been observed that up to 30% of LACs can initially correct dRVVT 31 seconds (NL = less than
and then exhibit the time-and-temperature-dependent effect confirmatory: 35 sec; mean =
on the incubated mixing study.'°*'°° In addition, aPTT 28 sec)
reagents are manufactured with variable sensitivities to the Screening ratio: 50/33 = 1.52
LAC; some institutions do not desire a sensitivity to LACs, as Confirmatory ratio: 31/28 = 1.11
determined by the medical director, since these usually do not dRVVT ratio: 1.52/1.11 = 1.37 (NL = less than
represent a surgical risk. The LAC may also affect aPTT-based 1.20)
factor assays; a prolongation of the clotting time in factor
assays translates to a decreased factor activity. The patient The commercially available dRVVT reagents contain an
could possibly be misdiagnosed with a factor deficiency/ anti-heparin agent to prevent interferences by this frequent
inhibitor. For this reason, factor assays must be performed on contaminant and therapeutic agent. In addition, this test can
at least two to three serial dilutions of the patient’s plasma to be performed on patients with a normal aPTT because these
demonstrate an increase in factor activity with the dilutional reagents can vary in their sensitivity to LACs; the dRVVT is
decrease of the inhibitor’s concentration. The presence of mul- a more sensitive test than the aPTT.
tiple factor assays with this “inhibitor effect,” non-correction The PNP/high phospholipid aPTT method is a modified
on mixing studies with pooled normal plasma, and no clinical aPTT in a two-step process: the phospholipid is an extract of
evidence of bleeding are most suggestive of a nonspecific human platelets. The first step is an aPTT with added buffer,
inhibitor. producing a prolonged aPTT. The second step is an aPTT with
Because LACs are such a heterogeneous group of anti- the added platelet extract. This increases the phospholipid
bodies, using only one type of assay is insufficient to diagnose content and will shorten the aPTT if an aPL antibody is pre-
the condition; i.e., no one assay is specific. It is possible that sent. The difference between the two clotting times should be
one type of assay is positive while others are negative. Testing at least 3 to 5 seconds, the actual value determined in studies
for, and confirmation of the LAC should include the use of test by the performing laboratory. In addition, the patient’s aPTT
systems that have different types, concentrations, and configu- must be abnormal before this test can be performed, in order
rations of phospholipids, to demonstrate the specificity and to demonstrate the effect of the added phospholipid. False-
neutralization or bypassing of the inhibitor. These tests can positive tests may occur in heparinized patients due to platelet
include the diluted Russell’s viper venom time (dRVVT), factor-4, a potent anti-heparin substance contained in platelets.
platelet-phospholipid neutralization/high phospholipid aPTT Patients with factor deficiencies of the intrinsic and common
procedure (PNP), and the hexagonal phospholipid aPTT pathways will show equal prolongations in both steps.
(Staclot-LA®, Diagnostica Stago, Parsippany, NJ). These are Methodologies using altered phospholipid configurations
the most sensitive assays currently in use. Other less sensitive (i.e., hexagonal phospholipids) have also been used as confir-
assays have included the kaolin clotting time, and diluted matory techniques. By changing the epitope (antigenic deter-
thromboplastin inhibition time (Tables 26-10 and 26-11). minant) to which the antibody may be directed, increases the
The dRVVT method utilizes a two-step test system. odds of detection (see Table 26-11). Altered configurations
Russell’s viper venom, an extract from the venom of Vipera display different antigenic determinants than the other lamel-
russellii, directly activates F X, so only the common pathway lar, or flat, molecular structures. The Staclot LA® method is
is focused upon. The first step or “screening” reagent contains similar to the PNP principle of a two-step aPTT, however, it
the diluted venom and a low concentration of buffered utilizes an LAC-sensitive aPTT reagent and incorporates
cephalin (phosphatidylethanolamine) as the cofactor for pro- pooled normal plasma as part of the assay system, simultane-
thrombinase production; this increases the sensitivity to ously eliminating the effects of a factor deficiency. A shorten-
inhibitors of the prothrombinase complex, and a prolonged ing of more than 8 seconds with the addition of the hexagonal
clotting time is produced. A mixing study with pooled normal phospholipid versus the buffer is suggestive of a LAC. This
plasma and this reagent will correct in the presence of a factor assay also contains anti-heparin agents.
deficiency of the common pathway, e.g., during warfarin ther- The continuing association between aPL antibodies and
apy. The next step, if the screening dRVVT is abnormal, con- thrombotic episodes has supported advances in the diagnosis
sists of a “confirmatory” dRVVT: the same reagent containing of this syndrome. The most recent guidelines for LAC testing
a higher concentration of the same phospholipid is used. This were developed by the International Society on Thrombosis
will neutralize or bypass the inhibitor, providing additional and Hemostasis (ISTH), Scientific and Standardization
631
Disorders of Plasma Clotting Factors

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 Chapter 26 Disorders of Plasma Clotting Factors 633

Committee (SSC), Subcommittee for the Standardization of

Lupus Anticoaguiant/Antiphospholipid Antibodies. The crite- The history of bruising, epistaxis, and dental bleeding
ria for the laboratory diagnosis of LAC are as follows: with a prolonged aPTT, BT, and reduced F VIII:C, vWF:
Ag, VWFR:Co, and RIPA are indicative of classic von
1. The prolongation of at least one phospholipid-dependent Willebrand disease (type 1).
clotting test (aPTT, dRVVT);
2. Evidence of inhibitor activity shown by the effect of patient QUESTIONS
plasma on pooled normal plasma (non-correction on
1. Which blood product could correct the RIPA value in
mixing study);
this case?
3. Evidence that the inhibitor activity is dependent on phos- 2. Which laboratory values reflect a type 1 von Willebrand
pholipid (dRVVT, PNP, Staclot-LA®); disease?
4. It must be distinguished carefully from other coagu- 3. What form of therapy would be indicated here?
lopathies that may give similar results or occur concur-
rently with LA (exclusion of specific factor inhibitor).'°7 ANSWERS
A complete personal and family history is of utmost — . Concentrates of F VHI:C, cryoprecipitate, and von
importance in beginning a workup for a hemostatic defect. Willebrand factor can correct the RIPA response in this
This should also include a list of any prescription or over- case,
tO. Reduced levels of F VHI:C (30%), vWf:Ag (45%), and
the-counter medications.
Treatment of thrombosis in patients with APLS involves vWER: Co (41%) along with the prolonged bleeding
time and depressed RIPA are classically seen with type
long-term anticoagulation (greater than 6 months to life-long)
1 von Willebrand’s disease.
with warfarin, except in pregnancy where it is contraindi-
Ww. DDAVP would be advantageous as a prophylactic treat-
cated. If the presence of antibodies resolves, treatment should ment prior to any future dental extractions or minor surgi-
continue for at least 6 months to ensure no recurrence of the cal procedure. This avoids exposure to any blood products
antibodies. Heparin therapy is instituted in a hospital setting by causing a transient increase in F VIII:C and vWF.
as treatment after a thrombotic event, to prevent extension of
the existing thrombus or additional thrombosis, until warfarin
therapy is in the therapeutic range (INR 2.0 to 3.0; see Chap.
28). Other options can include low-molecular-weight heparin
(expensive; useful during pregnancy), or low-dose (baby) Case Study 2
aspirin therapy in asymptomatic patients, as prophylaxis
against thrombosis. A full-term male infant was born by normal vaginal deliv-
ery. Thirty hours postpartum, there was new onset of oozing
of blood at the umbilical cord stump. Laboratory analysis
included:

Test Result Ref. Values

Case Study 1 CBC and platelet count Normal Normal
PAL 14.0 sec 10.0-14.0 sec
A 12-year-old boy underwent tooth extraction in preparation APTT 36 sec 23-36 sec
for orthodontia. Persistent bleeding followed. The history was Fibrinogen 400 mg/dL 200-400 mg/dL
remarkable for bruising and frequent epistaxis. The patient’s
mother also experienced easy bruising and menorrhagia.
Physical examination revealed several medium-sized ecchy- Clinical presentation did not indicate a hemorrhagic ten-
motic lesions on the lower extremities. Laboratory findings dency. Late onset of umbilical cord stump bleeding prompted
were as follows: the evaluation of F XIII. A recalcified plasma clot exposed to
a5 M urea solution dissolved within 4 hours. This is conclu-
Test Result Ref. Values sive for F XIII deficiency.

Py 12.0 sec 10.0-14.0 sec

QUESTIONS
APTT 40 sec 23-36 sec
BT >15 min 2-9 min 1. Is the 5-molar urea lysis test a qualitative or quantitative
Platelet count B00 107A 1504502107 determination?
F VIII:C 30% 50-150% 2. What treatment is indicated for this patient?
vWF: Ag 45% 50-150%
vWER:Co 41% 50-150%
RIPA* Depressed Normal ANSWERS
response response
1.5 M urea lysis for F XIII deficiency is a qualitative
Multimers Normal Normal
determination; however, the test is positive when activ-
*Ristocetin-induced platelet aggregation. ity 1s less than 1%.
2. Treatment would consist of fresh frozen plasma or cryo-
continued precipitate infusion.
634 — Chapter 26 Disorders of Plasma Clotting Factors

This patient has F VII deficiency. F VII is not measured

Case Study 3 by the aPTT and is normal, whereas the PT is sensitive to
deficiencies of the extrinsic system (factors I, II, V, VI, and
A 5-year-old boy presented in the emergency department with X) and is prolonged in these cases.
a hemarthrosis of the right knee after falling off a playground
swing. Physical examination revealed a_well-nourished QUESTIONS
child with no fractures or other ecchymoses. He was afebrile.
1. What material was used to perform the mixing study?
Laboratory findings were as follows:
2. What treatment is indicated here?
3. Why was the PT prolonged and the aPTT normal in this
Test Result Ref. Values
case?
CBC and platelet count Normal Normal
[PAB 13.0 sec 10.0—14.0 sec ANSWERS
ALPARIE 87 sec 23-36 sec
Fibrinogen 325 mg/dL 200-400 mg/dL |. The mixing study was performed with pooled normal
BT 6 min 2-9 min plasma.
2. This patient would require fresh frozen plasma or
prothrombin-complex concentrate. However, if the defi-
Further studies included: ciency can be attributed to poor diet, liver disease, or
warfarin therapy, vitamin K supplementation may be
aPTT 1:1 mix 32 sec 23-36 sec preferable to using blood products.
F VILI:C 10% 50-150% 3. The PT was prolonged because the extrinsic pathway
vWF:Ag 90% 50-150% could not proceed normally, caused by the deficiency of
F VII. The aPTT was normal because F VII does not
participate in the intrinsic pathway and therefore the test
This patient was known to have hemophilia A.
is not sensitive to this defect.

QUESTIONS
1. What material did the laboratory professional use when
performing this mixing study?
2. What treatment is indicated for this patient? Case Study 5
ANSWERS A 29-year-old woman was seen by her obstetrician for pre-
natal care. She had three previous pregnancies, two resulting
1. The mixing study in this case would have been per- in spontaneous first-trimester miscarriages and the third in
formed with pooled normal plasma or adsorbed plasma. fetal demise at 28 weeks’ gestation. Laboratory values were
Aged serum would not have corrected the aPTT. as follows:
2. This patient requires infusion of factor VIII concentrate.
Test Result Ref. Values

CBC and platelet count Normal Normal

[Pap 11-5.sec 10.0-14.0 sec
aPTT 45 sec 23-36 sec
Case Study A Fibrinogen 375 mg/dL 200-400 mg/dL

A 40-year-old woman was scheduled for an elective surgi-

cal procedure. Preadmission testing revealed the following Further studies included:
results:
aPTT mixing study 43.0 sec 23-36 sec
Test Result Ref. values DRVVT ratio 1.60 <1.20
PNP difference 15 sec <5 7Sec
CBC and platelet count Normal Normal Anticardiolipin Ab positive Negative
[eae 18.0 sec 10.0-14.0 sec
alien 30 sec 23=3 01SEC
This patient has a lupus anticoagulant with positive anti-
cardiolipin antibodies. These are known to cause recurrent
After further examination, the patient stated that she had spontaneous abortion, as well as thrombotic events such as
episodes of epistaxis and easy bruising. Additional labora- stroke, deep vein thromboses, and pulmonary embolism.
tory testing included:

QUESTIONS
Piel 13.0 sec 10.0-14.0 sec
F VII 30% 50-150% 1. What complex is inhibited by LAC, causing a prolonga-
tion of the clotting time?
continued continued
 Chapter 26 Disorders of Plasma Clotting Factors 635

2. Why does the PNP show a significant difference in this

patient? Case Study 7
3. What form of treatment is indicated here?
A 65-year-old woman with liver disease was admitted to the
ANSWERS medical intensive care unit. Admission laboratory data were
as follows:
1. The complex that is inhibited by LAC is the Prothrombi-
nase complex (factors Xa, V, II, calcium, and phospho-
Test Result Ref. values
lipid), delaying the formation of fibrin and producing a
prolonged clotting time. Pr 14.5 sec 10.0—14.0 sec
2. The high concentration of phospholipid in the PNP aPTT 39 sec 23-36 sec
bypasses or “neutralizes” the inhibitor thereby creating a Fibrinogen 190 mg/dL 200-400 mg/dL
greater difference versus the aPTT without the addi- Thrombin time 18 sec 4-12 sec
tional phospholipid.
3. Treatment of a patient with LAC would require antico-
Prolongation of the aPTT and thrombin time could be
agulation with warfarin or, in some circumstances,
caused by the presence of heparin. This should be ruled out
heparin may be used (e.g., during pregnancy).
before further testing is performed.
Reptilase time = 29 seconds (ref. value: 18—22 sec)
Fibrinogen antigen = 380 mg/dL (ref. value: 200-
400 mg/dL)
A PT and aPTT mixing study produced a correction for
Case Study 6 both tests.
Factors VIII:C, IX, XI, and XII were within normal
A 25-year-old man was admitted for hernia repair. Admis- limits.
sion laboratory data were as follows: This patient was neither receiving heparin nor was the
specimen contaminated by heparin, as evidenced by the pro-
Test Result Ref. Values longed reptilase time. The combination of a decreased fib-
rinogen level and prolonged thrombin and reptilase times
CBC and platelet count Normal Normal are suggestive of a dysfibrinogenemia, especially in patients
Bae 1D Dasee 10.0-14.0 sec
with liver disease. Slight prolongation of the PT may also be
ap il 115 sec 23-36 sec
noted and will not be attributable to any factor deficiency.
aPTT mixing study 25 sec 23-36 sec
F VUL:C 97% 50—150% Antigenic assay of fibrinogen usually results in higher
F Ix 104% 50-150% values than the functional (clottable) assay.
F xI 110% 70-130%
F XI 96% 70-140% QUESTIONS
1. How can it be known that this patient is not on heparin
Interval incubations of the patient’s plasma with the aPTT therapy?
reagent, containing silica as the activator, produced the 2. Differentiate between the thrombin time and reptilase
following results: time with regard to heparin.
3. What treatment is indicated here?
5-min incubation aPTT 115 sec 23-36 sec
10-min incubation aPTT Sisee 23-36 sec ANSWERS
15-min incubation aPTT 35 sec 23-36 sec
Prekallikrein activity <1% 30-100% ey. The prolonged thrombin time and reptilase time prove
that the specimen does not contain any heparin.
2. Heparin, even in relatively small amounts, will prolong
This patient has a prekallikrein deficiency. Contact sys- the thrombin time. The reptilase time, which is unaffected
tem deficiencies (F XII, HMWK, prekallikrein) do not pose by heparin, will be normal.
any hemorrhagic risk. 3. Treatment for this patient could include anticoagulants if
there is evidence of thrombosis.
QUESTIONS
1. Why was the aPTT abnormal, but the PT remained normal?
2. What treatment would be indicated in this case?

ANSWERS
Case Study 8
1. The PT measures the extrinsic pathway, in which PK is A 60-year-old woman was admitted to the emergency
not involved. Therefore, only the aPTT will reflect an department with hemorrhage into the right arm and right
abnormal clotting time. breast. No previous history of bleeding or medication was
2. No treatment is indicated: there is no associated risk of indicated. Laboratory data upon admission were as follows:
bleeding. continued
636 Chapter 26 Disorders of Plasma Clotting Factors

Test Result Ref. Values

Case Study B_cont'a
CBC and platelet count Normal Normal
Pa 12.0 sec 10.0-14.0 sec
Test Result Ref. Values ALPALE 60 sec 23-36) Sec
Thrombin time 5 sec 410esec
Pa 12.0 sec 10.0-14.0 sec
aPTT 58 sec 23-36 sec
Fibrinogen 400 mg/dL 200-400 mg/dL The patient was asymptomatic for any bleeding. Testing
aPTT mixing study 40 sec 23-36 sec continued and produced the following results:
DRVVT ratio 1.1 <2
PNP difference 1 sec ESO seG
aPTT mixing study 82, Sec 23-36 sec
F VUE:C 90% 50-150%
FIX 771% 50-150%
This patient does not have a lupus anticoagulant, but still
F XI 45% 70-130%
does not correct in a mixing study with pooled normal
plasma. Further studies must be performed. Incubation of
the patient’s plasma with pooled normal plasma for 2 hours This patient was found to have F XI deficiency. The ini-
at 37°C, demonstrates time-and-temperature dependency, tial history revealed that he was of Eastern European-
indicating the presence of an inhibitor. In addition, factor Ashkenazi Jewish background. This factor deficiency is
assays indicate decreased activity of a single factor. prevalent among this population. The patient’s factor level
was not depressed enough to produce symptoms, but could
PVILEC 10% 50-150% have posed a significant surgical risk. Heterozygous F XI
FIX 90% 50-150% deficiency has a variable presentation and bleeding risk may
FAL, 95% 70-130% not correlate with factor levels.

Factor inhibitor analysis (Bethesda assay) for F VIII:C QUESTIONS

produced a titer of 8 Bethesda units of inhibitor activity 1. Why was the aPTT normal but the PT remained normal
(normal is less than 0.5 units). One Bethesda unit of in this case?
inhibitor is equivalent to 50% residual factor activity after 2. What does the aPTT mixing study result indicate?
incubation at 37°C for 2 hours. This patient developed a 3. What treatment is indicated here?
spontaneous inhibitor to F VII:C that resulted in the mas-
sive bleeding into the tissue of her arm and breast.
ANSWERS
QUESTIONS 1. The PT measures the extrinsic pathway, in which F XI is
not involved. Therefore, only the aPTT will reflect an
1. Which test(s) rule out the presence of a lupus anti-
abnormal clotting time.
coagulant? 2. The aPTT mixing study shows a correction of the aPTT
2. What treatment is indicated here? into the normal range, indicative of a factor deficiency
of the intrinsic pathway, most likely factor VIII, IX, XI,
ANSWERS XI, HMWK, or PK (a normal PT rules out factor II, V,
1. The presence of a lupus anticoagulant is ruled out by the or X, and a normal TT rules out fibrinogen deficiency).
negative DRVVT and PNP tests. 3. No treatment is necessary for F XI deficiency unless the
2. Treatment of a F VII:C inhibitor can include porcine patient requires surgery and the F XI activity is less than
F VIII:C concentrates; prothrombin-complex concen- 20% to 30%. Fresh-frozen plasma or the supernatant
trates; steroids (alone or in combination); immunosup- plasma after the removal of cryoprecipitate may be used
pressive therapy; cytotoxic agents; high-dose intra- in severe deficiencies to control bleeding.
venous immunoglobulin, immunoadsorption or
plasmapheresis if the inhibitor titer is very high.

Case Study 9
Case Study 10
A 37-year-old man was seen by his physician for a routine
physical examination. A full blood workup was drawn and A 66-year-old man with a diagnosis of amyloidosis was
sent to a reference laboratory for analysis. Results were as admitted to the hospital with severe gastrointestinal bleed-
follows: ing. Admission laboratory data included:
continued continued
 Chapter 26 Disorders of Plasma Clotting Factors 637

Test Result Ref. Values

This patient was determined to have F X deficiency sec-
ondary to amyloidosis. Other vitamin K—dependent factors
CBC Anemia; Normal were normal, as was F V, which ruled out a nutritional
microcytic/ deficit or liver disease.
hypochromic
Platelet count Normal Normal
PAE 45.0 sec 10.0-14.0 sec
QUESTIONS
apy | SEINE 23-36 sec 1. Why were both the PT and aPTT prolonged in this case?
Fibrinogen 400 mg/dL 200-400 mg/dL 2. What test results indicate a factor deficiency as opposed
Thrombin 5 sec 4-12 sec to a circulating anticoagulant?
time
PT mixing 13 sec 10.0-14.0 sec
study ANSWERS
aPTT Mixing 30 sec 23-36 sec
1. F X participates in both the intrinsic and extrinsic path-
study
ways (also known as the common pathway), which will
Stypven time 55 sec 14-20 sec
FI 15% 50-150% cause a prolongation of both the PT and aPTT if deficient.
FY 93% 50-150% 2. A factor deficiency is indicated rather than a circulating
F VII 90% 50-150% anticoagulant, demonstrated by the correction of both
FIX 100% 50-150% the PT and aPTT in the mixing studies, and the normal
FX 5% i 50-150% levels of other factors except F X. The Stypven time will
be prolonged in F X deficiency.
continued

Oi ceticans
L Which of the following can lead to impairment of the a The factor deficiency that produces a normal aPTT and
coagulation system? an abnormal PT is:
a. Decreased factor synthesis a. Afibrinogenemia
b. Interference by abnormal molecules b. Prekallikrein
c. Loss, consumption, or inactivation of factors c. Factor VII
d. All of the above d. Factor X
. Which of the following shows decreased activity of F
i) 6. A patient with amyloidosis can have a deficiency of
VII:C? which factor?
a. Hemophilia A a. Factor V
b. Hemophilia B b. Factor X
c. Parahemophilia c. Factor VIU:C
d. Hemophilia C d. Factor I
. Which coagulation disorder has decreased activity of F 7. The reptilase time will be normal in the presence of:
VUI:C, vWF:Ag, and vWFR:Co, and a prolonged bleed- a. Heparin
ing time test? b. Dysfibrinogenemia
a. Thrombocytopenia c. Afibrinogenemia
b. von Willebrand disease d. Hypofibrinogenemia
c. Hemophilia A 8. Which of the following is not considered a test for iden-
d. Acquired hemophilia tification of a lupus anticoagulant?
. The lysis of a clot in a 5 M urea solution is an indication a. Platelet neutralization procedure (PNP)
of which disorder? b. Anticardiolipin antibodies
a. Dysfibrinogenemia c. Diluted Russell’s viper venom time (dRVVT)
b. F XIII deficiency d. Hexagonal phospholipid aPTT
c. Antiphospholipid antibody syndrome
d. Factor V Leiden
638 Chapter 26 Disorders of Plasma Clotting Factors

9. All of the following statements are true regarding the 10. Activated protein C resistance:
detection of a lupus anticoagulant (LAC) except: a. Is the result of a single point mutation of the F V gene
a. Platelet-poor plasma samples are required. in 90% of cases;
b. The aPTT is frequently prolonged and is usually the b. Leads to increased thrombin generation as a result of
first indication of a LAC. impaired F V inactivation;
c. A mixture of patient plasma with pooled normal c. Is found in 20% to 60% of patients with venous
plasma will correct the prolonged aPTT. thrombosis;
d. The diluted Russell’s Viper Venom Time may be used d. All of the above
to confirm the presence of LA.
See answers at the back of this book.

e" oe See ¢ The thrombin time assesses thrombin-fibrinogen inter-

J SUMMARY — actions and fibrin polymerization. It will be abnormal
in the presence of heparin, afibrinogenemia, hypofib-
m Screening tests for coagulation abnormalities should
rinogenemia, dysfibrinogenemia, and fibrinogen/fibrin
include:
degradation products (FDPs).
* Complete blood count (CBC), platelet count, differential A reptilase time will be normal in the presence of
smear heparin.
¢ Prothrombin time (PT) For specimens that have both an abnormal PT and
* Activated partial thromboplastin time (aPTT) aPTT that corrects with mixing studies, deficiencies of
¢ Fibrinogen factors I, II, V, and X should be considered.
¢ Thrombin time If an abnormal PT and aPTT does not correct with mix-
¢ Bleeding time ing studies, an inhibitor to factors I, V, or X should be
w When the platelet count and morphology are normal, the considered.
results of the PT and aPTT should be evaluated as follows: Laboratory findings in patients with hemophilia A
¢ For an abnormal! PT with a normal aPTT, a mixing study include a prolonged aPTT, normal PT, and normal
of the PT should be performed using pooled normal bleeding time; mixing studies with normal or adsorbed
plasma; if the PT is corrected, a factor VII deficiency plasma will correct the aPTT.
should be considered and subsequent factor assay is Laboratory studies for von Willebrand disease should
indicated. include a bleeding time, platelet count, aPTT, mixing
* For an abnormal PT that does not correct with a mixing study, F VIII:C activity, von Willebrand factor antigen
study, inhibitors to factors II, V, VII, or X should be (vWF:Ag), ristocetin cofactor activity (VWFR:Co), ris-
considered. tocetin-induced platelet aggregation, and multimer
¢ For a normal PT with an abnormal aPTT, a mixing analysis.
study of the aPTT should be performed using pooled The most common inhibitor to a specific clotting factor
normal plasma. If the aPTT is corrected, a deficiency is factor VII:C (FVII:C); the inhibitor is predomi-
of factor(s) VIII:C, IX, XI, XII, prekallikrein (PK), nantly IgG and time-and-temperature-dependent; the
and/or high-molecular-weight kininogen (HMWK) laboratory assay for inhibitors is the Bethesda Assay;
should be considered, with subsequent testing by spe- they are measured in Bethesda units, where neutraliza-
cific factor assay performed. tion of 50% of the FVIII:C activity is considered |
* For abnormal aPTTs that do not correct with a mixing Bethesda unit.
study, inhibitors to factor(s) VIII:C, IX, XI, XII, PK, The lupus anticoagulants may be IgG, IgM, or IgA in
and/or HMWK should be considered as well as the nature, and can be detected by the platelet neutraliza-
presence of the lupus anticoagulant or the anticoagulant tion procedure (PNP), diluted Russell’s viper venom
heparin. time (ARVVT), or Hexagonal Phospholipid aPTT.
¢ The presence of heparin can be ruled out if the throm-
bin time is normal;

Acknowledgment
The author gratefully acknowledges the contributions of
Judith Brody, MD.
 Chapter 26 Disorders of Plasma Clotting Factors 639

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 Chapter |

Interaction
of the Fibrinolytic,
Coagulation, and Kinin
Systems; Disseminated
Intravascular
Coagulation; and
Kelated Pathology
John Lazarchick, MD
Melanie Oswald, MHS, MT(ASCP)SH

Systems and Related OBJECTIVES

Pathology
At the end of this chapter, the learner should be able to:
Molecular Components:
Physicochemical and 1. Name the components of the coagulation and fibrinolytic systems.
Functional Properties 2. Describe the physiologic interactions of these proteolytic systems.
Plasminogen
Plasminogen Activators 3. Have an understanding of the clinical and laboratory abnormalities associated with
Plasminogen Activator disseminated intravascular coagulation.
Inhibitor-1 4. Describe the laboratory abnormalities associated with primary fibrinolysis versus dis-
Plasmin seminated intravascular coagulation.
Plasmin Inhibitors
Thrombomodulin
Thrombin-Activatable
Fibrinolysis Inhibitor
Congenital Abnormalities
Disseminated Intravascular
Coagulation
Triggering Mechanisms:
Associated Clinical
Disorders
Clinical Presentation
Laboratory Diagnosis
Therapy
Related Disorders
Case Study
644 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

Systems and Related Pathology FIBRINOLYTIC SYSTEM

Plasminogen
Normal hemostasis is the result of the balanced interaction of Activated protein C
Xlla, Xlla fragments
the vascular endothelium and platelets with four biochemical
Paes ee
Kallikrein
systems'”: the coagulation, the fibrinolytic, the kinin, and, to Endothelial cells

a lesser extent, the complement systems. The interrelationship Plasminogen Plasminoge .

proactivator activator Tissue plasminogen
between these systems is illustrated in Figure 27-1. The
activator
endothelium not only provides procoagulant, fibrinolytic,
Plasmin
anticoagulant, and inflammatory functions, but in addition
these activities vary among the different vascular beds and in
some cases are organ specific.* Detailed discussion of the Inactivation Xlla —> Xila fragments
ever-emerging central role of the vascular endothelium in of Factors V
and VIII
hemostasis and inflammation is beyond the scope of this Fibrin/Fibrinogen
chapter and the reader is referred to several excellent reviews
on the topic.*°
FDPs
When a stimulus initiates activation of the coagulation
system with resultant fibrin formation and the establishment COAGULATION SYSTEM
of a hemostatic barrier, a series of enzymes comprising the
Kinins HMWK
fibrinolytic system are simultaneously activated to lyse the
fibrin thrombus and reestablish vessel lumen integrity and
Kallikrein Prekallikrein
blood flow. This chapter deals with the biochemistry of the
components of this fibrinolytic system, its associated patho-
HMWK
physiologic disorders, and laboratory tests available to evalu- XII Xlla
ate individual components and overall function (Fig. 27-1).
Xl cia
1

Molecular Components: Tissue i

(Intrinsic cascade)
Physicochemical and Functional thromboplastin

Properties Vil ——~ Vila- - : (Extrinsic cascade) —> Thrombin

The molecular components of the fibrinolytic system consist of:

Protein C cofactor
1. The plasma protein plasminogen
2. Its active enzymatic form, plasmin Protein C> ! Activated
protein C
3. A group of plasminogen activators that convert plasmino-
gen to plasmin Platelet
4. Plasmin inhibitors, most prominently «,-antiplasmin aggregation and Fibrinogen
inhibitor release |
5. Thrombomodulin, an endothelial cell surface membrane Fibrin
protein that binds thrombin +

Fibrinopeptides
6. Thrombin-activatable fibrinolysis inhibitor that cleaves A and B
plasminogen/plasmin binding sites on fibrin
7. Fibrin/ fibrinogen, which serve as substrate for the active Figure 27-1 m Summary of the interaction of the coagulation, fibri-
nolytic, and kinin systems. High-molecular-weight kininogen (HMWK)
enzyme plasmin (Table 27-1).
and prekallekrein catalyze the activation of factor XII to factor Xlla.
Factor Xlla then promotes the conversion of prekallekrein to kallekrein.
The latter liberates the kinins from HMWK, thus completing the posi-
Plasminogen tive feedback loops of the contact phase of coagulation. These compo-
nents stimulate endothelial cells to release plasminogen activator.
Native plasminogen is a single-chain plasma zymogen of Thrombin generated through the extrinsic and intrinsic coagulation
approximately 90 kD that circulates in two molecular forms, cascades then converts fibrinogen to fibrin, induces platelet activation,
differing only in their carbohydrate content.’ It is synthesized activates protein C, and stimulates both tissue plasminogen activator
by the liver and has a half-life of 2 days. The plasma content (tPA) and urokinase-like plasminogen activator (uPA) from the en-
dothelium, which then can convert plasminogen to plasmin. Thrombin
is approximately 20 mg/dL. Each form of this molecule has
exerts a negative feedback by simultaneously stimulating plasminogen
an amino acid terminal glutamic acid (Glu-plasminogen) and activator inhibitor 1 (PAI-1) release from the endothelial cells. PAI-1 will
is capable of undergoing limited proteolytic cleavage of this serve to bind tPA and uPA to dampen plasmin generation. Plasmin,
region to an incomplete molecule with lysine as the new ter- once formed, will initiate clot lysis by proteolytically cleaving fibrin and
minal amino acid (Lys-plasminogen). This latter form is more fibrinogen into fibrin/fibrinogen degradation products (FDPs). In addi-
tion, plasmin will inactivate a number of coagulation factors including
readily converted to active plasmin by plasminogen activators factors V and VIII and convert activated factor XII into Xlla fragments.
than the Glu-plasminogen form and is probably of greater Excess plasmin in circulation is prevented by the presence of
physiologic significance. a-antiplasmin which forms a complex with plasmin, thus inactivating it.
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 645

kallikrein, and high-molecular-weight kininogen interact to

yield plasminogen-activating ability.®
The exact biochemical steps involved in the formation of
this intrinsic activator are not completely understood. The
activator activity generated by this pathway slowly converts
1. Plasminogen plasminogen to plasmin. The primary source of activators,
with molecular mass of however, is the vascular endothelium, the site of synthesis of
90 kD both endothelial cell urokinase, a single-chain precursor, and
2. Plasminogen activators: tissue plasminogen activator (t-PA). The former probably
Tissue plasminogen Endogenous activator
plays a minor role in in vivo fibrinolysis but is the primary
activator (t-PA) liberated from endothelial
cells by the action of activator within the genitourinary system. Single-chain uroki-
thrombin nase (scu-PA) is converted to its active form, two-chain uroki-
Factor XIla, kallikrein, Contact phase activator nase, by either plasmin or kallikrein and is rapidly inhibited
factor XIla fragments, generated by the initiation by plasminogen activator inhibitor-1 (PAI-1).
factor Xla of coagulation
t-PA is the primary plasminogen activator within the vas-
Single-chain urokinase Endogenous activator
synthesized in endothelial cular circulation. Its greater efficiency in thrombolytic ther-
cells apy and potential for pharmacologic manipulation have led to
Converted to two-chain its widespread use as a therapeutic fibrinolytic agent. This
active form by plasmin protein is probably the major physiologic activator of plas-
and kallikrein
minogen. t-PA is an endothelial cell product with a molecular
Streptokinase Bacterial cell product; forms
a complex with plasmino- mass of approximately 68 kD.
gen that has intrinsic Although a number of biochemical stimuli, including
activating activity histamine, vasopressin, bradykinin, and adrenaline can induce
. Plasminogen activator
ios) Endogenous inhibitor of t-PA release, thrombin, generated as a result of activation of
inhibitor-1 (PAI-1) tissue plasminogen the coagulation system, is the most important release inducer.
activator; synthesized and
: secreted by endothelial
Thrombin thus serves as a coagulant component (converting
cells and platelets fibrinogen to fibrin, aggregating platelets, causing platelet
4. Thrombin-activatable Plasma procarboxypeptidase factor V to relocate to the cell surface); as a stimulant to
fibrinolysis inhibitor B, activated by thrombin, dampen the coagulant process by binding to its endothelial
(TAFI) which suppresses cell receptor, thrombomodulin, thus allowing for the genera-
plasminogen binding to
fibrin tion of activated protein C; and, finally and paradoxically, as
5. Plasmin Active serine protease of an initiator of fibrinolysis by stimulating endothelial cells to
70-75 kD release tPA and uPA (see Fig. 27-1). It is now realized that
6. a,-Antiplasmin Primary inhibitor of plasmin; thrombin, in addition to causing endothelial cell release of
inhibitor forms an irreversible PAI-1, also acts to inhibit fibrinolysis through a second mech-
complex with plasmin
a,-Macroglobulin Serves as a plasmin inhibitor anism. Thrombin in complex with soluble thrombomodulin
only when q,-antiplasmin fragments converts procarboxypeptidase B, a plasma protein,
inhibitor binding sites are to its active form which proteolytically cleaves the plasmino-
saturated gen binding site (lysine residue) on fibrin and thus prevents
7. Fibrinogen, fibrin Plasmin substrates;
plasmin formation and progressive clot lysis. This function is
proteolytic cleavage
results in the generation
termed thrombin-activatable fibrinolysis inhibitor (TAFI).'°
of degradation products Activated protein C exerts a negative feedback control on
the coagulation process by proteolytically cleaving activated
coagulant factors Va and VIIa, thus limiting further clot
formation.'':'* This latter function of activated protein C is
Plasminogen Activators
accelerated by its formation of a complex with protein S,
The conversion of either form to active plasmin can be ini- which serves as a cofactor. t-PA has a high affinity for fibrin,
tiated through a variety of direct or indirect mechanisms.’ and its adsorption to fibrin clots greatly enhances plasminogen
The group of activating proteins is collectively known conversion to plasmin. Because of a high affinity of both the
as plasminogen activators. Regardless of the initiating plasminogen activator and plasminogen for fibrin rather than
mechanism, activation of plasminogen to yield plasmin pro- fibrinogen, the effect of this reaction is accentuated on the sur-
ceeds through the cleavage of the same arginine 560—valine face of and within the clot. Release of t-PA from endothelium
561 bond in the Glu and Lys forms of plasminogen. These is also responsive to a variety of other stimuli, including
activators are either endogenous or exogenous in origin. venous occlusion, strenuous exercise, and treatment with
Endogenous activators are serine protease present in the vasoactive drugs, such as the vasopressin derivative DDAVP
blood and a variety of other tissues, particularly the vascu- t-PA activity is increased several-fold under these conditions.'*
lar endothelium. With the initiation of the contact phase of Exogenous activators have been available for clinical use
coagulation (see Chap. 24, factor XIla, Xa fragments, for a number of years. One of these, urokinase, is synthesized
646 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

by the kidney in addition to the vascular endothelium, as pre- The earliest recognized component is fragment X,
viously mentioned, and is excreted in the urine.'* It can also be which is still capable of clotting. A recent finding has been
identified in vitro using kidney cell cultures and is a potent the identification of a small peptide fragment from the B-B
direct activator of plasminogen. Its major drawbacks are its chain of fibrinogen, which is released simultaneously with
expense and its relatively lower affinity for fibrin as compared the formation of the X fragment. Measurement by radioim-
with t-PA. A consequence of the latter property 1s that the plas- munoassay of the B-B 15-42 related peptide may prove of
min generated will not only digest fibrin but also circulating value in the documentation of early fibrinolytic states.*!*
fibrinogen and, therefore, the development of severe hypofib- The X fragment undergoes further plasmin attack to yield
rinogenemia is not uncommon with its use. The other exoge- unclottable Y and D fragments. The Y fragment is further
nous activator, streptokinase, is a product of beta (8)-hemolytic digested to yield an additional D fragment and a single
streptococci. It is not a serine protease and has no intrinsic pro- E fragment. It is now realized that the proteolytic cleavage
teolytic activity but is capable of forming a 1:1 stoichiometric of cross-linked fibrin (i.e., fibrin transaminated through the
complex with plasminogen. This interaction results in a confor- action of factor XIa and calcium) results in other interme-
mational change of the plasminogen molecule and exposure of diate degradation products (e.g., D2E without the genera-
its active serine site.'° The streptokinase—plasminogen complex tion of fragment D or E). This proteolytic product is
can then undergo autocatalysis to yield other activators namely, referred to as the D-dimer.
streptokinase-Glu-plasmin and streptokinase-Lys-plasmin. Any These breakdown products have specific inhibitory
of these forms will readily convert free plasminogen to plas- effects on the coagulation system and thereby suppress fur-
min. Because streptokinase is a bacterial protein, a major limi- ther clot formation. Fragment X is capable of clotting slowly
tation with its use in thrombolytic therapy is the induction of an and exerts an anticoagulant effect by competing with fib-
immune response, with resulting antibody development and an rinogen for thrombin. It also forms slowly polymerizing
inhibition of its activity. complexes with fibrin monomer and inhibits the polymer-
ization step. Fragment D forms abnormal complexes with
fibrin monomers as it polymerizes. Fragment E is not known
Plasminogen Activator Inhibitor-1 to have any specific anticoagulant effect. In high concentra-
tions (more than 100 pg/mL), the degradation products are
Plasminogen activator inhibitor-| is a member of the
capable of inhibiting platelet aggregation and release. Plas-
family of protease inhibitors that includes antithrombin-HI,
min also exerts a direct limiting effect on the coagulation
a,-macroglobulin, a,-antitrypsin, and a,-antiplasmin, and
process by being able to proteolytically cleave and render
that are collectively referred to as serpins (serine protease
inactive factors V and VII, in addition to proteolysing fac-
inhibitors). PAI-1 is a53-kD glycoprotein also synthesized by
tor XII and platelet glycoprotein Ib, the von Willebrand’s
vascular endothelium and released primarily in an inactive,
factor receptor.
latent state.'® It is an acute-phase reactant and can be induced
by a variety of stimuli, including interleukin-1 (IL-1), endo-
toxin, and thrombin. PAI-1 is the primary inhibitor of both Plasmin Inhibitors
tPA and uPA. Thus, regulation of fibrinolysis is dependent on
Although plasmin formation characteristically takes place
the interaction of t-PA and uPA with PAI-1. Under basal con-
in the area of fibrin deposition with little free plasmin cir-
ditions, most of the t-PA released is bound to PAI-1.
culating, this enzyme if unchecked by the presence of spe-
Excess levels of this inhibitor have been associated with
cific inhibitors, would result in circulating fibrinogen being
thrombotic disease. A common diallelic polymorphism has
digested and the blood being rendered unclottable. The pri-
been described for PAI-1 in which the prevalence of the 4G
mary physiologic inhibitor of plasmin in vivo is a,-plasmin
allele is significantly higher in patients with myocardial
inhibitor.* It rapidly binds to the lysine binding site on
infarction who are younger than 45 years old.'’? However,
plasmin in a 1:1 molar ratio in an irreversible manner. Mea-
other studies have failed to find excess PAI-1 levels to be
surement of these plasmin—a,-antiplasmin inhibitor com-
associated with an increased risk of thrombosis.'®
plexes has been suggested as an indicator for activation of
the fibrinolytic system. Plasmin adsorbed onto fibrin during
Plasmin the fibrinolytic process appears to be protected from this
inhibitor because it binds to fibrin through the same lysine
The pivotal serine protease generated through these complex binding site. Because this binding site on plasmin is
biochemical processes is plasmin. This protein has a molecu- occupied, the inhibitor cannot bind, and clot lysis can pro-
lar weight of 77 to 85 kD, depending on whether Lys-plasmin ceed. The overall effect is to ensure that plasmin activity is
or Glu-plasmin is formed, and has a transient plasma half-life limited to the area of fibrin deposition and to prevent free
measured in seconds.'? Plasmin has the ability to proteolyti- plasmin from circulating. Other protease inhibitors in plasma
cally degrade both fibrin in clots and native fibrinogen in the include a,-macroglobulin, C1 inactivator, and q,antitrypsin.
circulation into a series of well-characterized end products Of these, only a,-macroglobulin has a role in plasmin inhi-
collectively known as fibrin/fibrinogen degradation products bition during normal hemostasis, but it participates only
(FDPs). This process results in an asymmetric, progressive when q,-antiplasmin inhibitor binding sites for plasmin are
breakdown of fibrin and fibrinogen.”° saturated.
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 647

Thrombomodulin elevated TAFI levels are felt to increase the risk for recurrent
venous thromboembolism.” Acquired abnormalities of the
Thrombomodulin is an endothelial cell membrane glycopro- fibrinolytic system are much more common and are discussed
tein that is a receptor for thrombin and forms a 1:1 stoichio- in the next section on disseminated intravascular coagulation
metric complex with thrombin.** Thrombin, once bound to and related disorders.
thrombomodulin, no longer has proteolytic activity to convert In summary, an integrated system of serine proteases is
fibrinogen to fibrin but retains esterolytic function. The com- brought into play once the coagulation process is initiated in
plex serves to convert protein C to activated protein C after the response to disruption of blood vessel integrity (Fig. 27-2).
zymogen binds to endothelial cell protein C receptor (EPCR). The response is balanced so that the same reaction that initi-
The rate of this reaction is accelerated by the presence of free ated thrombin formation and fibrin deposition also initiates a
protein S complexed to the protein C. Activated protein C series of reactions to lyse the clot. Factor XIla, with other
(aPC) then dampens the coagulation process by proteolytically components of the contact phase of coagulation, converts plas-
cleaving factor Va and VIIa. The thrombin—-thombomodulin minogen to plasmin; thrombin stimulates endothelial cells to
complex also serves to convert procarboxypeptidase B to its release t-PA with subsequent plasmin generation. Excess t-PA
activated form, thrombin-activatable fibrinolysis inhibitor activity is controlled by the presence of plasminogen activator
(TAFI). The number of thrombomodulin sites on the vascular inhibitor. Because of the high affinity of plasmin for fibrin,
endothelium is down-regulated by proinflammatory cytokines most of these fibrinolytic processes are taking place at the site
including IL-6 and tissue necrosis factor (TNF) and by other of fibrin deposition within the damaged blood vessel. The
inflammatory modulators released from activated neutrophils presence of plasmin inhibitors further ensures that the prote-
during the inflammatory process. olytic process is limited to this area.

Thrombin-Activatable Fibrinolysis Inhibitor

Thrombin-activatable fibrinolysis inhibitor (TAFT) circulates in
plasma in an inactive form (zymogen) as procarboxypeptidase
B and is converted to its active form by thrombin in complex Endotheli
FTN eMUtERC PAIS RPA t-PA uPA
with thrombomodulin. Carboxypeptidases are enzymes that = pee eee

hydrolyze carboxypeptidase bonds. The protein is synthesized

in the liver and has a molecular mass of 53 to 60 kD. TAFI Plasminogen -"------- > Plasmin Plasminogen
inhibits fibrinolysis by hydrolyzing lysine (lysyl residues) from
the carboxy] end of fibrin. These same residues serve as binding Plasmin
«#2 <— (/o-antiplasmin
sites on the fibrin clot for plasminogen-tPA complex to initiate Oo-antiplasmin
fibrinogen/fibrin degradation.” complex
Fibrin clot

Congenital Abnormalities
Congenital abnormalities of the fibrinolytic system causing
either a hemorrhagic or thrombotic diathesis are rare.** Only
three cases of an abnormal plasminogen have been reported in Lysyl residue —_Lysyl residue

which the patients had a history of recurrent thrombotic GB) TAFI Plasmin
episodes. In essentially all other cases reported, patients have ><
I
been asymptomatic even though a hypercoagulable state v
would have been expected.”’ Low levels of t-PA activity have FDPs FDPs
been documented in two families and were associated with a D-dimers D-dimers

similar thrombotic tendency. Whether deficiencies of tPA or Figure 27-2 @ Control of fibrinolysis. Plasminogen activators tissue
uPA cause a hypercoagulable state is controversial. A recently plasminogen activator (t-PA) and, to a lesser degree, urokinase (u-PA)
described platelet disorder, Quebec platelet disorder, leading are synthesized in the endothelium. Both t-PA and u-PA are rapidly
to a bleeding diathesis associated with moderate to severe inhibited by activated plasminogen activator inhibitor-1 (PAI-1), also
synthesized in the endothelium. The interaction of t-PA with PAI-|
thrombocytopenia, has been found to be due to excess uPA
regulates fibrinolysis, and under basal conditions most t-PA released
content in the a granules of platelets.** As mentioned previ- is bound to PAI-1. Plasminogen-tPA binds to fibrin in clots generat-
ously, allelic polymorphism has now been documented for ing plasmin at the LYSYL (lysine) residue site and clot lysis occurs
PAI-1, with the presence of the 4G allele being associated resulting in the formation of fibrin degradation products (FDPs)
with premature coronary artery disease.'’ Deficiencies of including D-dimers. The plasmin inhibitor «,-antiplasmin binds to
free circulating plasmin at the same lysine binding site, thus limiting
a,-antiplasmin inhibitor have been reported in four families plasmin activity to the area of fibrin deposition. Excess levels of
to date and, in contrast, are associated with a severe hemor- thrombin activate the plasma protein thrombin activatable fibrinoly-
rhagic tendency. Deficiencies of TAFI have not been reported sis inhibitor (TAFI), which binds with fibrin at the plasminogen bind-
to be associated with a hemorrhagic diathesis, however, ing site, thus preventing plasmin formation and fibrinolysis.
648 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

Disseminated Intravascular Coagulation Triggering Mechanisms: Associated Clinical

Disorders
In brief, under normal physiologic conditions when there is
damage to the vascular endothelium, platelets adhere to the The diverse stimuli that are capable of triggering the coagula-
site of injury, undergo activation, and aggregate. This reaction tion cascade in this manner all act through one or more of
provides a rich phospholipid environment in which coagula- three mechanisms**:
tion can proceed. Under most circumstances, the initial coag-
1. Activation of the extrinsic coagulation pathway by the
ulant response involves endothelial cell tissue factor (TF)
release of tissue factor or tissue thromboplastin-like
complexing with factor VII or Vila to form TF—VHa. This
substance
complex then rapidly converts factor X to Xa and factor IX to
2. Direct activation of factors X or II
IXa. The TF—VIHa complex is short lived because it is quickly
3. Activation of the intrinsic pathway.
inhibited by the naturally occurring inhibitor tissue factor
pathway inhibitor (TFPI). The latter forms an inactive quater- Tissue factor release/expression is the primary initiator in
nary complex of TF—VIla—Xa—TFPI. Further coagulant most cases of DIC either due to tissue thromboplastin
response then depends on the formation of [Xa—VIIla through entry into the circulation, extensive injury to the vascular
the activation of the intrinsic pathway to convert more factor X endothelium exposing tissue factor in the subendothelium
to Xa resulting in a sustained burst of thrombin generation. or enhanced expression of tissue factor by monocytes in
With the generation of thrombin, fibrinogen is converted to response to endotoxin or cytokines (tissue necrosis factor
fibrin, factor XIII to its activated form to make the clot insol- [TNF], Il-1 and Il-6) secondary to sepsis involving both
uble, and further coagulant activity is limited by thrombin gram-negative and gram-positive organisms or inflamma-
converting protein C to its activated form. The latter prote- tion. In certain conditions, specifically obstetrical compli-
olytically cleaves any factor Va and VIIa present, resulting in cations or cranial injuries, release into the circulation of
a dampening of further coagulant response. Endothelial cells thromboplastin-like substances that have functional proper-
are stimulated by thrombin to release t-PA and single-chain ties similar or identical to tissue factor activate the extrin-
urokinase to initiate the fibrinolytic process. Excess t-PA and sic pathway and lead to DIC. Macrophage stimulation
single-chain urokinase are rapidly inhibited by PAI-1, and results in increased TNF being released with the subsequent
excess plasmin is bound to a,-antiplasmin (Fig. 27—3). Thus, down-regulation of thrombomodulin sites on the endothe-
further hemorrhage is prevented and vascular repair is initi- lial cells and impairment of fibrinolysis. The intrinsic sys-
ated to restore normal homeostasis.°°*! tem involvement is usually indirect and occurs secondary to
Consequently, when there is damage to a blood vessel, activation of the coagulation process by initiators of the
an ordered, integrated series of reactions involving the coag- extrinsic system. DIC caused by direct activation of factor
ulation, fibrinolytic, kinin, and complement systems occurs VII, as seen after massive injury or in certain obstetrical
on endothelial cells and platelets at the site of injury, as out- complications, results from release of tissue thromboplastin
lined in the previous chapters, with the initial formation and from the injured tissue or from amniotic fluid entering the
subsequent lysis of fibrin deposits. The initial formation of the circulation. Certain tumors, particularly mucinous adeno-
fibrin clot prevents further hemorrhage and initiates vascular carcinomas, are rich in thromboplastin-like material and
repair. The subsequent clot lysis serves to reestablish blood may act through the same mechanism. The coagulopathy
flow and vascular integrity. This process is normally self lim- seen with red cell lysis following mismatched blood trans-
ited and localized. Under certain pathologic stimuli, however, fusions may be caused by the release of thromboplastin-like
the coagulation response may be accentuated and the normal activity from the stroma of these cells; however, evidence
inhibitory mechanisms overwhelmed. Activation of the coag- suggests that immune complex formation may be the prime
ulation system under these circumstances causes consumption initiator of the coagulant response.
of the coagulation factors and platelets, with subsequent Direct activation of coagulation factors X and II can
thrombus formation not only at the site of endothelial dam- also occur in the presence of proteolytic enzymes. The
age, but in a random manner throughout the microcircula- venoms of certain snakes act through this mechanism (e.g.,
tion.** This hemorrhagic syndrome has been referred to as Russell’s viper venom activates factor X whereas venom
disseminated intravascular coagulation (DIC), defibrination from the sand rattlesnake causes direct conversion of pro-
syndrome, or consumptive coagulopathy. thrombin to thrombin). Certain malignancies have also been
Simultaneous with and secondary to the activation of reported to have TF or direct factor X activation by a cal-
the coagulation cascade, the fibrinolytic system is activated. cium-dependent cysteine protease, and these properties may
Regardless of the nature of the inciting stimulus, the patho- account for the DIC seen in these states.** Although the hem-
physiologic effect of this process will be reflective of the orrhagic disorder noted in acute promyelocytic leukemia
balance between fibrin deposition (action of thrombin) and (APL) is listed as secondary to DIC, evidence suggests that
fibrinolysis (action of plasmin). The clinical manifestations, the bleeding diathesis seen in these patients may also involve
thus, can be one of diffuse hemorrhage (Fig. 27-4), owing primary fibrinolysis in addition to DIC. Leukemic promyelo-
to depletion of platelets and coagulation factors, ischemic cytes are rich in annexin II, a surface phospholipid, which
tissue damage caused by vascular occlusion, or the occur- can bind t-PA and plasminogen. Conversion of the plasmino-
rence of both simultaneously in different areas of the gen to plasmin then initiates the fibrinolytic process without
microvasculature. thrombin generation.»
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 649

Subendothelium

Endothelium

Protein C
MMalceyonleyial

Tissue plasminogen activators

Activated protein C
(t-PA and u-PA)

Tissue factor (TF)

ae 5 Extrinsic Plasminogen
Activated 6) patnway
platelets

ile
Plasmin
Platelet © Thrombin (small amounts)
Platelet

Fibrinogen 44 Fibrin clot

Thrombin
(large amounts)
Fibrin degradation products
D-dimer
Activated
platelets

@
Intrinsic pathway

Figure 27-5 @ Integrated system of hemostasis. 7. Disruption of endothelial continuity releases tissue factor (TF). 2. In the presence of ion-
ized calcium, TF forms a complex with factor VII or Vila leading to the conversion of factor X to Xa and rapid generation of small amounts of
thrombin and fibrin formation via the extrinsic pathway. In addition, the TF-factor Vila-Xa complex is rapidly inhibited by the presence of
tissue factor pathway inhibitor (TFPI). 3. The Vila-TF complex also activates factor IX to IXa, initiating the intrinsic pathway and the formation
of larger amounts of thrombin on the surface of activated platelets. 4. Enhanced expression of TF by monocytes also occurs in response to
endotoxin or cytokines secondary to sepsis. 5. Thrombin stimulates platelet activation and the release of platelet agonists. 6. Activated
platelets undergo shape change, resulting in the exposure of phospholipids and cofactors V and VIII on the surface of the platelet membrane.
7. Secondarily, and simultaneously, thrombin complexes with thrombomodulin (TM) on the endothelial surface. 8. Protein C, once bound to
this complex, is rapidly converted to its activated state. 9. Activated protein C indirectly causes release of tissue plasminogen activator in cells
from the subendothelium, in addition to direct stimulation and release of this glycoprotein by thrombin. 70. Plasmin-induced proteolysis of
the fibrin clot results in the formation of fibrin degradation products.

Although activation of the intrinsic system can result in may be more complicated, with endotoxin inducing release of
DIC, this is an uncommon mechanism. The exact sequence of a number of cytokines, including tumor necrosis factor-alpha
intermediary events by which certain stimuli initiate coagula- (TNF-a) and IL-1, and the primary activation of coagulation
tion is well understood, but with other stimuli this process occurring through the extrinsic (tissue factor-dependent) path-
remains uncertain. All pathologic stimuli that result in activa- way rather than the intrinsic pathway.*° TNF-a is capable of
tion of the intrinsic system probably do so indirectly by means inducing tissue factor activity in monocytes and on both the
of first inducing endothelial cell damage with subsequent expo- luminal and subendothelial surfaces of endothelial cells. Tissue
sure of the subendothelium. Platelet adherence and aggregation factor can bind and activate factor VII; the complex of TF-VIla
and factor XII activation can then occur. This is the proposed can then activate factor X, with subsequent thrombin forma-
mechanism of DIC associated with anoxia and immune com- tion. Binding of granulocytes to the endothelial receptors
plex formation, however, activation through the extrinsic sys- (selectins) induces the release of granulocyte cathepsins and
tem may also play a role. New insight regarding sepsis now elastases, which then lead to organ damage. The participation
suggests that the mechanism of DIC induced by endotoxins, the of the intrinsic system of coagulation in the DIC process then
lipopolysaccharide constituents of gram-negative organisms, involves activation of the kinin system through the interaction
650 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

Clinical Presentation

To a great extent, the clinical presentation depends on which of

the proteolytic processes (coagulant or fibrinolytic) is dominant.
This allows for a wide spectrum ranging from an acute, severe
hemorrhagic disorder (acute DIC) to a low-grade disorder with
predominantly thrombotic manifestations or laboratory abnor-
malities only (chronic DIC or compensated DIC) without clini-
cal evidence of hemorrhage or thrombosis. A number of factors
are important in determining the final clinical picture, including
the magnitude and duration of the triggering stimulus; the func-
tional ability of reticuloendothelial system, particularly the liver,
to remove from circulation activated coagulation factors, fibrin
monomers, fibrin/fibrinogen products, as well as immune com-
plexes; the compensatory ability of the liver and the bone mar-
Figure 27-4 @ Diffuse hemorrhage, a clinical manifestation in a row to accelerate clotting factor and platelet production; and,
patient with disseminated intravascular coagulation (DIC). Note the finally, the extent to which any particular organ is involved with
multiple cutaneous ecchymoses. hemorrhage or thrombus.®!

of factor XIla and prekallekrein, with high-molecular-weight

kininogen being converted to kinins. Kinins can then cause Laboratory Diagnosis
decreased vascular tone, increased vascular permeability,
To confirm a laboratory diagnosis of DIC, it has been sug-
hypotension, and, if severe, lead to shock.*! A list of clinical
gested that there must be confirmation of procoagulant and
conditions associated with these triggering mechanisms is
fibrinolytic component activation, evidence of inhibitor
shown in Table 27-2.
consumption, and biochemical proof of end-organ damage
or failure.** The laboratory findings in patients with DIC
“Table27-2Clinical Conditions reflect the direct or indirect effects of excess thrombin and
re - Associated with plasmin generation (Table 27—3). The constellation of labo-
ratory abnormalities in any particular patient, however, will
_ Disseminated
depend on the nature, magnitude, and duration of the trig-
Intravascular gering stimulus, the compensatory capacity available, and
Coagulation the underlying disease state. Although the ultimate confir-
matory test would be the direct demonstration of fibrin
Thromboplastin Release—Factor VII Activation
Placental abruption deposition in biopsy material from an involved blood ves-
Trauma sel, this is not practical. As a result, a multitude of tests
Fat emboli syndrome have been utilized by various laboratories to make this
Mucin-secreting adenocarcinoma diagnosis.*?"’
Sepsis*
It should be pointed out that no single test is diagnostic of
Promyelocytic leukemia
Retained dead fetus syndrome DIC; however, in the appropriate clinical setting (patient history
Acute intravascular hemolysis* and the type of bleeding and/or thrombosis) a battery of tests can
Amniotic fluid embolus* ensure a diagnosis of DIC. Indirect tests that lack specificity for
Cardiopulmonary bypass surgery thrombin action include the prothrombin (PT), activated partial
Endothelial Cell Damage-TF Release thromboplastin (APTT), and thrombin/reptilase clotting times;
Immune complex disease however, these tests are not always abnormal either individually
Intravascular hemolysis* or as a group if the process is chronic and the liver and bone
Liver disease* marrow can compensate. Utilization and degradation of the clot-
Heat stroke
ting factors in the DIC process result in prolongation of each of
Sepsis*
Burns these global tests. Because platelets are also consumed during
Vasculitis the coagulation process and their contents released, the finding
Anoxia of thrombocytopenia and elevated plasma levels of the platelet-
Acidosis specific proteins B-thromboglobulin and platelet factor 4 would
Factor X/II Activation be expected. The plasma inhibitor, antithrombin III, will
Snake venoms complex with thrombin and activated factor X and result in
Acute pancreatitis diminished plasma levels of antithrombin III. The presence of
Liver disease* thrombin-antithrombin III complexes formed can also be deter-
Fat emboli syndrome*
mined using an enzyme-linked immunosorbent assay (ELISA)
*More than one mechanism may be involved. system. If the fibrin deposition does not completely occlude
the lumen of the damaged blood vessel, red cells may undergo
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 65]

Excess Protease Effect Laboratory Tests

Fibrinogen utilization Fibrinogen concentration, thrombin/reptilase

time, prothrombin and activated partial
thromboplastin times
Utilization/degradation of other Prothrombin and activated partial
coagulation factors thromboplastin times, coagulation factor
assays
Fibrin monomer generation Soluble fibrin monomer complexes
Fibrinopeptide A/B release Fibrinopeptides A and B
Platelet aggregation/release Platelet count, B-thromboglobulin, and
platelet factor 4
AT-II complex formation Thrombin-antithrombin II complexes,
AT-III level
Thrombin/plasmin Proteolysis of fibrinogen/fibrin Fibrin degradation products, D-dimer
concentration, B-B 15-42 peptide assay
Release/activate Tissue plasminogen
Plasminogen activators and inhibitors Activator level, plasminogen activator
inhibitor level
Soluble fibrin monomer/fibrinogen Soluble fibrin monomer complexes
FDP complexes
Plasmin _ Plasminogen utilization Plasminogen concentration
- Proteolysis of fibrinogen/fibrin FDPs, thrombin/reptilase times, platelet
aggregation and release tests
Complexes with inhibitor a>-Plasmin, inhibitor concentration,
a,-plasmin inhibitor-plasmin complexes
Proteolysis of factors V/VIII Factor assays

a shearing effect as they traverse this area, with resultant Specific tests for direct evidence of thrombin activity
fragmentation and the development of a microangiopathic relate to the action of thrombin on fibrinogen. Other than cer-
hemolytic anemia (Fig. 27—5) or the lumen may be completely tain snake venoms, thrombin is the only enzyme that releases
occluded by the thrombus negating the formation of fragmented the specific peptides fibrinopeptide A (FPA) and B from the
RBCs in the circulation (Fig. 27-6). fibrinogen molecule. Both ELISA and radioimmunoassays
Conversion of prothrombin to thrombin is the result of are available commercially to measure each fibrinopeptide.**
cleavage from the parent molecule of a small, inactive peptide The major drawback to measuring these peptides, paradoxi-
referred to as prothrombin fragment F1.2. Measurement of cally, is the extreme sensitivity of the assay such that elevated
this activation peptide has thus far been utilized primarily for
diagnosis of a hypercoagulable state; however, it may have
equal utility as a molecular marker for DIC.

Figure 27-6 ® DIC (skin biopsy). Note both partial (small arrow)
Figure 27-5 m DIC (peripheral blood). Note presence of schisto- and complete (/arge arrow) occlusion of blood vessels by RBC/
cytes (arrows) and nucleated red cell (top border). fibrin clot.
652 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

levels can be seen in clinical conditions in which thrombin is tory Tests e a

ee
Table D7ek Labora
only transiently generated. As a consequence of fibrinopeptide
release, soluble fibrin monomers are formed that are capable a

of forming complexes with intact fibrinogen molecules or with

Acute Chronic
fibrin/fibrinogen degradation products. In our laboratory, we
(Decompensated) (Compensated)
are currently measuring soluble fibrin monomer complex for-
mation using a commercially available qualitative hemagglu- Routine Test
tination assay of human erythrocytes coated with fibrin Prothrombin time I N
Activated partial I N/D
monomers. Positive tests are indirect indications that thrombin
thromboplastin
was generated; therefore, the coagulation system had to have Thrombin time/ I N/I
been activated. reptilase time
Tests for the secondary activation of the fibrinolytic Fibrinogen D N
system in DIC are primarily directed at demonstrating the Platelet count D N/D
action of plasmin on fibrin/fibrinogen. As has already been D-Dimer/ I N/I
fibrin(ogen)
mentioned, a series of cleavage products are formed, the
degradation
fibrin/fibrinogen degradation products. The anticoagulant products
action of these fragments has been noted in the previous Euglobulin N/I/D N/D
section. lysis test
A number of immunologic tests are available to mea- Soluble fibrin IP P/Ng
monomer
sure one or more of the fibrinogen fragments and can yield
complexes
quantitative information on the degree of fibrinolysis. Antithrombin II N/D
Because of its ease of performance and specificity (..e.,
evidenge of plasmin action on cross-linked fibrin, therefore Special Test
Coagulation factor D N/D
coagulation has occurred), the D-dimer latex microparticle
levels
agglutination assays are the immunologic assays of choice Fibrinopeptide A I I
and automated techniques are now available to quantitate Plasminogen D N/D
levels. Direct measurement of the plasminogen concentra- Plasmin-a,-plasmin I N/I
tion in plasma can also be performed, and commercial 8-Thromboglobulin/ I I
platelet factor 4
assays are available.
levels
An indication of increased plasminogen activator activ- Thrombin— I I
ity seen in early stages of DIC can be obtained by performing antithrombin III
a euglobulin lysis time. The euglobulin fraction of plasma complexes
contains plasminogen, plasminogen activator, plasmin, and Prothrombin I I
fragment F1.2
fibrinogen. The rapidity of lysis of the fibrin clot is directly
related to plasminogen activator levels. The sensitivity of this I = increased; D = decreased; N = normal; P = positive;
global assay, however, is limited. As discussed, it is now pos- Ng = negative.
sible to quantitate each component directly with commer-
cially available kits that have largely replaced the euglobulin
lysis time. Assays for a,-antiplasmin inhibitor levels and for with the rate of destruction. Because of this balance the PT,
circulating plasmin—a,-antiplasmin inhibitor complexes are APTT, thrombin time, and platelet count are usually normal
available for clinical use. or only mildly abnormal. Confirmation of DIC is then based
Laboratory tests available to diagnose DIC and the con- on finding evidence of coagulation activation peptides (FPA,
stellation of results one can find in this syndrome, depending prothrombin fragment 1.2), complexes of activated coagulant/
on the balance between thrombin and plasmin activities and fibrinolytic components (thrombin—antithrombin HI, plasmin—
the compensatory capacity of the patient, are summarized in a,-antiplasmin) in addition to increased fibrin(ogen) degrada-
Table 27-4. Three generalized clinical states of DIC, and the tion products and elevated B-thromboglobulin or platelet
typical laboratory abnormalities associated with each, are factor 4 (PF4) levels.
described in the table. The hypercoagulable state is the result of excess thrombin
The acute (decompensated) DIC state refers to a condi- present in the plasma, with a delayed or lessened plasmin
tion in which active hemorrhage is evident and in which the response. In this condition, evidence of coagulation activation
consumption of the coagulation factors and platelets exceeds is apparent (increased levels of FPA, thrombin-antithrombin III
the capacity to increase the synthesis of these components. In complexes, prothrombin fragment 1.2, 8-thromboglobulin),
the chronic (compensated) state, laboratory evidence of an but all fibrinolytic activation markers are absent or minimally
accelerated coagulation and fibrinolytic process is evident increased. A characteristic finding in this form of DIC is a
(increased FPA, soluble fibrin monomer complexes, increased shortened APTT. It should be realized that these clinical states
8-thromboglobulin, increased FDPs or D-dimer levels, pres- are not static and it is not unusual for one to evolve into one of
ence of plasmin—a,-antiplasmin inhibitor complexes) but the the others, depending on the nature of the underlying disease
rate of synthesis of the coagulation components is balanced process and the response to therapy.
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 653

Therapy thrombosis, especially of small vessels, is the process that

most affects morbidity and mortality, heparin should theoret-
Therapy for DIC is essentially twofold: treatment or removal ically be indicated to slow or stop the coagulation process by
of the underlying pathologic stimulus, and maintenance of complexing with antithrombin III to inhibit thrombin or Xa.
blood volume and hemostatic function.*?*? Dramatic improve- This ameliorating effect has been noted with the use of sub-
ment in the patient’s clinical status with abrupt cessation of cutaneous low-dose heparin therapy in mild to moderate
bleeding and normalization of the coagulation abnormalities DIC.*° Its use can result in increased bleeding, and because
can be seen in certain cases of DIC with removal of the under- heparin itself affects a number of coagulation tests, it is often
lying pathologic stimulus alone (e.g., DIC associated with difficult to monitor the effect of conventional therapy.
retained dead fetus). In cases of DIC associated with sep- Heparin should be utilized and is most effective in cases of
ticemia, appropriate antibiotic therapy is imperative to control DIC that present with clinical evidence of a hypercoagulable
the pathologic process (i.e., bacterial or endotoxin-induced state with evident vascular thrombosis.
vascular damage). Because the inflammatory and coagulation systems are
Blood component replacement therapy with transfusion intricately involved in the response to sepsis-induced DIC
of packed red blood cells, fresh frozen plasma, and platelets with multiorgan failure, an additional therapeutic agent that
‘ to maintain blood volume and to support hemostatic function has been used in this group of patients is recombinant acti-
is indicated in patients with active bleeding or those whose vated protein C. A reduced mortality and improvement in
compensatory capacity is limited. In addition to fresh frozen organ function compared to control subjects has been
plasma, cryoprecipitate (enriched in fibrinogen, factor VIII, reported.*!
and fibronectin) and prothrombin complex (enriched in When major peripheral vessels are occluded as part of
vitamin K-—dependent clotting factors) are often used as the hypercoagulable process, the use of fibrinolytic agents
supplemental sources of blood component therapy. (recombinant t-PA, streptokinase, or urokinase) may be indi-
The administration of heparin in DIC has been advo- cated as an initial management choice with subsequent
cated by a number of investigators, but its use is still contro- heparinization, but clinical experience with this form of ther-
versial.*°** On the premise that the underlying pathologic apy is minimal. The previously described profile of DIC is
basis for DIC is generation of excess thrombin, and that summarized in Table 27-5.

le
27-5
tab Profile of DIC
Conditions Suggested Clinical
Associated Triggering Clinical Laboratory Sequential
Synonyms with DIC Mechanisms Manifestations Findings Therapy

1. Consumptive Obstetric Amniotic fluid, 1. General signs: Hypofibrinogenemia 1. Remove or

coagulopathy accidents which possesses significant treat triggering
thromboplastic hemorrhaging process
activity (usually from
3 unrealted sites:
melena and
hematemesis,
epistaxis, or
hemoptysis)
fever, hypotension,
acidosis, hypoxia,
proteinuria,
hematuria
2. Defibrination Intravascular Abnormal PT
syndrome hemolysis
Septicemia Retained fetus, Abnormal PTT 2. Stop or slow
which possesses coagulation
thromboplastic process:
activity a. Miniheparin
b. Heparin
c. Antiplatelet
drugs
d. AT-HI
concentrates
Viremia Abnormal
(varicella) thrombin time
continued
654 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

Table 27-5 Profile of DIC—cont’d

Conditions Suggested Clinical

Associated Triggering Clinical Laboratory Sequential
Synonyms with DIC Mechanisms Manifestations Findings seatl
Therapy. bin

Leukemias: Byproduct of red Abnormal platelets

cell hemolysis count
(phospholipid)
Acute Abnormal
tourniquet test
Promyelocytic Antigen/antibody Abnormal clot 3. Blood
complexes retraction component
replacement
a. Platelets
b. Cryopre-
cipitate
c. Prothrombin
complex
Other Endotoxin release 2. Specific signs:
Petechiae,
purpura,
gangrene,
wound bleeding,
venipuncture
‘ bleeding,
subcutaneous
hematomas
Solid Chronic stasis Abnormal factors
malignancy Vand VII
Acidosis Complement Positive fibrin(ogen)-
alkalosis activation split products
Burns
Crush injury Positive protamine 4. Antifibrinolytic
and tissue sulfate test therapy*
necrosis a. Epsilon
aminocaproic
acid (EACA)
Vascular 3. Microthrombi Positive ethanol
disorders gelatin test
4. End-organ
dysfunction AT-II consumption
Leukocytosis
Schistocytosis
Thrombocytopenia
Reticulocytosis

*Sequential therapy is used only after clotting is stopped (3% of patients may require this therapy).
PT 5 prothrombin time; PTT 5 partial thromboplastin time; AT-HI 5 antithrombin HI.

Related Disorders urologic procedures. The fibrinolytic state seen with metasta-
tic prostatic carcinoma is another example of this mechanism.
Primary fibrinolysis is an unusual situation in which plasmin The basis for the hemorrhagic state seen after cardiopul-
is formed in the absence of activation of the coagulation cas- monary bypass surgery is complex, with platelet dysfunction
cade. The clinical presentation in this disorder is similar to and hemodilution of coagulant proteins as the primary
that in DIC, with diffuse hemorrhage occurring as a result of defects; activation of the plasminogen—plasmin system with
increased plasma fibrinolytic activity. Several mechanisms increased fibrinolytic activity is also well documented.
can initiate this process. The presence of proteolytic enzymes The failure of the hepatic clearance mechanism to remove
in plasma that are capable of either directly or indirectly con- plasminogen activator accounts for the increased fibrinolytic
verting plasminogen into plasmin can occur in certain disease activity seen in a variety of hepatic disorders, particularly cir-
states. The genitourinary system is enriched in urokinases, rhosis. Under normal circumstances the hepatic reticuloen-
which can enter the systemic circulation following various dothelial system removes not only activated clotting proteins,
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 655

but also plasminogen activator from the systemic circulation. Thrombotic thrombocytopenic purpura is the syndrome
When this function is impaired because of hepatic disease or in in which fibrin and platelet thrombi are formed diffusely
patients who have portocaval shunting procedures, the removal throughout the microvasculature, in contrast to the localized
of plasminogen activator is less than adequate and hyperplas- thrombus formation seen in DIC.*° The clinical picture con-
minemia occurs, with resultant hemorrhage. Increased fibri- sists of a pentad of findings:
nolytic activity can also be seen in patients undergoing liver
lhever
transplantation, especially during the reperfusion phase follow-
2. Microangiopathic hemolytic anemia
ing reanastomosis of vessels.
3. Thrombocytopenia
The occurrence of DIC with secondary fibrinolysis is well
4. Azotemia
documented in patients with acute promyelocytic leukemia,
5. Vacillating neurologic deficits.
although as previously discussed, this disorder clearly causes
primary fibrinolysis through surface membrane annexin II bind- Despite fibrin and platelet deposition, this disorder is not typ-
ing of t-PA and plasminogen. It is now recognized, however, ically associated with excessive activation of the coagulation
that the coagulopathy these patients develop may also result system. The pathologic mechanism in this disorder is
from a primary fibrinolysis. The mechanism(s) is unsettled but either a hereditary/familial deficiency of von Willebrand
may involve direct activation by the leukemic cells with release cleaving enzyme (ADAMTS-13) in endothelial cells or the
of a urokinase-type or tissue-type plasminogen activator. development of an acquired IgG inhibitory antibody to the
The coagulation abnormalities seen in these fibrinolytic enzyme that results in the secretion and circulation of ultra-
disorders are similar to those in DIC, with prolonged PT, large high-molecular-weight von Willebrand factor multimers
APTT, and thrombin times. These defects result from the that spontaneously bind to circulating platelets and form
hypofibrinogenemic state induced by the proteolytic cleavage occlusive platelet thrombi in the vasculature.’ An abnormal-
of fibrinogen by excess plasmin, in addition to the catabolic ity of the fibrinolytic system can also be present in some
effect of this enzyme on factors V and VII. FDP concentra- patients with diminished or absent fibrinolytic activity, partic-
tions are increased and, as previously noted, will further ularly of t-PA, in plasma and blood vessels affected with
interfere with coagulation by acting as antithrombins and microthrombi. It is likely these are secondary changes. Ther-
inhibitors of fibrin polymerization. With the excess plasmin apy has not been standardized, but antiplatelet drugs (e.g.,
activity, the euglobulih lysis time is typically shortened. aspirin or dipyridamole), plasmapheresis, and exchange trans-
Because thrombin is not generated during this pathologic fusion have been used either singularly or in combination
process those laboratory finding that are the direct result of with variable success (see Chap. 25). The other causes of fib-
thrombin activity will be absent. Several laboratory tests can rinolytic activation are summarized in Table 27-7.
serve to readily distinguish primary fibrinolysis from DIC. In
primary fibrinolysis, the platelet count is typically normal;
fibrinopeptides A and B levels are not elevated, and circulat-
ing fibrin-monomer complexes and elevated D-dimer levels
are absent in contrast to the results in DIC (Table 27-6).

Primary
Laboratory Test Fibrinolysis

Prothrombin time
Activated partial
thromboplastin time
Thrombin time
Fibrinogen
Platelet count
Fibrinopeptide A
SFMC
TAT
FDPs
D-Dimers

SFMC = soluble fib rin monomer complexes; TAT = thrombin-

antithrombin complexes; FDPs = fibrin/fibrinogen degradation
products.
656 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

Table 27-7 Other Causes of Fibrinolytic Activation

Clinical Other Hemostatic

Clinical Condition Mechanism Manifestation Alterations

1. Chronic liver disease Abnormal fibrinolytic Often fulminant hemorrhage ite Hypofibrinogenemia
inhibitor (a@,-macroglobulin) with massive hemoptysis, (due to lysis)
hematachezia, melena, or
epistaxis
Abnormal hepatic clearance May also demonstrate . Elevated FDP (X, Y, D,
of plasminogen activators petechiae, purpura, spider and E) a. Defective fibrin
telangiectasia, ecchymoses monomer/polymerization
b. Platelet dysfunction
. Proteolysis of factors V,
VU, [X, XI
. Platelet defects
a. Thrombocytopenia
b. Platelet dysfunction
(EDPAPRE3)
. Coagulation protein defects:
a. Decreased synthesis of
factors II, VII, IX, and X
b. Decreased synthesis of
Fletcher factor
c. Decreased or dysfunc-
tional synthesis of AT-HI
2. Cardiopulmonary Unclear; possibly direct Hemorrhage; hematuria, . Hyperfibrinolysis results in
bypass (CPB) activation of fibrinolysis petechiae/purpura, a. Elevated FDP
by the oxygenation system and oozing from b. Hypofibrinogenemia
of pump-induced acceler- intravenous site in c. Low levels of cofactors V
ated flow rates may after conjunction with and VIII
endothelial plasminogen increased chest tube loss . Functional platelet defect
a. CPB-induced
b. Drug-induced
. Thrombocytopenia
. Hyperheparinemia-heparin
rebound(?)
. DIC(?)
3. Malignancy Poorly understood; in Thrombosis/hemorrhage . Thrombocytopenia
several instances tumor . Platelet function defects
extracts possess the . Elevated FDP
ability to activate directly . Decreased AT-III
or indirectly the fibrinolytic — . DIC(?)
MN
ABWN

system (e.g., gastric

carcinoma, sarcomas, and
prostatic carcinoma)
4. Acute promyelocytic Release of urokinase-type Severe hemorrhage . Hypofibrinogenemia (due
leukemia and tissue-type plasminogen to fibrin (ogen) lysis
activators from leukemia . Marked elevation of FDP
cells (X,Y, D, and E)
a. Defective fibrin
monomer/polymerization
b. Platelet dysfunction
oS). Proteolysis of factors V,

VU, XI, XII

. Decreased plasminogen
. Plasmin-a,-antiplasmin
complexes
FDP = fibrin/fibrinogen degradation products; PF3 = platelet factor 3; AT-II I = antithrombin II.
 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology 657

A diagnosis of DIC was made based on the patient’s clin-

ical presentation and the supportive laboratory data. The
patient was placed on intravenous fluids to maintain her
A 32-year-old white woman, gravida 3, para 2, in her blood pressure and was given 2 units of fresh frozen plasma
36th week of gestation noted the sudden onset of lower and 10 units of platelets. She was taken to the operating
abdominal pain and profuse vaginal bleeding. She was room and underwent a caesarean section. Her bleeding
rushed to the emergency department. On examination she abated postoperatively and all coagulation parameters
was noted to be hypotensive, with a blood pressure of 70/40 returned to normal within 36 hours.
and marked tachycardia. Large ecchymoses and continuous This case is illustrative of an obstetric complication, pla-
oozing of blood from venipuncture sites were evident. A cental abruption, which resulted in an acute DIC syndrome.
fetal heart tone was barely audible. Births of her other chil- Several triggering mechanisms have been postulated as an
dren were uncomplicated, and the family history was nega- explanation for the underlying coagulopathy in this disorder,
tive for a hemorrhagic diathesis. including the release of thromboplastin-like material from
Initial coagulation studies revealed PT of 26 seconds the amniotic fluid and tissue necrosis in the area of the
(normal is 11 to 13 seconds); APTT of 84 seconds (normal retroperitoneal hemorrhage. The laboratory parameters are
is 24 to 30 seconds); platelet count of 20,000/uL (normal is consistent with a consumptive coagulopathy and secondary
150,000 to 400,000/uL); fibrinogen, 85 mg/dL (normal fibrinolysis. With the delivery and removal of the placenta,
is 145 to 350 mg/dL); FDPs, greater than 40 ug/mL (normal the source of the triggering mechanism was removed and
is less than 10 ug/mL); and protamine sulfate test, positive the pathologic process stopped. Restoration of normal
(normal is negative). A blood smear showed numerous red hemostatic parameters occurs within hours postoperatively
cell fragments present. and usually no further blood component replacement ther-
continued apy is required.

Ore sti cas

1. D-Dimer formation is the result of the action of plas- 6. The primary tissue source for tissue plasminogen activa-
min on: tor (tPA) is:
a. Fibrin monomer a. Neutrophils
b. Fibrinogen b. Mast cells
c. Cross-linked fibrin c. Endothelial cells
d. FDPs d. Pluripotential stem cells
N . Inactivation of factors Va and VIIla is caused by: 7. DIC associated with malignancy can be due to:
a. Thrombin a. Tumor tissue factor-like activity
b. Protein S b. Factor X activation by tumor cysteine protease activity
c. t-PA c. Endothelial cell disruption by tumor invasion
d. Activated protein C d. All of the above
3. In primary fibrinolysis, which of the following labora- 8. Prothrombin fragment F1.2 is formed by the action of
tory tests will be abnormal? which protease action on prothrombin:
a. Platelet count a. Plasmin
b. D-Dimer level b. TAFI
c. Fibrinopeptide A level c. Factor Vila
d. Thrombin time d. Factor Xa
4. In DIC presenting clinically as a hypercoagulable state, 9. Clinical conditions associated with DIC include:
it is not unusual for which of the following coagulation a. Snake bites
times to be paradoxically shortened? b. Sepsis
a. Reptilase time c. Acute promyelocytic leukemia
b. Euglobulin lysis time d. All of the above
cCTAPTT 10. Which of the following laboratory tests is diagnostic
d. Thrombin time of DIC:
5. The primary inhibitor of the fibrinolytic system 1s: a. Prolonged thrombin time
a. Antithrombin III b. Elevated D-dimers
b. a,-Antiplasmin c. Hypofibrinogenemia
c. Protein C d. None of the above
d. a,-Macroglobulin
See answers at the back of this book.
65 8 Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; DIC; and Related Pathology

mw Triggering mechanisms of DIC may include activation of

the extrinsic coagulation pathway by release of tissue
thromboplastin, direct activation of factor X or II by cys-
= Normal hemostasis is the result of the balanced interac- teine protease and occasionally activation of the intrinsic
tion of the vascular endothelium and platelets with four system by thromboplastins.
biochemical systems: coagulation, fibrinolytic, kinin, and
complement systems. = Acute DIC (decompensated) state refers to a condition in
which active hemorrhage is evident and in which the
g The molecular components of the fibrinolytic system con- consumption of the coagulation factors and_ platelets
sist of plasminogen, plasmin, plasminogen activators, exceeds the capacity to increase the synthesis of these
plasmin inhibitors, thrombomodulin, thrombin-activatable components.
fibrinolysis inhibitor and fibrin(ogen).
wg Chronic DIC (compensated) state refers to laboratory
= Native plasminogen is a single-chain plasma zymogen of evidence of accelerated coagulation and fibrinolysis; how-
approximately 90 kD that circulates in a lysine and glu- ever, the rate of synthesis of the coagulation components
tamic acid form. is balanced with the rate of destruction.
w Plasminogen activators include tissue plasminogen acti- m The hypercoagulable state of DIC is the result of
vator (t-PA), urokinase, and streptokinase. excess thrombin present in the plasma, with a delayed or
g Thrombin complexed with soluble and cell-bound throm- weakened plasmin response.
bomodulin activates procarboxypeptidase B, which binds m Therapy for DIC is aimed at treatment or removal of the
to the plasminogen binding site on fibrin, preventing plas- underlying pathologic stimulus and maintenance of blood
min formation; this is known as thrombin-activatable volume and hemostatic function.
fibrinolysis inhibitor. é
w Primary fibrinolysis is a condition in which plasmin is
m Plasmin is a serine protease that has the ability to degrade formed in the absence of coagulation processes; pro-
fibrin in clots and native fibrinogen in circulation into a thrombin time (PT), activated partial thromboplastin time
series of well-characterized end products known as (APTT), thrombin time, and FDP are increased, and
fibrin/fibrinogen degradation products (FDPs). euglobulin lysis time is shortened.
m The primary physiologic inhibitor of plasmin in vivo is m= The FDP fragment X is capable of clotting and exerts an
a,-antiplasmin; it binds to the lysine binding site on plas- anticoagulant effect by competing with fibrinogen for
min in a 1:1 molar ratio in an irreversible manner.
thrombin; fragments D and Y are unclottable. D-Dimer
g Disseminated intravascular coagulation (DIC) occurs when represents a specific proteolytic product of fibrin degrada-
the coagulation response has been accentuated and the tion by plasmin.
normal inhibitory mechanisms are overwhelmed and @ Clinical conditions associated with DIC may include pla-
cannot stop thrombus formation; coagulation factors and cental abruption, trauma, sepsis, promyelocytic leukemia,
platelets are consumed with subsequent thrombus forma- cardiopulmonary bypass, immune complex disease,
tion throughout the microcirculation. burns, anoxia, liver disease, and snake bites.

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Henry, JB (ed): Clinical Diagnosis and properties of an endothelial cell cofactor
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ting, kinin forming, complement and fib-
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. Sundsmo, JS, and Fan, DS: Relation-
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of fibrinolytic activity by the interaction 1984.
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Springer Semin Immunopathol 6:231,
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1983.
9. Bachman, F, and Kruithof, IEKO: Tissue Haemorrh 34:386, 1975.
. Rosenberg, RD, and Aird, WC: Vascular-
plasminogen activator: Chemical and . Brogden, RN, et al: Streptokinase: A
bed-specific hemostasis and hypercoagu-
physiological aspects. Semin Thromb review of its clinical pharmacology,
lable states. N Eng J Med 340:994,
Hemost 10:6, 1984. mechanism of action and therapeutic
1999.
. Hosaka, Y, et al: Thrombomodulin in uses. Drugs 5:357, 1973.
. Wu, KK, and Thiagarajan, P: Role of
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endothelium in thrombosis and hemosta-
fibrinolysis through acceleration of of plasminogen activator inhibitor-1
sis. Annu Rev Med 47:315, 1996.
thrombin-dependent activation of plasma activity. Semin Thromb Hemost 20:319,
. Michiels, C: Endothelial cell functions.
procarboxypeptidase. Thromb Haemost 1994.
J Cell Physiol 196:430, 2003.
DEST, USS. . Vaughn D: Update on plasminogen acti-
6 . Castellino, FJ: Recent advances in the
. Owen, WG: The control of hemostasis. vator inhibitor-1. Can J Cardiol.
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Arch Pathol Lab Med 106:209, 1982. 14(Suppl D):14, 1998.
Chem Rev 81:431, 1981.
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18. Juhan-Vague, I et al.: Fibrinolytic factors 2 Demarmeis Biasiutti, F, et al: Is plas- 3 Menell, J, et al: Annexin II and bleeding
and the risk of myocardial infarction or minogen deficiency a risk factor? A in acute promyelocytic leukaemia.
sudden death in patients with angina study on 23 thrombophilic patients and N Engl J Med 340:994, 1999.
pectoris. ECAT Study Group. European their family members. Thromb Haemost 36. Dempfle, CE: Coagulopathy of sepsis.
Concerted Action on Thrombosis and 80:167, 1998. Throm Haemost 91:213, 2004.
Disabilities. Circulation 94:2057, 1996. 28. Kahr, WH, et al.: Platelets from patients . Fareed, J, et al: Impact of automation on
. Gonzalez-Gronow, M, et al: Purification with Quebec platelet disorder contain the quantitation of low molecular weight
and some properties of the glu-and lys- and secrete abnormal amounts of markers of hemostatic defects. Semin
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@hemZa2-2Wsn 977: Blood 104:159, 2004. . Hirsh, J: Blood tests for the diagnosis of
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Oe Kudryk, B, et al: Measurement in human nate intravascular coagulation: Clinical 40. Sakuragawa, N, et al: Clinical evaluation
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taining the B-beta 15—42 sequence. SOS), WON). ((FR-860) on disseminated intravascular
Thromb Res 25:277, 1982. . Bick, RL: Disseminated intravascular coagulation—a multicenter co-operative
. Ockelford, A, and Carter, J: DIC: Appli- coagulation: Pathophysiological mecha- double-blind trial in comparison with
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Semin Thromb Hemost 8:198, 1982. Thromb Hemost 24:3, 1998. 4]. Vincent, JL, et al: Effects of drotrecogin
. Aoki, N, and Harpel, PC: Inhibitors of . Bick, RL: Disseminated intravascular alfa (activated) in organ dysfunction in
the fibrinolytic enzyme system. Semin coagulation: Objective criteria for clini- the Prowess Trial. Crit Care Med 31:834,
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Med Clin N Am 56:163, 1972. NOOTe
 Chapter

Introduction
to Thrombosis
and Anticoagulant
Therapy
Aamir Ehsan, MD
Jennifer L. Herrick, MD

Introduction OBJECTIVES
Regulation of Coagulation At the end of this chapter, the learner should be able to:
andFibrinolysis
The Role of Endothelium iD Name natural anticoagulants and inhibitors present in plasma.
Platelets . Describe the role of endothelium in thrombogenesis.
Procoagulant Factors and
Generation of Thrombin . Describe the mechanism of thrombin/thrombomodulin.
Natural Inhibitors of . Compare and contrast the protein C and protein S systems.
Coagulation Factors
(Plasma Components) . List the inherited causes of thrombophilia in order of frequency of occurrence.
Fibrinolytic System . Name the risk factors and acquired conditions with hypercoagulable states.
Inherited Thrombophilia
. List the laboratory tests to evaluate patients with hypercoagulable states.
Activated Protein C
Resistance . Describe the issues to consider in laboratory testing in patients with thrombosis.
Protein C Deficiency
. List laboratory tests for evaluating a lupus anticoagulant and the antiphospholipid
Sey
fee
Na)
SSS)
SS
a
Protein S Deficiency
syndrome.
Antithrombin Deficiency
Prothrombin Nucleotide . Describe the mechanism of heparin-induced thrombocytopenia.
G20210A Mutation
. Name laboratory tests for evaluating heparin-induced thrombocytopenia.
Hyperhomocysteinemia
Tissue Factor Pathway . Explain the mechanism of action and define the conditions in which heparins, oral
Inhibitor Deficiency anticoagulants, direct thrombin inhibitors and thrombolytic agents are used.
Factor XII Deficiency
. List the most common laboratory tests to monitor oral anticoagulant therapy.
Heparin Cofactor Il
Deficiency . List laboratory tests to monitor heparin therapy.
Dysfibrinogenemia
Elevated Plasma Factor VIII
Coagulant Activity
Lipoprotein a and
Thrombosis
Other Coagulant Factors
Associated with
Thrombosis

660
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 66]

Acquired Thrombotic
Disorders
Lupus Anticoagulant/
Antiphospholipid
Syndrome
Heparin-Induced
Thrombocytopenia
Other Acquired Conditions
Associated with
Thrombosis
Thrombosis with Pregnancy
and Use of Oral
Contraceptives
Thrombosis and Nephrotic
Syndrome
Thrombosis and
Medications
Thrombosis in Cancers and
Other Conditions
Thrombosis and Major
Trauma

Diagnostic Approach and

issues in Laboratory
Testing in Patients with
Thrombosis
Complete History and
Physical Examination
Conditions That Can
Interfere with Test Results
Testing in the Appropriate
Clinical Setting
Functional Assays
In Arterial Thrombosis,
Consider the Additional
Evaluation of
Hyperhomocysteinemia
and Lipoprotein a
Repeat Testing Prior to
Diagnosis
Performing Testing and
Making Evaluation
Worthwhile
Anticoagulant Therapy
Unfractionated Heparin
Therapy
Administration and
Monitoring
Low-Molecular-Weight
Heparin
Oral Anticoagulation
Alternative Anticoagulants
Antiplatelet Agents
Thrombolytic Therapy
Case Study 1
Case Study 2
662 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

Introduction several of these conditions. These are addressed later in the

discussion of inherited and acquired thrombophilia.
Hypercoagulable states have a considerable impact on soci- The description of a Norwegian family suffering from
ety, not only in the form of primary pathologic thromboses thrombotic diatheses was first linked to a congenital deficiency
but also in the medical management of seemingly unrelated of antithrombin III (AT), by Egeberg? in 1965. Since then,
conditions to prevent devastating secondary thrombotic com- rapidly progressive discoveries of the association between
plications. Although thought to be underreported, it has been thromboembolism and alterations in factors that inhibit coag-
estimated that more than 200,000 individuals in the United ulation ensued. On further study, abnormalities of hemostasis
States suffer from venous thromboembolism each year. These manifesting in venous thrombosis have been shown to primar-
blood clots carry a 30% mortality rate, and approximately ily reflect disturbances in two regulatory mechanisms: the
40,000 of the fatalities are sudden death due to pulmonary physiologic coagulation inhibitors (e.g., protein C, S, and AT)
embolism (PE)!. When one couples the incidence of PE with and the fibrinolytic system (e.g., plasminogen). Hereditary
the thrombotic aspect of arterial disease as manifested by deficiencies in activated protein C resistance (due mainly to
stroke, myocardial infarction (MI), and peripheral vascular the factor V Leiden mutation), prothrombin gene G20210
disease (PVD), thrombosis becomes overwhelmingly the mutation, AT, protein C (PC), protein S (PS), and defects of
most common mechanism of death encountered in western the fibrinolytic system, as well as the existence of acquired
countries. antiphospholipid antibodies, are the abnormalities most directly
More than 150 years ago, Virchow called attention to the associated with thromboembolic disease in patients younger
fundamental processes involved in the pathogenesis of throm- than 40 years of age. In patients older than 40, malignant neo-
bosis. These include (1) the role of the blood vessel, (2) the plasms are commonly the etiologic stimulus.* Recently, eleva-
flow of the blood within the vessel, and (3) the chemistry of tions in factor VII and fibrinogen have been shown to be risk
the blood itself. Physicians and the public alike are well factors in venous thrombosis.*
versed regarding risk factors for the arteriovascular disease of In the discussion that follows, the risk factors for throm-
atherosclerosis that manifests primarily as stroke and MI boses are addressed individually; however, the reader is
(hypercholesterolemia, hypertension, and cigarette use). In encouraged to recall that the development of thrombosis? is a
these arterial vasculopathic disorders, thrombosis is often the complex process involving the vessel wall, the flow of blood,
final mechanism of occlusion, usually secondary to an ather- and the interactions of coagulation factors and their endoge-
osclerotic lesion. The lesions demonstrate Virchow’s triad, nous regulators as a composite system. Although some condi-
with a disrupted vessel lining, a turbulent flow of blood, and tions exist that are sufficient to cause disease alone, most
alterations in the concentrations of coagulation factors all inherited and acquired risk factors compound one another to
contributing to the risk of thrombus formation in this setting. create an optimal environment for thrombosis.
Altered flow within the blood vessel is a well recognized
problem, particularly in the diseased vessel and in the immo-
bilized or postoperative patient. The slowing of blood flow by Regulation of Coagulation
prolonged sitting as in long airplane or automobile trips (or and Fibrinolysis
writing a book chapter on hypercoagulable states), or the
creation of turbulent flow that is present in atherosclerotic The regulation of hemostasis is complex and involves the
vessels can precipitate thrombosis, especially if added risk interaction of many components of the coagulation system. In
factors for thrombophilia exist. If large blood clots form in the the normal physiologic state, procoagulant and anticoagulant
veins of a patient, an unstable segment may break off and systems are in equilibrium. This balance may be tipped in either
float to the lung vasculature. This potentially deadly pul- direction by a change in physiologic or clinical circumstances.
monary embolism, among other complications, is the main Because the concentration of regulatory and procoagulant pro-
reason for the widespread use of prophylactic anticoagulation teins varies, a patient may vacillate among a prothrombotic, an
medication within inpatient settings. Other conditions that anticoagulated, and a balanced state. Endothelial cells (and
promote a hypercoagulable state and therefore are indications subendothelial structures), platelets, procoagulant factors that
for anticoagulation are chronic atrial fibrillation, artificial participate in the generation of thrombin, natural inhibitors of
heart valves, and connection to bypass machines in patients coagulation (plasma components), and the fibrinolytic system
undergoing cardiac surgery. Altered blood flow and some all play important roles in maintaining this equilibrium. Each of
platelet activation mechanisms contribute to the hypercoagu- these roles is discussed.
lable state in these conditions.
This chapter focuses on thrombophilia caused by abnor-
The Role of Endothelium
malities in the plasma and the laboratory identification of
these abnormalities. Until the mid-1980s, the laboratory had The endothelium plays a key role in the regulation of hemosta-
little to offer in the identification of etiologic or risk factors in sis and has anticoagulant and procoagulant functions. The dual
patients who suffered from thrombosis. Initial interest in these roles lie in the needed properties of maintaining a fluid liquid
states of risk dates back to the 1960s with the identification of physiologic state as well as aiding instant clotting in traumatic
the relationship of lupus anticoagulant to thrombosis and has situations to prevent exsanguination. As arteries become smaller
proceeded to the understanding of the molecular aspects of in their path through the tissue, a given volume of blood is
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 663

forced into sequentially narrower spaces (the surface area to plasminogen activator inhibitor (TPAI) to block degradation
volume ratio increases) to allow the exchange of oxygen from of a newly formed clot. Interestingly, endothelial cells can be
the red blood cell to the tissues. The peak of this activity induced by various cytokines and endotoxins to produce tis-
is reached in the capillary bed where, at a vessel diameter sue factor (also known as thromboplastin), which initiates
as small as 4 um, the surface area to volume ratio approaches coagulation through the activation of factor VII. This is a
1000 mM*/uL*’ More graphically, it is the same as spreading mechanism that may explain the procoagulant state seen in
1 mL of liquid uniformly over the surface of an average game septic and neoplastic conditions.
table, allowing no cell or molecule to pass through a capillary
without contacting the endothelial cells lining the vessel. As a
result, opportunities for ligand-receptor interactions are pro- Platelets
vided and, if needed, endothelial cell activation resulting in a Platelets are activated in response to trauma to the vessel wall.
slowing of velocity within these areas that allows for an Platelet activation has three steps: (1) adhesion, (2) secretion,
increased chance of unnecessary coagulation. In addition, as in and (3) aggregation (see Chap. 24). Adhesion to the underly-
rivers, both large- and medium-diameter vessels have slower ing collagen, which anchors the activity to the needed area
blood flow near the vessel wall, creating a need for endothe- and stimulates a change in platelet shape, defines the first step
lial cells to have anticoagulant properties along the surface to of platelet activation. Functional aspects of platelet shape
prevent pathologic clot formation. However, if the lining of change lie in the rearrangement of the phospholipid platelet
the vessel is breached, agonists contained in the damaged membrane. This not only provides a surface for the surface-
endothelial cell stimulate immediate hemostatic mechanisms dependent component of coagulation (contact factors, which
that localize clot formation to the area of trauma to prevent regionalizes the coagulation enzymes to enhance enzyme—
excessive blood loss and thus the cell contains procoagulant substrate encounters) but also exposes binding sites for
properties when needed. These prothrombotic and anticoagu- platelet-to-platelet binding (aggregation). The catalytic ability
lative roles are discussed subsequently. of the platelet surface may in part explain why thrombin gen-
eration is greatly accelerated in the presence of platelets. The
ANTICOAGULANT ROLE
second step is the secretion of agonists that function in
In the normal state, intact endothelium physically prevents recruiting additional platelets and promote further adhesion
adhesion of platelets to underlying collagen, preventing an and aggregation. The platelet release reaction (secretion)
initiating event of platelet activation. Endothelial cells dis- contributes to thrombogenesis through the release of the
courage platelet aggregation by the continual release of alpha (a) granule contents in response to various stimuli (e.g.,
prostacyclin (PGI,) and nitric oxide (both potent aggrega- thrombin). These granules are visible via light microscopy
tion inhibitors). A product of arachidonic acid metabolism, and contain the platelet-specific proteins, platelet factor
the PGI, molecule is the most important natural inhibitor of 4 (PF4), beta (8)-thromboglobulin, platelet-derived growth
platelet function. This is the mechanism by which aspirin factor, and a variety of other proteins, including fibrinogen,
and nonsteroidal anti-inflammatory medications (NSAIDs) fibronectin, factor V, factor VII, and vWF. These con-
exert their platelet inhibitory effects. The endothelial cell tribute to platelet aggregation, thrombin generation, as
affects secondary hemostasis by binding the endogenous well as local platelet adhesion. The other type of storage
anticoagulants, thrombomodulin and heparin-like mole- granule, called dense bodies, are visible only via electron
cules, to its surface, thereby inhibiting the coagulation cas- microscopy and contain ADP, ATP, calcium, histamine,
cade (described in more detail later). Lastly, endothelial serotonin, and epinephrine. The third step in platelet activa-
cells synthesize tissue plasminogen activator (t-PA), which tion is aggregation, which is linked through fibrinogen
by promoting fibrinolytic activity is instrumental in the and creates the end product of primary hemostasis: the
degradation of any wayward fibrin deposits on endothelial platelet plug (the main physical barrier). These three steps
surfaces. of platelet activation are the basis of the platelet function
assays. How platelets respond enables assessment of the
PROTHROMBOTIC ROLE etiology of platelet disorders.
When endothelial cells are torn, platelets adhere to the newly
exposed subendothelial collagen, become activated, and
secrete substances to recruit and activate additional platelets. Procoagulant Factors and Generation
The cells synthesize von Willebrand factor (VWE), which is of Thrombin
the bridging molecule essential for this platelet adhesion to
collagen. vWF is packaged in cytoplasmic secretory granules As stated earlier, endothelial cells express binding sites for acti-
called Weibel-Palade bodies. It also serves as a carrier mole- vated factors [Xa and Xa. They also provide vWF, which
cule for factor VIII. This coupling of vWF to factor VIII bridges platelets to collagen and also facilitates efficient clotting
serves two functions: (1) protection of factor VIII from degra- by binding and stabilizing factor VII, localizing this powerful
dation and (2) localization of factor VIII to the area of trauma. potentiator of clot formation to the required area of hemostasis.
The endothelial cell also expresses binding sites for activated The resultant environment of activated platelets and clotting
factors [Xa and Xa on its surface, further localizing coagula- factors generates thrombin (factor Ia) through both the extrin-
tion activity to the site of injury while producing tissue sic and intrinsic pathways of the coagulation cascade. Thrombin
664 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

then is principally responsible for the resultant generation of Natural Inhibitors of Coagulation Factors
fibrin monomers which become the insoluble fibrin strands (Plasma Components)
needed for blood clot formation. Thrombin is the last and
most important enzyme of the clotting cascade. The active Normal plasma contains a sophisticated system of serine pro-
site of the enzyme is buried deep within a groove that is pro- tease inhibitors capable of inhibiting many of the activated
tected by surrounding structures. Selective access accounts proteases generated during coagulation that slow the genera-
for the specificity of the enzyme. In addition, thrombin has tion of thrombin (Fig. 28-1). Three types of natural anticoag-
two positively charged regions that are sites for macromole- ulants are present in the plasma:
cule ligand binding (exosite I and exosite II). Exosite I is the
|. Serine-protease inhibitors, AT and heparin cofactor II
binding site for the thrombin receptor, fibrinogen, factor V,
(HC-II).
protein C, and thrombomodulin. Owing to its versatility, NO . Protein C (PC), which upon activation becomes APC and
thrombin is one of the most fascinating enzymes in the coag-
degrades factor Va and VIIa in the presence of its cofactor
ulation system. Among the various activities of thrombin are
Protein S (PS).
the following (Table 28-1):
3. Tissue factor pathway inhibitor (TFPI).
1. It proteolytically cleaves fibrinogen to produce two mole-
cules of fibrinopeptide A and two molecules of fibrinopep-
ANTITHROMBIN (ANTITHROMBIN-IID)
tide B from the Aa and BB fibrinogen chains, respectively.
According to the current international nomenclature, AT-III
This results in the conversion of fibrinogen to a fibrin
is renamed antithrombin (AT). AT combines with heparin as
monomer. The released fibrin monomers spontaneously
a cofactor to become the most powerful anticoagulant in the
polymerize to form fibrin.
circulation. On serum protein electrophoresis, it is an
2. It converts factor XIII (in plasma and platelets) to an active
a>-glycoprotein composed of a single chain of 432 amino
transglutaminase, which cross-links fibrin with covalent
acids with a molecular mass of 58 kD and an in vivo half-
amidé bonds that render the fibrin insoluble in order to
life of approximately 2 to 3 days. AT is synthesized by the
become a physically binding structure.
liver and belongs to the serine protease inhibitor (serpin)
3. It activates the procoagulant factors V, VIII, and XI, to par-
superfamily. Normal plasma levels are approximately
ticipate in amplifying its own generation.
150 ug/mL. AT is a major inhibitor of thrombin (factor Ila)
4. It also binds platelets at low concentrations and initiates
and factor Xa. Other coagulation factors such as XIla, XIa,
shape change, aggregation, and secretion promoting pri-
and [Xa are also inhibited but to a lesser extent.
mary hemostasis.
Normally, AT is a relatively weak inhibitor of the serine
5. It participates in self-regulation by binding to thrombo-
proteases, but it is activated in vivo by heparin released from
modulin which then activates PC and PS to inhibit exces-
granules within mast cells and heparan sulfate, a glu-
sive thrombin formation.
cosaminoglycan which lines the endothelial layer. Heparin,
The complexity of the thrombin molecule is demonstrated which is highly negatively charged, interacts with a short pen-
by its participation in self-regulation and although it is tasaccharide ___domain in the AT molecule containing a high
generally considered a powerful procoagulant, has the density of basic amino acids, particularly lysine. As a result of
capacity to become an anticoagulant. This property will be the conformational change in the AT molecule precipitated by
discussed in more detail in the following section. Interest- the binding of heparin, an arginine residue is made readily
ingly, in addition to these functions in coagulation, throm- available to the active site of a serine protease. A locked com-
bin has been shown to play a role in inflammation and cell plex that blocks enzymatic activity is formed between AT and
growth.* the factor, tying up the availability of the protease. As the
complex forms, the heparin molecule disassociates and is
ready to react with another AT molecule. Therefore, heparin
administered even in small doses converts AT from a slow,

. Cleaves fibrinogen to form fibrinopeptides A and B and Xlila

Xla
fibrin monomers.
IXa
. Activates factors V, VII, XI, and XIII and protein C. Xa < ~
. Activates platelets to aggregate and secrete granule con- AT Vila TFPI
tents which generates platelet procoagulant activity. Va 5 ge ds +PS
. Induces endothelial release of platelet-activating factor, Villa
prostacyclin, von Willebrand factor, interleukin-1, throm- Thrombin
bospondin, tissue plasminogen activator, and plasminogen
activator inhibitor-1. HC wa
. Induces granulocyte chemotaxis and adherence to
endothelial monolayers and macromolecules. Figure 28-1 @ Physiologic inhibitors of coagulation.
. Depresses reticuloendothelial clearance of activated prod- AT = antithrombin; APC = activated protein C; Xlla = activated
ucts of coagulation. factor XII; HC-Il = heparin cofactor Il; PS = protein S; TFPI = tissue
factor pathway inhibitor.
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 665

relatively ineffective inhibitor to a fast, effective one. The active form in human platelets and that platelet HC-II could
conformational changes due to this pentasaccharide sequence play a role in the regulation of thrombin generated on the
accelerates by several hundred-fold the inhibition of factor Xa.° platelet surface. A number of clinical studies including a large
This effect has been exploited in the development of scale effort with a decade of follow-up have failed to demon-
the relatively new anticoagulant medication, fondaparinux, strate evidence of a correlation between HC-II deficiency and
which is a synthetic form of this pentasaccharide sequence!” arterial or venous thrombosis.'?
and a direct Xa inhibitor. However, to induce inhibition of
thrombin, longer heparin chains are required. Longer chain THROMBIN/THROMBOMODULIN INTERACTION
heparins such as those found in unfractionated heparin, Thrombomodulin is a transmembrane glycoprotein synthe-
inhibit through approximation (binding and linking the target sized mainly by endothelial cells with a crucial role in the reg-
serine protease to antithrombin via a second heparin binding ulation of coagulation. When thrombin is generated at a
site), as well as the short-chain pentasaccharide-induced con- remote ___location and is released into the circulation, it comes
formational change. into contact with and quantitatively binds to the thrombomod-
ulin on the surface of the endothelium.'*'° When bound, it
HEPARIN COFACTOR I (HC-ID changes its enzymatic specificity, no longer recognizing fib-
HC-II is a minor inhibitor of thrombin and was first identified rinogen as substrate and, paradoxically, acts on protein C
by Birginshaw in 1974"! and isolated by Tollefsen in 1981.!* (PC), generating activated protein C (APC). APC, in the pres-
The primary amino acid structure of HC-II is quite distinct ence of its cofactor, protein S (PS), forms the APC proteinase
from that of AT. The specificity of HC-II is narrowly restricted complex. This complex has powerful anticoagulant effects by
to thrombin and does not inhibit other coagulation factors. cleaving factors Va and VIIla and promoting fibrinolysis by
HC-I also possesses an affinity for heparin that is significantly inactivating plasminogen activator inhibitor-1 (PAI-1). The
less than that of AT; therefore, a higher concentration of APC-proteinase complex constitutes, on a molar basis, the
heparin is necessary to inhibit thrombin by this cofactor. How- most active inhibitor of plasma coagulation identified, and
ever, other mucopolysaccharides, such as dermatan sulfate, are this factor Va-protein C pathway plays a significant role in the
able to dramatically accelerate the action of this protease regulation of thrombus formation (Fig. 28-2). Therefore, as
inhibitor. Indeed, it appears likely that the observed anticoag- previously mentioned, thrombin indirectly possesses antithrom-
ulant effects of mucopolysaccharides other than heparin are botic activities, limiting the extent of its own generation.
primarily a result of interactions with HC-II. HC-II probably A soluble form of thrombomodulin is present in plasma
plays a minimal role when heparin is used clinically as an with physiologic levels of 3 to 50 ng/mL.'° Increased soluble
anticoagulant. Tollefsen has suggested that HC-II may func- TM levels are seen in conditions with vascular damage (i.e.,
tion as a second-line inhibitor of thrombin after its activation inflammation and sepsis).'’ The relationship between soluble
by dermatan sulfate present on the surface of the vessel wall. TM levels and coronary artery disease are controversial and
It has also been suggested that HC-II is present in a functional remain under debate.'*

Activation of APC
Endothelium

Va to Vi

——» Inactivates PAI-1

Villa to Vili

Inhibition of APC occurs by

PCl-1 (PAI-3)
PCI-2 (alpha 1 antitrypsin)

Figure 28-2 # Thrombin binds to thrombomodulin on the surface of endothelial cell. Thrombin/thrombomodulin complex acts on protein C,
resulting in the formation of activated protein C (APC). APC cleaves factors Va and Villa in the presence of its cofactor, protein S. APC also inac-
tivates plasminogen activator inhibitor-1 (PAI-1). Inhibition of APC occurs by PCI-1 and PCI-2. TR = thrombin; TM = thrombomodulin;
PC = protein C; PS = protein S; PCI = protein C inhibitor.
666 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

THE PROTEIN C AND S SYSTEM formation. This activity is enhanced in the presence of heparin.
PC is a vitamin K-dependent zymogen with anticoagulant A second inhibitor of PC, named protein C inhibitor-2 (PCI-2),
properties that, once activated by the thrombomodulin-altered was found to be q,-antitrypsin by amino acid analysis and is
thrombin molecule, proteolytically degrades factors VIIa and believed to be heparin independent.°!
Va.'° PC is a glycoprotein with heavy and light chains linked
by a disulfide bond with a molecular weight of 62 kD. The TISSUE FACTOR PATHWAY INHIBITOR
anticoagulant role of PC is also described under thrombin/
TFPI circulates and is associated with a lipoprotein that
thrombomodulin interaction, previously, and is depicted in
inhibits plasma coagulation in a dual manner: (1) it serves to
Figure 28-2.
bind the activated form of either factor X or factor IX, thereby
Thrombin/thrombomodulin activation of PC is modu-
inhibiting their enzymatic activity and (2) the TFPI-factor
lated by activated factor V (Va) concentrations. Factor Va
Xa/or [Xa complex then binds to the membrane-bound factor
concentrations above a certain level enhance the activation VIla-tissue factor complex, competitively inhibiting further
rate of PC, whereas lower concentrations inhibit PC activa-
activation of factor X or factor IX through the extrinsic path-
tion. This provides a necessary feedback mechanism allowing way.”* These actions are depicted in Figure 28-3. The role of
factor Va activation to occur (favors coagulation) until the
TFPI in clinical thrombosis has been studied in animal mod-
level becomes high enough to necessitate control of the els. The absence of circulating TFPI activity has been shown
process (to avoid hypercoagulation) through PC activation.
to result in the reversal of aspirin inhibited intravascular
PC function is significantly enhanced in the presence of its
thrombus formation. TFPI plasma levels appear to be acutely
cofactor, PS, and phospholipids. In the absence of adequate
consumed in acute myocardial infarction from intracoronary
quantities of PS, PC function is inadequate to control the
thrombosis. Most intriguing is recent data regarding arterial
generation of thrombin. A role in inflammation has been
wall gene transfection with that coding TFP with promising
established by the favorable clinical response to infused
therapeutic implications in the prevention of intravascular
recombinant APC in patients with sepsis.°°
thrombus formation. Certainly TFPI is an important compo-
In the presence of calcium, protein S (PS) binds to the
nent of dysregulated coagulation and may represent a pivotal
phospholipid surface of endothelial cells and activated
protein in novel therapeutic interventions.”
platelets and associates with activated protein C. This local-
izes the PS-APC complex at the needed site of clot formation.
PS is a vitamin K—dependent protein that is produced in the Fibrinolytic System
liver, as well as, in megakaryocytes and endothelial cells. PS
is a single chain with a molecular mass of 69 to 84 kD. It is The fibrinolytic system in vascular hemostasis is critical for
unique among the vitamin K—dependent coagulation proteins controlling the amount of clot formation and for resolving
in that it is not the zymogen of a serine protease. Plasma PS clots no longer needed. The plasminogen inhibitors contribute
circulates in two forms: one is bound with a 1:1 ratio to the to clot stability by blocking premature dismantling of the
C4b-binding protein (C4bBP) from the complement system, thrombus. Among the components of the fibrinolytic system
the other is free. The free PS form represents about 40% of are plasminogen/plasmin and the activator system: urokinase-
total PS in normal individuals and is the only functional form plasminogen activator (u-PA) and tissue-type plasminogen
of PS. Platelets also contain PS; therefore, the activation of activator (t-PA), which convert plasminogen into the func-
platelets provides not only a surface for procoagulant reactions, tional form, plasmin. The major inhibitors are a,-antiplasmin,
but also a cofactor for eventual control of thrombin generation. and plasminogen activator inhibitors | and 2 (PAI-1, PAI-2).
Decreased PS synthesis 1s seen in many conditions, the most Physiologic fibrinolysis results in the proteolytic degradation
common being oral anticoagulant therapy; however, vitamin K of polymerized fibrin. The central reaction in this system is
deficiency, liver disease, chemotherapy, and L-asparaginase the conversion of a proenzyme, plasminogen, to the prote-
therapy are also known etiologies. Consumption of all factors olytic enzyme plasmin. Plasminogen is a 90 kD glycoprotein
including protein S can occur with acute thrombotic events or synthesized in the liver with a circulating half-life of 2 to
disseminated intravascular coagulation (DIC). The carrier mol- 3 days. Plasminogen circulates both in the free state or bound
ecule C4b-BP acts as an acute phase reactant with fluctuating to histidine-rich glycoprotein (HRGP), «,-antiplasmin, or fib-
concentrations associated with inflammatory states. An increase rinogen itself. The physiologic activation of plasminogen is
in C4b-BP correspondingly increases the percentage of bound achieved through the extrinsic pathway initiated by t-PA
PS antigen and a relative decrease in free PS antigen and there- release from the endothelial cells after stimulation, and by the
fore PS activity. Conditions that are associated with an increase intrinsic pathway through a factor XIIa-dependent activator
in C4b-BP concentrations are pregnancy, oral contraceptive and urokinase.
use, diabetes mellitus, and systemic lupus erythematosus. Free A specific group of proteins control fibrinolysis. They
PS antigen and activity are also often decreased in nephrotic are serine protease inhibitors and are members of the serpin
syndrome. superfamily. They are structurally homologous to many
Two PC inhibitors have been described. The first is inhibitors of the serine proteases of the coagulation system
protein C inhibitor-1 (PCI-1), also known as plasminogen (Table 28-2). Plasmin activity is regulated and inhibited by
activator inhibitor-3 (PAI-3). PCI-1 inhibits the thrombin— a number of plasma proteins such as a,-antiplasmin,
thrombomodulin complex which indirectly inhibits APC Q-macroglobulin, o,-antitrypsin, AT (especially in the
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 667

Lipoprotein = Inactive enzyme

Active enzyme

Factor VilaV_|

Tissue factor ——~>

Active complex
Inactivated complex

Figure 28-5 ™ Shown here is the interaction of tissue factor pathway inhibitor (TFPI) with factors VII, IX, and X. (Courtesy of John D. Olson,
MD; PhD, Department of Pathology, University of Texas Health Science Center, San Antonio, TX.)

presence of heparin), and Cl esterase inhibitor. Physiologi- Plasminogen activators such as uPA and tPA, are inhibited
cally, «,-antiplasmin, the main plasmin inhibitor, is a 60 kD by a number of specific proteins. PAI-1 and PAI-2 are found in
glycoprotein and circulates in the plasma at a concentration of plasma and urine (Table 28-3). A third, tentatively termed
1 uM. Its properties include: PAI-3, was subsequently shown to be the previously described
nonspecific serine protease heparin-binding APC _ inhibitor,
1. Drect inhibition of plasmin
PCI-1. These inhibitors are nonspecific in nature and deacti-
2. Interference with absorption of plasminogen to fibrin
vate enzymes extrinsic to the coagulation system. The most
3. Susceptibility to factor XIII-catalyzed cross-linking of
important inhibitor in the plasminogen activator system is
antiplasmin to fibrin which incorporates the enzyme into
PAI-1, the primary fast-acting serpin inhibitor of t-PA. The
the structure of the clot providing stability by inhibiting
physiologic plasma concentration of PAI-1 varies widely
intrathrombotic plasmin activity.
from 0 to 60 ng/mL, with an average range of 5 to 20 ng/mL
The combined effects of these three characteristics render and many patients with myocardial infarction have elevated
a,-antiplasmin much more specific and effective in inhibition levels. Because the role of this enzyme is to dampen the
of fibrinolysis than any of the other major inhibitors. degradation of a clot, there has been interest in defining a role
in myocardial infarction and stroke, especially in atypical
patients (younger than 45 years of age). Although a case

of theDeerine Grotensa |
Inhibitors _se
PAI-2
Pay, abibitory, Effects
Origin Plasma Placenta tic
ee Ahiplenin Plasmin
Platelets Leukocytes Plasma
a>-Macroglobulin Thrombin, plasmin, kallikrein
Endothelial Macrophages
a,-Antitrypsin Thrombin, trypsin,
cell
chymotrypsin, factor XIa,
Granulocytes
elastase, activated protein C
MW 54,000 47,000 50,000
AT Thrombin; factors Xa, XIIa,
(daltons)
IXa, and Xa; plasmin;
Inhibitory TRA TPA UK
kallikrein effect
C1 inhibitor Kallikrein, plasmin, factors
UK UK APC
XIJa and XIa, C1 esterase
Concentration O-1.3nM 2 uM -
Antichymotrypsin Chymotrypsin
_ Grd trimester)
HC-II Thrombin
Protein C inhibitor Activated protein C UK = Uiadee APC = = activated protein C; MW = molecular
PAI-1 and PAI-2 Plasminogen activators weight; nM = nanomoles; uM = micromoles.
668 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

control study reported a correlation of younger patients with The exogenous addition of activated protein C (APC) to
myocardial infarction and a PAI-1 genetic polymorphism, a test tube of normal plasma creates an anticoagulant effect
subsequent prospective studies did not support this finding. that can be measured as a prolongation of the activated partial
More recently, the noncoagulant role PAI-1 plays in adipocyte thromboplastin time (aPTT). The absence of this anticoagu-
formation has received attention. Some researchers have lant response reflects the decreased ability of APC to inacti-
induced weight loss in mice despite a high-calorie diet by vate factor Va and results in a thrombophilic state called APC
inhibiting PAI-1.** Additional studies suggest a role between resistance (APC-R). The concept of APC-R was first
the upregulation of PAI-1 and ethanol-induced liver injury.” described by Dahlback and associates in 1993.*7 In the major-
Another enzyme under investigation is thrombin acti- ity of cases, APC-R is the result of a genetic defect. The pre-
vatable fibrinolysis inhibitor (TAFI), also known as procar- dominant etiology of APC-R is a defect in the factor V gene
boxypeptidase B. This clot-stabilizing 55 kD glycoprotein involving the point mutation at codon 506 of exon 10 which
is synthesized in the liver and platelet alpha granules and substitutes a glutamine residue for the wild-type arginine
circulates in plasma bound to plasminogen. TAFI cleaves amino acid and is termed “factor V Leiden” (or FV:Q°”°).”8
C-terminal lysine groups essentially removing the binding This codon is the site where APC cleaves and inactivates
sites for plasminogen and tPA to fibrin strands. The exact factor Va. The activity of factor V and its role in coagulation
role in hypercoagulable states and inflammation is not clear are not affected. The mutated factor Va is resistant to cleavage
at the present time.*° by APC and results in prolonged procoagulant activity; there-
The fibrinolytic system is as complex as the coagulation fore, it increases the risk of pathologic thrombus formation.
system. The cause-and-effect relationship between the fre- Mutations at the arginine 306 residue in factor V, the second
quency of deficiencies in the physiologic fibrinolytic system and APC site, have been reported as well but are much less
the occurrence of thromboembolism is not fully understood. common.” Other possible mechanisms of APC-R_ include
However, it seems the most common abnormality is the pres- inhibitors (autoantibodies) to APC, functional PS deficiency,
ence of excess PAI-1 leading indirectly to a decreased functional and mutated forms of factor VIII; however, no genetic abnor-
availability of t-PA, which results in a hypercoagulable state. malities in the factor VIII gene have been identified to cause
APC-R in humans.
Among Caucasians, APC-R is the most common risk
Inherited Thrombophilia factor associated with inherited venous thrombosis.. It can be
detected in 20% to 60% of patients with recurrent venous
Inherited thrombophilia is a group of congenital hematologic
thrombosis and shows autosomal-dominant inheritance. The
disorders that includes a variety of hypercoagulable states
heterozygous state is relatively common in Western countries
that usually present clinically as venous and/or arterial
and occurs in approximately 6%*° of the general popula-
thrombosis. Hypercoagulability is usually defined as an alter-
tion.*'** The prevalence in Hispanic, African American,
ation of the blood coagulation mechanism that predisposes to
Asian, and Native American populations appears to be low.
thrombosis.
The thrombotic risk for patients carrying heterozygous factor
The homozygous deficiency of natural inhibitors of
V mutation is increased five to ten times, while the risk for
coagulation (e.g., deficiencies of PC and PS, factor V Leiden
homozygous patients is 50 to 100 times.
mutation) substantially increases the risk of thrombosis. For
Factor V Leiden—associated thrombotic risk is dependent
example, virtually all neonates with homozygous deficiency
on the underlying clinical setting. Although oral contracep-
of PC and/or PS deficiency suffer from purpura fulminans in
tives and pregnancy significantly increase the risk for venous
the perinatal period. In contrast, the heterozygous condition
thrombosis in women with the mutation, the increased throm-
for PC deficiency and factor V Leiden mutation is widely
botic risk in patients with preexistent cancer or recent surgery
variable in the expression of thrombosis. There is little ques-
cannot be demonstrated.*° There is no convincing data that
tion that these conditions impart a risk; however, it is notable
factor V Leiden mutation confers an increased risk for arter-
that the majority of people who carry heterozygous mutations
ial thrombosis. Data for factor V Leiden as an independent
for many of the genes associated with thrombosis never man-
risk factor for myocardial infarction are controversial.
ifest a thrombotic disease. For thrombosis to manifest clini-
Most of the screening tests for APC resistance are func-
cally, more than one and presumably several, risk factors need
tional aPTT—based assays. They can be used as a screening
to be present simultaneously to overcome the natural
method to identify patients in need of the confirmatory factor
processes in the blood that are normally protective against
V Leiden molecular test. The functional APC resistance assay
thrombosis.
is performed by measuring an aPTT both in the absence and
then the presence of a standardized amount of APC, with a
Activated Protein C Resistance calculation of the resultant ratio (the APC ratio, Fig. 28-4). In
a normal person, the addition of APC to plasma induces a pro-
When thrombin is generated at a remote ___location and is longed aPTT because APC cleaves and inactivates factors Va
released into the circulation, it passes to the capillary bed and VIIIa. In patients with APC resistance, the aPTT is rela-
where it will bind quantitatively to thrombomodulin on the tively shorter than in normal plasma. Factor Va is not cleaved
surface of the endothelium. This mechanism was described and therefore is not inactivated. The test results can be inter-
earlier and is depicted in Figure 28-2. preted by comparing the ratio to the normal range, or by
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 669

Hidsthabieststecliia

Note: The sensitivity of the test is increased by comparing the ratio to the normal
range or by normalizing it to the APC resistance obtained using normal pooled
plasma.

Figure 28-4 @ Data on the functional assay for activated protein C are shown here. This is an APTT-based assay and the APC-R ratio can be
calculated by measuring the patient clot time with and without activated protein C (APC) in the presence of CaCl,. In a normal person, the
ratio is more than 0.9. In case 1, the final ratio is 1.2 and thus the test is negative for activated protein C resistance (APC-R). In case 2, the
ratio is less than 0.9 and the test for APC-R is positive. In case 2, therefore, the patient should be tested for factor V Leiden mutation by poly-
merase chain reaction (PCR). APC-V = activated protein C-factor V ratio.

normalizing it to the APC resistance ratio obtained using sample DNA is analyzed, allowing specific genotypes to be
normal pooled plasma. By diluting the patient’s plasma with identified (normal, heterozygous, or homozygous).
an excess of factor V—deficient plasma, the sensitivity and The genetic test is usually performed to confirm the
specificity of the aPTT-based APC-R assay can be increased factor V Leiden mutation and has several advantages over
and allow the analysis of plasma from patients who are taking plasma-based functional assays. The genetic test is not
oral anticoagulants or have other factor deficiencies without affected if the patient is receiving anticoagulants or has
interference. Factor V—deficient plasma is made of normal plasma inhibitors (such as lupus anticoagulant). It does not
plasma with all the moieties needed for coagulation except fac- have a threshold range as does the functional plasma-based
tor V (which the patient’s plasma must contribute to the mix- assays, and it can reliably differentiate between heterozygous
ture) and thus normalizes the concentrations of other plasma and homozygous states.
proteins involved in the formation and regulation of thrombin. One of the approaches in diagnosing patients with factor
The limitation of this aPTT-based functional assay (and, in fact, V Leiden is to perform an aPTT-based functional assay for
all aPTT-based assays) is that the results are unreliable when the APC-R. Patients with low APC-R ratios (results may vary
test is performed on patients treated with heparins or in the pres- among different laboratories) should be genotyped for the
ence of an underlying inhibitor such as a lupus anticoagulant factor V mutation. For example, in our coagulation labora-
that can artificially prolong the baseline aPTT. The test; how- tory, a normalized ratio (obtained using normal pooled
ever, may be performed during an acute thrombotic episode. plasma) of less than 0.9 is considered abnormal. Even in lab-
The genetic test for FV:Q°*° mutation is often referred to oratories in which there is an excellent concordance between
as the factor V Leiden assay. In this test, the genomic DNA is APC-R assays and the results of factor V Leiden mutation
isolated from blood mononuclear cells. Using allelic discrim- assays, Some patients with a low APC-R ratio and negative
ination methodology by polymerase chain reaction (PCR), the genetic results can still be identified. The significance of this
670 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

finding is uncertain and may be related to mutations other measured via a chromogenic assay. Using the clotting and
than FV:Q°°. amidolytic assays in tandem, useful information regarding the
nature of molecular defect in type I] PC deficiency may be
Protein C Deficiency detected. The cases have been described with normal PC anti-
gen level but reduction in PC anticoagulant activity and
PC is vitamin K—dependent glycoprotein which is synthesized normal amidolytic activity via chromogenic assay. For this
by the liver. Hereditary deficiency of PC shows an autosomal reason, clot-based PC assays are preferred (Table 28-4). High
dominant inheritance pattern. The prevalence for inherited PC levels of factor VIII (greater than 250%) may falsely decrease
deficiency among patients with venous thrombosis linked to PC levels in a clot-based assay.
inherited causes is estimated to be 6% to 10%. The frequency Acquired PC deficiency can be seen in a number of con-
of the heterozygous state varies between 1/300 and 1/1000, and ditions (Table 28-5), including liver disease, DIC, warfarin
the frequency of the homozygous state is estimated to be therapy, severe infection/septic shock, adult respiratory distress
1/50,000.** Homozygotes have a marked tendency for recurrent syndrome, postoperative states, acute thrombotic episode and
venous thrombosis and PE, neonatal purpura fulminans, and secondary to chemotherapy (e.g., L-asparaginase, methotrex-
warfarin-induced skin necrosis. Interestingly, heterozygotes ate, cyclophosphamide, and 5-fluorouracil). It is important to
commonly do not manifest thrombosis unless concomitant note that due to the vitamin K dependency of this factor, mea-
additional risk factors exist. surement cannot be taken while on oral anticoagulation and
Warfarin-induced skin necrosis has been associated with should be drawn only after an appropriate waiting period sub-
patients who have PC deficiency. It occurs during the first few sequent to the cessation of warfarin therapy (approximately
days of warfarin therapy. Warfarin ingestion results in a 4 weeks).
decrease in vitamin K—dependent factors (II, VU, IX, X, PC,
and PS). Because PC has a short half-life (six hours) as com-
pared to other vitamin K—dependent factors, the decrease in
PC level occurs before the decrease in factor II and X levels.
This causes a temporary imbalance between procoagulant and
anticoagulant factors, resulting in a transient hypercoagulable
state that can lead to thrombosis and subsequent skin necrosis.
Anticoagulant Amidolytic
Infants with inherited homozygous PC deficiency may Subtypes Antigen Activity Activity
develop thrombosis of smali capillaries including that of cere-
bral vessels and laboratory evidence of disseminated intravas- I Low Low
Ila Normal Low
cular coagulation (DIC). The condition is usually fatal and is
IIb Normal
called neonatal purpura fulminans. It is important not to mis-
interpret the physiologically low level of PC in a normal new-
born, which may be near zero in a sick preterm infant. The
level usually rises rapidly after birth.
The functional PC activity assay (mixing patient’s
plasma with protein C—deficient plasma and measuring the
aPTT) is used as the screening assay for PC deficiency.
The antigenic assay is used to determine the mechanism of
the deficiency (decreased production or abnormal protein) Inhibitors Causes of Acquired Defects
and is rarely necessary in diagnosis. Based on immunologic AT DIC
and functional assays, two subtypes of heterozygous PC defi- Liver disease
ciency have been described. Type I deficiency is the most com- Nephrotic therapy
mon, which shows a reduction in the activity due to a Oral contraceptives
L-Asparaginase
concomitant decrease in the amount of antigen (to approxi-
Protein C Oral anticoagulant treatment
mately 50% of normal). Type II deficiency is less frequent, and DIC
like type I, shows reduced functional activity but can be differ- Vitamin K deficiency
entiated by the measurement of normal antigen levels. This After plasma exchange
type is a result of defective molecular mechanisms that allow Postoperative state
expression of a protein that has lost functional capacity.***> L-Asparaginase
Liver disease
The procedures used for antigenic assays are enzyme- Protein S Oral anticoagulant treatment
linked immunosorbent assay (ELISA), electroimmunodiffu- Pregnancy
sion (Laurell rocket electrophoresis), and radioimmunoassay. Oral contraceptives
In the simpler functional assay, PC is activated in the presence Vitamin K deficiency
of the specific activator extracted from snake venom. The Liver disease
Diabetes type I
resulting APC inhibits the factors V and VIII, and thus pro-
Acute inflammation
longs the APTT. The enzyme’s anticoagulant activity can be Newborn infants
measured in a clotting assay or its amidolytic activity can be
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 671

Protein S Deficiency acts as a cofactor for APC, functional assays may show spu-
rious low PS levels in patients with APC resistance .
PS is a vitamin K—dependent nonenzymatic glycoprotein syn-
thesized in the liver, endothelial cells, megakaryocytes, testes,
kidney, and brain. It exists in an active free form that func-
Antithrombin Deficiency
tions as a cofactor for APC, and an inactive form that is bound As discussed earlier, because antithrombin serves to regulate the
to complement binding protein (C4bBP). The free form most powerful procoagulant, thrombin, a deficiency results in a
accounts for 40% of total PS and has a half-life of approxi- prothrombotic state. Homozygous states are largely incompati-
mately 45 hours.* ble with life except for a rare functional (type II) variant, and the
PS deficiency is inherited as an autosomal dominant majority of affected individuals are heterozygous with AT levels
disorder. The prevalence of PS deficiency in the general popu- 40% to 70% of normal. It is an autosomal dominant disorder
lation is 1 in 33,000. The incidence of PS deficiency in patients with a prevalence in the general population of approximately
with venous thrombosis is 6% to 10%. In contrast to PC defi- 1/2000 to 1/5000. One percent of all venous thrombosis patients
ciency, even heterozygotes have a strong tendency to develop and 5% of patients younger than 70 years of age with venous
DVT, PE, cerebral and mesenteric thrombosis, superficial thrombosis were found to have an AT deficiency.°°**
thrombophlebitis, and arterial thrombosis and may also develop The initial clinical manifestation of AT deficiency is com-
wartarin-induced skin necrosis. Patients with homozygous PS monly between the age of 10 and 50 years (peak 15 to 35), may
deficiency are severely affected and may develop neonatal occur spontaneously in half of the patients and are characterized
purpura fulminans just after birth, similar to that seen with PC by thrombosis of the deep veins of the lower extremities, mesen-
deficiency. teric veins, and PE. In the remaining patients, the thrombosis
Both immunologic and functional methods are available may occur in the presence of additional risk factors such as
to measure PS. Reliable measurements of total antigen are pregnancy, the use of oral contraceptive pills, surgery, or trauma.
performed using radioimmunoassay, ELISA, or electroim- The risk of arterial thrombosis is not well established but has
munodiffusion (Laurell rocket electrophoresis). Functional been reported as 2%.°°
assays are performed for the quantitative measurement of the Acquired antithrombin deficiency is common, arising
free functional PS level based on the ability of PS to serve as from conditions such as decreased synthesis (liver disease or
a cofactor for the anticoagulant effect of APC inhibition of L-asparaginase treatment), consumption (thrombosis, dissem-
factor Va. This inhibition is reflected by the prolongation of inated intravascular coagulation [DIC], surgery) or loss (pro-
the clotting time of a system that is enriched with factor Va, a teinuria in renal disease), and continuousheparin use (see
physiologic substrate for APC. Table 28—5).*°
Based on the immunologic and functional assays described Mild decreases can be seen with pregnancy, oral contra-
earlier, three subtypes of PS deficiency have been noted ceptive use, and colitis. Before a diagnosis of antithrombin
(Table 28-6). The most common, type I deficiency, is associated deficiency is rendered in any patient with these conditions, the
with decreased activity due to a low antigenic level of total and test should be repeated once the condition is no longer pre-
free PS levels. In type Ila deficiency, total PS is normal; how- sent. Confirmation of a hereditary antithrombin deficiency
ever, the free PS antigen levels are decreased and the functional may require documentation of a deficiency in a blood relative.
PS activity is low. In type IIb deficiency, total and free antigen Various methods have been described for the measurement
levels are normal, but activity level of PS is low. of AT. Immunologic assays such as radial immunodiffusion are
Acquired PS deficiency can be seen in liver disease, available that measure the antigen levels but cannot be used to
DIC, pregnancy, oral contraceptive use, vitamin K deficiency, detect dysfunctional molecules. To detect the qualitative abnor-
during an acute thrombotic event, and in therapy with mality of the AT molecules, the functional assay is preferred. A
warfarin and L-asparaginase (see Table 28-5). C4bBP is an known amount of thrombin is added to the patient’s plasma con-
acute-phase reactant and its increase will proportionately bind taining AT in the presence of heparin. The aliquot is removed
free PS; therefore, the amount of free PS may decrease in and added to a specific synthetic chromogenic or fluorescent
inflammatory conditions such as systemic lupus erythemato- thrombin substrate and the released compound is measured
sus (SLE), inflammatory bowel disease, pregnancy, and spectrophotometrically or via fluorometer, respectively.
acquired immunodeficiency syndrome (AIDS). Because PS Based on immunologic and functional assays, two types
of AT deficiency can be distinguished phenotypically. In the
common type I deficiency there is a proportionate decrease in
both the functional and antigen levels because the deficiency
is due to a decreased amount of a functionally sound molecule
(quantitative defect). Approximately 80 distinct mutations
have been described in patients with type I deficiency, and
Subtypes Total PS Free PS PS Activity most are a result of genetic mutations producing silent
alleles.*'** Type IL deficiency shows a more pronounced
I Low
Ila Normal
decrease in the functional level with a relatively normal anti-
IIb Normal gen level (qualitative or dysfunctional defect) because the
molecule is created but does not function properly. Type II is
672 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

associated with point mutations and can be further subdivided At present, the PCR test for G20210A mutation is avail-
into three subtypes: those characterized by abnormalities able in clinical laboratories. In this test, DNA is isolated from
primarily affecting (1) the serine protease inhibition site for blood mononuclear cells. Using allelic discrimination method-
thrombin/Xa, (2) the heparin-binding site, and (3) those with ology by PCR the sample DNA is analyzed, allowing specific
effects on both protease inhibition and heparin binding. The genotypes to be identified (normal, heterozygous, or homozy-
classification of hereditary AT deficiencies is summarized in gous). Among the Dutch population, the prothrombin gene
Table 28-7. mutation is the second most common genetic defect (the most
Based on functional assays, the prevalence of thrombo- frequent being the factor V Leiden mutation). Because of the
sis appears to be different in heterozygous patients with high prevalence of APC resistance (FV:Q**) and of the pro-
type II defects. Individuals with heparin-binding site defects thrombin gene mutation (G20210A), combinations of genetic
have infrequent thrombotic episodes while, in contrast, those defects are relatively common. Patients with both mutations
with thrombin-binding site defects sustain venous thrombosis (double heterozygotes) may have a high risk of thrombosis.
as often as type I patients. However, more studies are needed to improve our understand-
ing of the ethnicity-specific variability of the factor V Leiden
and prothrombin gene mutations.
Prothrombin Nucleotide G20210A
Mutation Hyperhomocysteinemia
Prothrombin (coagulation factor II) is a vitamin K—dependent
Hyperhomocysteinemia (an elevated plasma level of homo-
factor that is converted to thrombin by factor Xa in the pres- cysteine) is a risk factor for the development of early ather-
ence of factor Va, Ca**, and phospholipids. The combination
osclerotic vascular disease and venous thrombosis. Some
of factor Va, Xa, Ca**, and phospholipids is called the pro-
investigators include hyperhomocysteinemia under acquired
thrombinase complex. It is a macromolecular enzyme com-
disorders, whereas recent studies suggest that these should
plex in which prothrombin is a substrate and thrombin the
be included among the inherited causes of thrombophilia.
product. Hereditary disorders of prothrombin synthesis may
Homocysteine is an amino acid derived from metabolic
result in quantitative defects (decreased production) and qual-
conversion of methionine. It is usually metabolized within the
itative defects (dysfunctional prothrombin molecule). Clini-
cells to methionine (via the remethylation pathway) and to
cally, both of these defects may result in a bleeding diathesis.
cysteine (via the transulfuration pathway). Remethylation to
A 21-kb gene present on chromosome 11 codes for the
methionine involves two pathways. In the first remethylation
prothrombin molecule. This gene consists of 14 exons and
pathway, homocysteine accepts a methyl group from betaine.
13 introns. A mutation in the prothrombin gene has been
In the second remethylation pathway, homocysteine accepts a
described recently,** revealing the presence of a single gua-
methyl group from 5-methyltetrahydrofolate, with vitamin
nine (G) to adenine (A) mutation at the nucleotide position
B,, acting as a cofactor. This pathway is catalyzed by methio-
20210 to be associated with an elevated prothrombin level
nine synthase. The transulfuration pathway is catalyzed
and an increased risk of DVT.
by cystathione B-synthetase with vitamin B, as a cofactor*
A heterozygous G20210A mutation is found in 2% of
(Fig. 28-5).
healthy controls, 6% to 7% of venous thrombosis patients, and
18% of selected thrombophilia patients with a positive family
history. The relative risk for venous thrombosis in heterozygotes
HOMOCYSTEINE
is approximately threefold.“ Carriers of this mutation have
higher plasma prothrombin levels than those with a normal
20210 genotype. A high level of prothrombin alone is not a
reliable marker of disease predisposition, whereas detection of eet

the mutation by genetic testing appears to be a reliable prognos-

tic factor.
ihe

5,10-methylene
tetrahydrofolate

AT-Heparin
Subtypes AT Antigen Cofactor Progressive AT Figure 28-5 = Homocysteine can be metabolized to methionine
(via remethylation) and to cysteine (via transulfuration). The num-
I Low Low Low bers represent the enzymes involved in the reactions: (7) Betaine
Ila Normal Low Low homocysteine methyltransferase; (2) methionine synthase in the
IIb Normal Low Normal presence of cofactor vitamin B,.; (3) cystathione -synthetase in the
IIc Normal Normal Low presence of cofactor vitamin B,; (4) methylene tetrahydrofolate
reductase (MTHFR).
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 673

Hyperhomocysteinemia can be inherited or acquired. factor, after the patient in whom the deficiency was first identi-
Inherited forms have genetic defects that involve (1) the fied in Cleveland in 1955. Patients with factor XII deficiency
remethylation pathway, as a result of a deficiency of methylene have a prolongation of APTT but no bleeding diathesis. Instead,
tetrahydrofolate reductase (MTHFR) and (2) the transulfuration a number of cases with venous thromboembolism and myocar-
pathway, owing to the deficiency of cystathione B-synthetase. dial infarction have been described in factor XII-deficient
The mutation of MTHFR gene (alanine substitution to valine at patients. Mr. Hageman himself died of a PE. Because
amino acid 677) is more common. This mutation can vary from factor XIla is involved in activating plasminogen, the throm-
1.5% to 15%, depending on the population studied.*° The fre- bophilic tendency in factor XII deficiency patients has been
quent causes of acquired hyperhomocysteinemia are secondary attributed to reduced plasma fibrinolytic activity. The incidence
to deficiencies of folate, vitamin B,,, and/or vitamin B,, which of thrombosis in patients with heterozygous or homozygous
are cofactors in homocysteine metabolism. factor XII deficiency in the absence of other thrombotic risk
Possible mechanisms by which hyperhomocysteinemia factors is uncertain and, thus, an increased thrombotic risk asso-
acts as a thrombogenic and atherogenic risk factor include ciated with factor XII deficiency remains to be well defined.
vascular smooth muscle proliferation, inhibition of endothe-
lial cell growth and intimal thickening, activation of factor V,
and inhibition of PC activation. Severe hyperhomocysteine- Heparin Cofactor II Deficiency
mia is usually secondary to genetic defects and characterized
Heparin cofactor II is a single-chain glycoprotein present in
by mental retardation, premature atherosclerosis, venous
human plasma that inhibits only thrombin but does not inhibit
thromboembolism, and skeletal abnormalities. Mild to mod-
other coagulation factors. The affinity of heparin cofactor II
erate hyperhomocysteinemia can be secondary to genetic
for heparin is lower than that of AT and requires a higher con-
or acquired conditions and is an independent risk factor
centration of heparin to affect in vitro testing (more than the
for stroke, MI, and peripheral vascular disease. Levels of
therapeutic concentration, i.e., more than | U/mL of heparin).
homocysteine can be measured by high-pressure liquid chro-
Therefore, heparin cofactor II probably plays an insignificant
matography. A PCR-based genetic test is available to detect
role in the clinical setting.’ On the contrary, the activity of
the common Ala 677 Val mutation in the MTHFR gene.
heparin cofactor II can be increased several-fold by dermatan
«
sulfate, which has no effect on the activity of AT. The clinical
manifestations of hereditary heparin cofactor II deficiency
Tissue Factor Pathway Inhibitor Deficiency
range from an absence of symptoms to arterial and venous
Tissue factor pathway inhibitor (TFPI) may play a significant thromboses. More studies are needed to determine the signif-
role in preventing thrombus formation; however, deficiency icance of this deficiency.
of this inhibitor is yet to be associated with thromboembolic
disease. Evidence suggests that TFPI neutralizes two pro-
teases simultaneously, and the inhibitory actions of TFPI Dysfibrinogenemia
occur through three domains.*’ The first ___domain inhibits Dysfibrinogenemia is defined as a condition in which there is
factor VHa—tissue factor (TF) complex, whereas the second a structural or functional abnormality of the fibrinogen mole-
___domain inhibits factor Xa. The function of the third ___domain is cule, or both. If congenital, the inheritance is autosomal dom-
unclear. TFPI can be found in endothelial cells, plasma, and inant. The clinical presentation is variable and can include
platelets. The endothelial cell pool represents the majority of bleeding or clotting tendencies. Sixty percent of the patients
the TFPI. Most of the plasma pool is complexed with various are asymptomatic, a bleeding diathesis can be seen in 20% of
lipoproteins, whereas only 10% of the TFPI is present as a cases, and 20% of cases may present with recurrent arterial or
free form and is biologically active. The concentration of venous thromboembolism.*!
TFPI is increased in plasma in patients receiving heparin A number of functional and biochemical defects of the fib-
infusion. Recombinant TFPI is now available and has been rinogen molecule have been described. These include an abnor-
evaluated in a few studies, with results showing a possible role mality in binding of thrombin to fibrinogen, the release of
of TFPI in the interruption of thrombus formation. Whether or abnormal fibrinopeptides, defective fibrin polymerization and
not there is a congenital deficiency of this inhibitor leading to cross-linking, decreased activation of plasminogen, and resis-
thromboembolic conditions is not yet known.” tance to lysis by plasmin. These abnormalities are reflected in
Recent evidence suggests that autoantibodies to tissue fac- the routine coagulation assays as prolongation of PT, APTT,
tor pathway inhibitor (TFPI) and/or antiphospholipid antibodies fibrinogen, reptilase time, and thrombin time. The fibrinogen
(aPL) may contribute to upregulation of the tissue factor (TF) concentration via functional assay is low, but is normal when
pathway of blood coagulation and the development of throm- measured immunologically. An important point to consider is
botic complications in the antiphospholipid syndrome (aPS).”° that the abnormal fibrinogen may not be incorporated into the
clot, and the soluble molecules that remain in the serum can be
mistaken for fibrinogen degradation products in some assays,
Factor XII Deficiency
leading to the misdiagnosis of active fibrinolysis and thus DIC.
Factor XII is one of the contact factors that initiates the intrinsic Dysfibrinogenemia can be seen in acquired conditions
pathway of coagulation in vitro. It is also known as Hageman such as severe liver disease, paraneoplastic syndromes, and
674 — Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

with paraproteinemias.° Acquired dysfibrinogenemia is vascular thrombosis and pregnancy morbidity.” The thrombosis
rarely symptomatic and thrombotic complications have not may be arterial or venous and tends to recur in the same site.
been described. aPL syndrome requires the combination of at least one clinical
and one laboratory criterion (Table 28-8). aPL syndrome is con-
sidered secondary when it occurs in association with another
Elevated Plasma Factor VIII Coagulant disorder and primary when unassociated with any identified
Activity underlying disorder. Secondary aPL syndrome is most fre-
quently associated with systemic lupus erythematosus (SLE) or
The elevated plasma factor VII coagulant level (elevated func-
other autoimmune diseases, but has occasionally been seen
tional and antigenic levels) is considered an independent risk
after exposure to certain pharmaceuticals (such as phenoth-
factor for thrombosis.**>’This abnormality could not be attrib-
iazines, quinidine, hydralazine, procainamide) and in patients
uted to inflammation, because fewer than 10% of the patients
with underlying malignancies. The overall prevalence of aPL
with factor VIII:C levels of greater than 150% had elevations
syndrome is difficult to determine because of variations in
in an acute phase reactant (such as C-reactive protein, fibrino-
laboratory techniques and diagnostic criteria. In spite of this,
gen, and erythrocyte sedimentation rate [ESR]).°*°°
the prevalence of aPL syndrome in SLE patients has been
Studies have demonstrated that patients with factor VII
examined by many researchers. The frequency of lupus
greater than 150% had an increased risk of thrombosis when
anticoagulant (LA) and anticardiolipin (aCL) in nearly
compared to patients with factor VIII less than 100%.°°' One
2000 SLE patients reported in the literature is 31% and
study has demonstrated that an elevated factor VIII level may
40%, respectively. Patients with SLE and aPL antibodies
also be a strong thrombotic risk factor in the black popula-
had a 30% to 40% risk of thrombosis.”
tion. The clinical utility of routinely measuring factor VIII
The term /upus anticoagulant refers specifically to the lab-
levels in patients with thrombosis remains to be determined.
oratory in vitro phenomenon of prolongation of phospholipid-
dependent coagulation assays such as the APTT in the
Lipoprotein a and Thrombosis absence of an attributable factor deficiency. Laboratory
evidence of aPL antibodies can be in the form of either
Lipoprotein a (Lpa) represents a low-density lipoprotein immunologically demonstrated aPL antibodies or, more fre-
(LDL)-like particle having a protein moiety apoprotein B-100 quently, LA, or both.
linked by a disulfide bridge to a glycoprotein called apolipopro- The presence of aPL antibodies, either demonstrated by
tein a (Apo a). Elevated levels of Lpa are under genetic control LA or detected immunologically, is not sufficient for the diag-
and have been recognized as an atherothrombogenic factor. But nosis of aPL syndrome. Additional clinical features must also
the underlying mechanisms for this pathogenicity are not well be present to diagnose aPL syndrome (see Table 28-8) because
understood. There is a structural homology of Apo a with plas- aPL antibodies alone may not carry a similar clinical signifi-
minogen, and laboratory studies have shown that Lpa inhibits cance. In particular, infection-associated aPL antibodies are
fibrinolysis. It effectively competes with plasminogen for bind- usually transient and are rarely associated with thrombotic
ing to fibrin or endothelial cells. It also binds t-PA and may also complications.”°’’ Most aPL antibodies detected in children
stimulate the release of PAI-1 from endothelial cells. Studies do are of this type.
show evidence of increased levels of Lpa in atherogenesis/
thrombogenesis°* and an association with coronary heart dis- HISTORY OF ANTIPHOSPHOLIPID SYNDROME
ease and stroke. However, its role in venous thromboembolism The term antiphospholipid antibody is a misnomer. A more
is not established.°’ accurate term would be antiphospholipid-protein antibodies
because the antibodies have specificities for certain proteins
when those proteins are bound to phospholipids or other sur-
Other Coagulant Factors Associated
faces such as microtiter plates. “Lupus anticoagulant” is also
with Thrombosis not an optimal term because the anticoagulant phenomenon
High levels of factor XI, IX, fibrinogen, IL-8, and TAFI and seen in vitro is not always associated with SLE and causes
low levels of plasma fibrinolytic activity have been consid- thrombosis in vivo. However, for historical reasons, the terms
ered to be associated with venous thrombus. Assays for some aPL antibody and LA are well embedded in the literature and
of these proteins are not widely available, and the clinical util- clinical use.
ity of these measurements is currently uncertain.°*” In 1963, Bowie and colleagues published a paper calling
attention to a syndrome they had observed in patients with
SLE.” The syndrome, still unnamed, consisted of recurrent
venous or arterial thrombosis accompanied by paradoxical
Acquired Thrombotic Disorders
prolongation of clotting times in phospholipid-dependent
Lupus Anticoagulant/Antiphospholipid clotting assays. This phenomenon of prolonged clotting times
Syndrome in the absence of specific factor inhibitors or deficiencies came
to be known as lupus anticoagulant. As in many SLE patients,
Antiphospholipid (aPL) syndrome is one of the acquired throm- some of these patients were observed to have a false-positive
botic disorders. The clinical features of aPL syndrome include venereal disease research laboratory test (VDRL). The VDRL
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 675

aPL syndrome is present if at least one of the clinical criteria and one of the laboratory criteria are present.

Clinical

¢ Confirmed vascular thrombosis

¢ Pregnancy morbidity, to include one of the situations described below:
* One or more unexplained death at >10 weeks EGA. Morphologically normal neonate
* One or more premature birth at <34 weeks EGA due to severe pre-eclampsia, eclampsia, or severe placental insufficiency.
Morphologically normal neonate
¢ Three or more unexplained consecutive spontaneous abortions at <10 weeks EGA. Maternal and paternal chromosomes normal.
No maternal anatomic or hormonal abnormality
Laboratory
¢ Medium to high titer IgG and/or IgM aCL antibody in blood on two or more occasions, >12 weeks apart measured by standardized
ELISA
¢ Anti-82GPI antibody of !gG and/or IgM isotype in serum or plasma present in two or more occasions, at least 12 weeks apart mea-
sured by standardized ELISA

on Thrombosis and Haemostasis.

Source: Adapted from the International Consensus Statement on an update of the Classification Criteria for Definite
Antiphospholipid Syndrome. Eleventh International Congress on Antiphospholipid Antibodies.**

is a serologic screening test for syphilis that uses a mixture of antibodies bind prothrombin.*°** These antibodies usually do
cardiolipin (an acidic phospholipid found in beef heart), not deplete plasma levels of 82GPI and prothrombin because
lecithin, and cholesterol. Given the coexistence of the LA they bind only when these antigens are attached to phospholipid
phenomenon and the antiphospholipid antibodies demon- surfaces. Occasionally, patients with SLE have reduced pro-
strated by a positive VDRL, it seemed likely that LA antibod- thrombin levels and a resulting hemorrhagic diathesis. Other
ies were nonspecific antibodies that reacted with both platelet phospholipid-binding proteins may also be targets of thrombo-
and reagent phospholipids. The interference of such antibod- genic aPL antibodies.
ies with phospholipid-dependent coagulation reactions could
prolong clotting times. How these antibodies promote coagu- MECHANISM OF THROMBOSIS
lation in vivo was not understood and is still unproven, but It would be reasonable to question whether aPL antibodies are
eventually the concept of an aPL syndrome proposed by causative in aPL syndrome or merely an epiphenomenon that is
Harris in 1987 took hold and the terms are still with us.” useful for diagnosis. It is likely that multiple disturbances of
hemostatic mechanisms are required to result in thrombophilia.
IMMUNOLOGY One could assume that, if they were part of the etiology, they
In 1983, Harris and colleagues developed a solid-phase radioim- would affect phospholipid-dependent reactions in vivo and in
munoassay (RIA) for aCL antibodies.*° Recall that cardiolipin is vitro. In vitro evidence of such effects have been investigated
one of the components of the VDRL. High titers of these anti- and include inhibition of activated protein C, inhibition of
bodies correlated well with the presence of LA, thrombosis, and antithrombin-dependent anticoagulant mechanisms, inhibition
thrombocytopenia. The aCL assay, now usually done via ELISA of fibrinolysis, induction of increased soluble tissue factor, inhi-
rather than via RIA, is the most frequently used immunologic bition of prostacyclin secretion, promotion of platelet activation,
assay for the identification of aPL antibodies.*' Over the years it interference with the anticoagulant properties of 82GPI, and
has been shown that aPL antibodies can be detected by using displacement of annexin V from trophoblast.*’ Annexin V is a
various phospholipids, including phosphatidylinositol, phospha- protein that has potent anticoagulant properties in vivo and that
tidic acid, and phosphatidylserine. In addition, investigators may modulate thrombosis on the surfaces of cells lining placen-
have shown that LAs and aCL antibodies in a given patient can tal and systemic vasculatures. Interference with the function of
often be separated. In 1990, three groups working independently annexin V may play a role in aPL syndrome in which there is
found that the majority of aCL antibodies bind phospholipid- recurrent pregnancy loss.”
bound ,-glycoprotein I (B2GPI).*°* B2GPI, also known as
apolipoprotein H, is a 50-kD plasma protein present at a concen- THE LABORATORY’S CONTRIBUTION
tration of approximately 200 mg/mL that appears to have anti- TO THE DIAGNOSIS OF aPL SYNDROME
coagulant properties, although its physiologic role is uncertain. Overall, the presence of LA is a better predictor of thrombosis
Some LAs have also been shown to bind in a B2GPI-dependent and pregnancy loss than the presence of high-titer anticardi-
manner.® In addition, a large proportion of LAs and some aCL olipin antibodies.’' However, some patients with aPL syndrome
676 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

if This Screening Assay Is Positive ... One ofThese Techniques Should Be Used asa y Contiemainry Test

APTT-based assay Platelet neutralization Procenute

Hexagonal phase phospholipid test (e.g., Staclot LA)
Phospholipid dilutions
Dilute Russell’s viper venom (dRVV) time dRVV confirm
Platelet vesicles
Platelet neutralization procedure
Taipan snake venom time Platelet neutralization procedure
Dilute prothrombin time (thromboplastin inhibition test) Phospholipid dilutions of same
Kaolin clotting time Platelet vesicles
Or
Silica clotting time Inosithin
_ Bovine Phospholipids

Eon assays detect eSa eioniea Runestime. CG assays demonstrate whether or not the prolongation
is decreased by phospholipids capable of neutralizing the LA antibody.
Source: Horbach, DA, et al: Lupus anticoagulant is the strongest risk factor for both venous and arterial thrombosis
in patients with systemic lupus erythematosus. Thromb Haemostas 76:916, 1996.

do not have both LA and aCL antibodies. Therefore, the labora- can be in the form of excess phospholipids (dRVV con-
tory diagnosis of aPL syndrome should include both coagulation firm), hexagonal (II) phase phospholipids (Staclot test) and
assays for LA and immunologic assays for aPL antibodies. platelets or platelet vesicles (platelet neutralization test).
Some authors have suggested that a broad panel of aPL antibod- The excess phospholipid binds and neutralizes the LA,
ies should be included when investigating aPL syndrome as a leaving enough phospholipid to provide a surface on which
possible cause for recurrent fetal loss.”? In the following discus- coagulation reactions can proceed.”*” A confirmatory
sion, we concentrate on the hematology laboratory’s role in assay that corresponds to the initial type of assay and mix-
working up a suspected case of aPL syndrome. ing study that were positive should be used. Solid-phase
As in most diagnostic algorithms, initial assays with broad assays for aPL antibodies, such as the aCL assay, should
sensitivity are performed and, if positive, they are followed by not be considered confirmatory for LA activity.
confirmatory assays that are more specific (Table 28-9). The 4. In addition, the absence of clinical or laboratory evidence
Subcommittee on Lupus Anticoagulant/Antiphospholipid ofa specific inhibitor of any one coagulation factor may
Antibody of the Scientific and Standardization Committee of also be necessary, because specific factor inhibitors can
the International Society on Thrombosis and Haemostasis cause mixing studies to remain uncorrected. The clinical
has recommended criteria, paraphrased here, for the diagnosis presentation is very important and may be informative.
of LA’ (Table 28-10). Patients with factor inhibitors have a bleeding diathesis
rather than thrombosis.
1. Prolongation of at least one phospholipid-dependent clot-
ting assay. Examples of such assays using a lupus-sensitive These steps can be carried out in various ways, using various
reagent include the APTT, dilute PT (dPT), kaolin clotting discrete assays or by using integrated systems for the diagno-
time (KCT), and the dilute Russell’s viper venom time sis of LA that incorporate an initial assay, mixing study, and a
(dRVVT) and colloidal silica clotting time.°*°’ Because of
the heterogeneity of these antibodies, no single screening
test will detect all of them. A minimum of two “screening” ‘Table 28-10 Criteria for Lupus —
assays should be available in a laboratory offering LA test-
_ Anticoagulants —
ing. Having three screening tests available is optimal.
NO. Evidence of inhibition of clotting is demonstrated by mix-
A tested sample should show each of Ieee
ing studies. This involves mixing the patient’s plasma and
. Prolongation of at least one phospholipid-dependent clot-
pooled normal plasma and repeating the phospholipid- ting test
dependent clotting assay in which the prolongation of clot- . Evidence of inhibitory activity shown by the effect of
ting time was observed. If the prolongation disappears patient plasma on pooled normal plasma
(“corrects”) with mixing, this usually indicates that there is . Evidence that inhibitory activity is dependent on phospho-
lipid
a factor deficiency.
. Satisfactory exclusion of another coagulopathy that could
3. If the results of the mixing study suggest an LA, then evi- “give similar laboratory results
dence of phospholipid dependence is required for confir-
Source: Horbach, DA, et al: Tae mitecieeent: is the strongest
mation. Confirmatory assays demonstrate phospholipid
risk factor for both venous and arterial thrombosis in patients with
dependence by demonstrating a reversal of the LA effect systemic lupus erythematosus. Thromb Haemostas 76:916, 1996.55
when excess phospholipid is added to the test mixture. This
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 677

procedure that confirms phospholipid dependence into a single laboratory diagnosis of an LA as depicted in Figure 28—7.'°!
kit. Table 28-9 lists Screening and confirmatory assays that Many, but not all, patients with a LA will have a prolonged
have been used are listed in Table 28-9, and an approach to the APTT. Some APTT assays are more sensitive to LA than oth-
diagnosis of LAs is illustrated in Figure 28-6.” ers and, therefore, they vary in their value as initial tests.!0*'°°
In addition to the requirements for diagnosis already Remember that prolongation of the APTT also occurs with
mentioned, several other elements are crucial to arriving at heparin therapy, heparin contamination, insufficient volume
the correct diagnosis in cases suspicious for aPL syndrome, as of blood for the amount of anticoagulant in the sample tube
follows: (short draw), DIC, factor deficiencies, and specific coagula-
tion factor inhibitors. Clinical history elucidates the cause of
1. Care should be taken to use properly prepared platelet-poor
some prolonged APTTs, and depending on the clinical situa-
plasma in order to avoid false-negative results caused by
tion other assays such as the thrombin time and fibrinogen
the neutralization of LA by platelet membrane phospho-
level, may be employed to identify heparin contamination or
lipids.'°°
DIC. If the prolonged APTT is unexpected or not explained
2. Immunologic assays for antiphospholipid antibody should
by the causes mentioned earlier, the laboratory investigation
also be done.
for LA is warranted, especially in a patient with a history of
3. Testing should be repeated after a minimum of 6 weeks to
thrombosis or other findings indicating aPL syndrome.
demonstrate persistence of the LA. Transient aPL antibod-
If the APTT is prolonged and investigation of a possible
ies do not appear to be associated with the clinical compli-
LA is indicated, the next step is to perform an APTT using a
cations of aPL syndrome.
1:1 mix of the patient’s plasma and normal pooled plasma. If
4. Finally, the diagnosis of aPL syndrome should make sense
the APTT does not correct, then either antibody to a phospho-
based on the patient’s history and clinical findings.
lipid in the test system or antibody to a specific coagulation
Laboratory testing for LA is currently hindered by the lack of factor is responsible. Correction can be defined in different
standardized controls. Laboratories have no option other than ways, but probably the most useful approach is to consider an
to use patient plasmas that have proved positive for LA that are APTT corrected when it returns to within 5 seconds of the
available commercially. Trials of potential standardized con- APTT of the pooled normal plasma.'™ If the APTT does not
trols containing monoclonal anti-B2GPI and antiprothrombin correct in the 1:1 mix, then confirmatory testing is indicated,
antibodies are ongoing. as discussed earlier.
If the APTT does correct, then the abnormality is
A WALK THROUGH THE ALGORITHM attributable to coagulation factor deficiency or a time- and
In the hematology laboratory, detection of aPL antibodies temperature-dependent antibody. Specific coagulation fac-
usually begins with the APTT. In this discussion, we trace the tor inhibitors are usually time and temperature dependent.
It has also been observed that up to 30% of LAs are time
dependent.'*'° An incubated 1:1 mix allows for detection
PROLONGED APTT of a time- and temperature-dependent inhibitor. Interpreta-
tion of incubated mixing studies can be challenging. The
incubated mix should be used only if the immediate mix
Explore | No [Thrombin
time ].Y°S | Fibrinog corrects. The incubated mix helps to detect inhibitors, but
et MNES _ measurable? _ >100 cannot be used to determine their etiology. The use of
inspecmen} Lo unmixed incubation controls, as shown in Figure 28-7, is
suggested.'°' Assays for specific coagulation factors can
Explore detect factor deficiencies, and serial dilutions are helpful
hypofibrinogenemia_ in detecting antibodies to specific coagulation factors.

THERAPY AND MONITORING

Because LA can prolong coagulation tests, it may interfere
with the usual tests (APTT and sometimes prothrombin time
[PT]) used to monitor anticoagulation. When the baseline
APTT is prolonged in a patient with LA, then a heparin assay
Non-specific
is recommended to monitor unfractionated heparin therapy.
Procoagulant _ | Specific
factor (lupus-type) inhibitor Warfarin therapy is monitored by the PT assay, with an inter-
anticoagulant national normalized ratio (INR) of 2 to 3 being a typical
therapeutic window. However, this is a complex issue for two
Figure 28-6 In the hematology laboratory, detection of LA usu- reasons: (1) there is controversy regarding the optimal degree
ally begins with an elevated APTT. This figure is a flowchart that of anticoagulation and (2) some thromboplastins are more sen-
illustrates an approach to the diagnosis of LA. The shaded boxes sitive to the effects of LAs than others. Monitoring therapy
show the typical abnormalities that can be seen when LA is present with a PT using one of these thromboplastins can be mislead-
in the sample. (Courtesy of John D. Olson, MD; PhD, Department
ing even if the baseline INR of the patient with an LA is within
of Pathology, University of Texas Health Science Center,
San Antonio, TX.) the reference range. During anticoagulation, the INR may be
678 — Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

INCUBATED MIXING STUDY:

4:1 MIXTURE OF THE PATIENT PLASMA WITH POOLED NORMAL PLASMA

Test specimen: Control specimen:

Mix, then incubate, Incubate, then mix,
then test then test
Mix Patient Pooled
normal

: Incubate
Incubate Patient

Perform APTT
and compare results

Figure 28-7 @ Incubated mixing study can be performed as shown in this figure. For details, refer to the accompanying text. (Courtesy of
John D. Olson, MD; PhD, Department of Pathology, University of Texas Health Science Center, San Antonio, TX.)

prolonged by the LA and appear to be within therapeutic Heparin-Induced Thrombocytopenia

range, although the patient is actually insufficiently
anticoagulated. Heparin-induced thrombocytopenia (HIT) is another seem-
The effect of LA on the PT is not predictable, creating ingly paradoxical antibody-mediated cause of venous and
problems when determining the appropriate INR to use for oral arterial thrombosis and is a life-threatening disorder that fol-
anticoagulation. Because of the variable effect, it would be pru- lows exposure to unfractionated heparin (less commonly to
dent to determine the therapeutic INR for each patient with LA low-molecular-weight heparin). Studies have shown that
who receives oral anticoagulant therapy. The goal of therapy is between 1% and 5% of hospital patients exposed to heparin
to reduce the functional level of vitamin K—dependent factors for | to 2 weeks develop HIT. Of patients diagnosed with
in the plasma; therefore, an assay of one of the factors to con- HIT, approximately one-third will develop overt thrombosis
firm the therapeutic effect should be done after the patient is and of these, about one-third will suffer amputation or
receiving a stable dose of anticoagulant. The assay of factor X death.''' Hence, the overall chance of serious morbidity or
is the most frequently used, with a target of 20% to 25% activ- mortality as a result of a course of heparin therapy is about
ity. The LA can also affect clot-based assays, so the assay for 3 per 1000.''*:''° Early recognition and appropriate treatment
factor X needs to be an assay that uses a chromogenic substrate may reduce these numbers.
in a reaction that is not phospholipid dependent. Once the fac- Heparin is a widely used anticoagulant that can be
tor X is confirmed to be in the therapeutic interval, the corre- administered intravenously (IV) or subcutaneously (SC) both
sponding INR can be used for monitoring, if the INR is in a to prevent thrombosis in high-risk patients and to limit pro-
sensitive range (1.e., less than 6.0). For the rare patient whose gression of established thrombosis. It is also used as a flush to
LA has a profound effect on the INR, precluding its use, peri- keep IV lines open. Heparin is often used to prevent clotting
odic assay of factor X may be necessary. In aPL syndrome in extracorporeal circulation such as that in heart-lung bypass
associated with recurrent fetal loss, subcutaneous unfraction- machines, where it is infused or even present as the anticoag-
ated heparin or low-molecular-weight heparin (LMWH) plus ulant coating within the tubing system.
aspirin are often used.''° HIT is characterized by an unexplained decrease in
To summarize, features of aPL syndrome include venous platelet count, occurring 5 or more days after the initiation of
or arterial thrombosis, recurrent pregnancy loss, and thrombo- heparin therapy. This lag is consistent with an immune
cytopenia. The aPL antibodies, manifesting as LA or detected response related to heparin administration, unless the patient
via immunologic tests such as the aCL ELISA, may be seen has been previously exposed to heparin.
in the setting of aPL syndrome or be incidental and without Nomenclature seen in the literature regarding this syn-
apparent ill effect. The aPL syndrome can be associated with drome can be confusing. Over the years, various names have
SLE and other autoimmune disorders, as well as with drugs. been used for this syndrome, such as heparin-induced thrombo-
It can also occur in the absence of a known underlying disease cytopenia with thrombosis syndrome (HITTS) and HIT type II.
process. Laboratory diagnosis of aPL antibodies is complex These terms have been used to distinguish immune-mediated
and requires an approach that takes into account the hetero- HIT from the mild, non—immune-mediated thrombocytopenia,
geneity of the antibodies. Laboratories offering testing for which may occur within the first few days of heparin adminis-
LAs should have a systematic approach that ensures that an tration. This nonimmune (also called HIT type I) thrombocy-
adequate investigation takes place when there 1s clinical con- topenia resolves spontaneously and does not increase the risk
cern regarding aPL syndrome. for thrombosis. For the rest of this chapter, when the term H/T
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 679

is used, it signifies the immune-mediated disorder in which MECHANISM

there is a risk of thrombosis. When heparin complexes with PF4, it forms the antigen usually
responsible for initiating HIT''*''* (Fig. 28-8). PF4 is present in
CLINICAL MANIFESTATIONS the « granules of platelets and is released when heparin or other
Although HIT may present with simultaneous thrombocytope- agonists activate them. Antibodies to these heparin-PF4
nia and thrombosis, or sometimes with thrombosis preceding (H-PF4) complexes form in almost all HIT patients." IgM,
thrombocytopenia, the first manifestation of HIT is usually an IgA, and IgG specific for H-PF4 antigen have all been detected,
unexplained decrease in platelet count of 30% to 50% or to although IgG is most common.'”? When the H-PF4—antibody
less than 100 X 10°/L, occurring 5 or more days (usually 5 to immune complex is bound by platelet FeyRIIa receptors (a sub-
8 days) after the initiation of heparin therapy.'"' The platelet type of receptor found on platelets that binds the constant
count rarely decreases to 15 X 10°/L or less. The mean nadir portion of immunoglobulin molecules), two things may hap-
has been reported to be approximately 60 < 10°/L.'"? pen.'*! First, splenic macrophages may remove the platelets, as
HIT can cause both venous and arterial thrombosis, but they are now coated with Ig attached to the platelet Fc receptors;
venous thrombosis occurs about four times more often. DVT, second, the attachment of the immune complex to the Fe recep-
PE, lower limb arterial thrombosis, and coronary arterial throm- tor can result in platelet activation. The activated platelets
boses may occur.'!*''® Other sequelae can also be seen. Throm- release substances that attract and activate more platelets.
boses may be multiple. The occurrence of multiple arterial Levels of thrombin increase, which along with platelet-derived
thromboses is sometimes referred to as “white clot syndrome,” microparticles can ultimately result in thrombosis. Endothelial
because the thrombi formed in high-flow vessels have a high cell damage or activation probably also plays a role in the devel-
platelet and fibrin content and relatively few red blood cells. opment of thrombosis.!”?
Accurate diagnosis of HIT requires a high degree of suspi- The fact that many patients form these antibodies and do
cion on the part of the physician caring for the heparin-exposed not develop HIT'* suggests that other factors are required for
patient. Recommendations for monitoring platelet counts vary, the development of HIT besides H-PF4 antibodies. One fasci-
but it seems prudent to check a baseline platelet count at the nating and controversial possibility is that the His-131 polymor-
initiation of heparin therapy and to repeat platelet counts at phism in the gene for the platelet FeyRIla receptor may increase
intervals of several days. Because of the timing of the onset of susceptibility to HIT.'7°!4-!76
HIT, particular vigilance around day 4 after initiation of heparin Unfractionated heparin (UH), still the most commonly
(day O being the first day of heparin administration) and for used heparin, is the major medication associated with HIT
10 days thereafter is particularly crucial. Patients who have had which occurs less often with porcine UH than with bovine
previous exposure to heparin may have an amnestic response UH. Low molecular-weight heparin can also cause HIT,
and develop HIT rapidly after repeat heparin exposure. Many although at a much lower rate than UH.'’’
other causes for a decreased platelet count should be considered
before rendering a diagnosis of HIT. Among these are fever,
DIC, splenomegaly, and medications (other than heparin). Of
note, in spite of the thrombocytopenia, HIT patients rarely have Heparin
a bleeding diathesis.
If suspicion of HIT is high, all sources of heparin expo- (1) Heparin binds
sure should be discontinued immediately. Continued heparin to PF4
exposure greatly increases the risk of thrombosis and LMWH
should not be administered, as there is a cross-reactivity rate
of 93%. Assays are available to assist in the diagnosis of HIT,
but discontinuation of heparin should not wait for these (2) Antibody binds
results. Alternative anticoagulant therapy—direct thrombin to platelet and PF4
inhibitors (hirudin analogs or argatroban) should be consid-
ered because of the high risk of thrombosis even after heparin
is discontinued. Platelet count often rises rapidly after the dis-
continuation of heparin. The return of the platelet count to
(3) Fe portion binds
normal within 5 to 7 days of discontinuation of heparin is to platelet’s receptor
consistent with a diagnosis of HIT, although some have been releasing more PF4
observed to take up to a month to recover completely. As with
most immune reactions, heparin antibodies may remain in the
plasma for extended periods of time. However, testing should Figure 28-8 ® Heparin complexed with PF4 is the antigen usually
occur within 6 weeks of a thrombocytopenic event. responsible for initiating HIT (7). An antibody is formed against
The risk of HIT appears to be greater in patients exposed heparin-PF4 complexes (2). The antibody complexes with heparin-
PF4 and the Fc portion of the antibody binds to the platelet FcRila
to large amounts of heparin, such as when systemic anticoag- receptor (3), resulting in platelet activation and release of more PF4
ulation is required. However, patients with exposures to very (4), which then binds to more heparin. (Adapted from Van Cott, EM:
small amounts of heparin, such as that used to keep IV lines How to diagnose and manage heparin induced thrombocytopenia.
from clotting when not in use, have also developed HIT." CAP Today March:50, 1997.)
680 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

LABORATORY DIAGNOSIS Another functional assay takes advantage of the release

Two types of assays are commonly used to assist in the diag- of ATP from platelet-dense granules when H-PF4 antibodies
nosis of HIT: functional assays and antigen assays. The anti- stimulate the release reaction.'** The indicator reagent con-
gen assays are ELISAs that use the H-PF4 complex as the tains both luciferin and luciferase. The substrate luciferin is
target antigen to detect HIT-type immunoglobulin in the converted to a product by luciferase in an ATP-dependent
patient’s serum. Serologic assays detect IgG, IgA, and IgM reaction that emits photons. Therefore, if the platelet release
antibodies and have high sensitivity (more than 95%) but the reaction occurs, the ATP from the platelets will result in a
specificity is low (74% to 86%). In other words, the positive detectable light emission referred to as lumi-aggregometry.
predictive value of serologic assays is low but the negative This technique is also used to test for platelet secretion
predictive value is high. Functional assays may simply look (release reaction) in response to a variety of agonists. The
for platelet aggregation or may be a variation on a platelet same controls as used in the SRA are also appropriate for this
aggregation method that detects products of the platelet method. This assay is somewhat less labor intensive than the
release reaction, such as serotonin or adenosine triphosphate SRA because it can be done with platelet-rich plasma rather
(ATP). These assays use the patient’s serum, heparin, and than with washed platelets, and radioisotopes are not used.
donor platelets. Either bovine or porcine heparin may be used Flow cytometric methods for diagnosing HIT have also
in the assays. There is no need for the laboratory to determine been developed. They employ fluorescent antibodies against
whether the patient has received bovine or porcine heparin. It platelet glycoproteins and forward angle light scatter (indicat-
is important to use platelets from donors whose platelets are ing cell size) to detect platelet microparticles generated when
known to be reactive to HIT sera. It is unknown why some donor platelets are exposed to patient sera that are able to
donors’ platelets are reactive and others are not. Occasional cause platelet aggregation in the presence of therapeutic con-
combinations of known HIT sera and known reactive HIT- centrations of heparin. Adequate data comparing the results of
reactive platelets do not aggregate. Therefore, it has been sug- flow cytometry assays with clinical outcomes are not avail-
gested that platelets from two donors whose platelets are able, but these methods seem promising in their ease of
known to react to HIT sera should be used. If platelet aggre- performance, and have been observed to correlate well with
gation occurs or if there is evidence of the release reaction the SRA. 20"
only at a low concentration of heparin (0.1 U/mL), this is The clinical diagnosis of HIT often remains unconfirmed
evidence that the patient’s serum contains antibodies that by any of these laboratory techniques. Sensitivities and speci-
activate and aggregate platelets in the presence of a therapeu- ficities reported for these assays vary, but overall are subopti-
tic concentration of heparin. mal. No single technique is reliable enough to be considered a
In any of these functional assays, it is important to use gold standard for the diagnosis of HIT.'*° Sensitivity and speci-
appropriate controls to exclude the presence of non—heparin- ficity can be improved by using more than one methodology to
induced aggregation. In addition to using a control that lacks test for heparin-associated antibodies, including one H-PF4
heparin, the use of a control containing a high concentration of ELISA. In cases suspicious for HIT, repetition of the assays
heparin (100 U/mL) is included. When HIT serum is tested every few days may provide confirmation. Patients must be
using a high concentration of heparin, aggregation should not removed from heparin for up to 4 hours before a sample is col-
occur. It is reasonable that this might be caused by antigen lected. The presence of heparin in the sample may cause a
(heparin) excess, but nonspecific inhibition of platelet aggrega- false-negative result. Clinical decision-making should be based
tion in response to other agonists (collagen, adenosine diphos- predominantly on clinical impression. However, laboratory
phate [ADP], and epinephrine) has also been observed. In any evidence of HIT is often helpful in supporting the decision to
case, aggregation ofplatelets at both low and high concentrations discontinue heparin and use an alternative anticoagulant.
of heparin is considered to be evidence that the aggregation is
caused by a mechanism other than that of HIT.!?7-'*! THERAPY AND MONITORING
One of the functional assays is the serotonin release The effective management of patients with HIT is to prevent
assay (SRA). It is currently the primary comparison proce- thrombosis by limiting platelet activation and thrombin gen-
dure because most of the available data on clinical outcomes eration. Stopping all heparin exposure is the most important
have been gathered in studies using this assay. Donor platelets step in preventing or limiting thrombosis in patients with HIT.
are incubated with [*C]serotonin, to allow its uptake into However, even after cessation of heparin, the patient still has
their dense bodies. These platelets are then washed, removing a risk of approximately one in three for developing thrombo-
free ['*C]serotonin that has not become incorporated. They sis. LMWH should not be given to patients with HIT because
are then exposed to the serum of the patient to which is added of high cross-reactivity with PF4-heparin antibodies. There-
a therapeutic concentration of heparin. If the patient’s serum fore, the use of other anticoagulants should be considered. In
contains H-PF4 antibodies capable of activating platelets, addition, the substitution of another anticoagulant may be
then the radioactively labeled serotonin will be released from beneficial in patients with an established thrombus.
the dense granules. The test specimen is then centrifuged and Because of the risk of venous limb gangrene (distal
the supernatant tested for '*C activity.'?? This assay has high ischemic necrosis that is present despite palpable or Doppler-
sensitivity (88% to 100%) and high specificity (89% to identifiable arterial pulses) the oral anticoagulant warfarin
100%). However, it is time-consuming and requires the use of sodium should not be used in a patient with HIT unless the
radioisotopes. patient is also adequately anticoagulated by another nonheparin
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 681

anticoagulant for the first few days.'** The early warfarin- oral contraceptives prescribed in the United States contain
induced reduction of functioning protein C in the presence of low-dose estrogen. However, even the use of low-dose estro-
increased thrombin generation seen in HIT, puts the patient at gen contraceptives confers a fourfold increased risk of throm-
very high risk for this and other thrombotic complications. bosis when compared with nonusers. The risk may become
LMWH is also not recommended for the treatment of significant if the woman also has an inherited risk factor for
HIT. Although it is less likely to induce HIT once a patient has thrombophilia. The exact mechanism of the prothrombotic
a heparin-induced antibody, exposure to LMWH carries a risk state is not well understood, but it is thought that these med-
of thrombosis.!?’ ications may cause an acquired APC-R-like state.'*”
Anticoagulants available for use in HIT patients include
heparinoids such as danaparoid and direct thrombin inhibitors
such as hirudin, argatroban, and bivalirudin, which are dis- Thrombosis and Nephrotic Syndrome
cussed subsequently. The adjunctive use of medications that Nephrotic syndrome is characterized by heavy proteinuria
limit platelet function is also under investigation. Examples (more than 3.5 g/day), hypoalbuminemia, severe edema, and
include glycoprotein IIb/IIIa (GPIb/IHa) inhibitors such as hyperlipidemia and thrombosis of both venous and arterial
GPI 562 and ADP receptor antagonists, clopidogrel and systems may be seen. The renal vein is the most common
ticlopidine.'*°
site involved and is present in approximately one-third of
The current recommendation for HIT patients is therapy patients.'*° The incidence of thrombosis varies and correlates
with an alternate anticoagulant (direct thrombin inhibitors) to be
with the severity of the disease. Depending on the complex
followed by a transition to warfarin. The warfarin should be interaction between hepatic protein biosynthesis and the sever-
started at low doses and given concurrently with direct throm- ity of renal disease, the following changes of coagulation
bin inhibitors for 5 days until a therapeutic INR is achieved for parameters can be seen: AT levels are often decreased; PC and
2 days.'* Care should be taken in monitoring INR during this S are increased; and factors V, VII, VIII, X, and XIII are
period, as direct thrombin inhibitors may prolong the INR. increased, whereas factors XI and XII are decreased. In addi-
Obtaining a chromogenic X assay may help in transitioning a tion, platelet hyperactivity can also be seen in these patients.
patient from a direct thrombin inhibitors (DTI) to warfarin.
Warfarin is typically continued for 3 to 6 months; however, the
optimal duration of anticoagulation needs further study. Thrombosis and Medications
L-Asparaginase, used as a part of induction chemotherapy for
acute lymphoblastic leukemia (ALL), may cause venous
Other Acquired Conditions Associated thrombosis (intracranial thrombosis is observed most fre-
with Thrombosis quently). The incidence of venous thrombosis has been esti-
Thrombosis with Pregnancy and Use mated to be 1.2%.'*' This drug causes a significant reduction
in hepatic synthesis of both procoagulant (factors V, VU, VIII,
of Oral Contraceptives
IX, X, XI, and fibrinogen) and anticoagulant proteins (AT, PC,
There is a sixfold increased risk of thromboembolism during and PS). The alteration in the hemostatic balance in a particu-
pregnancy. The puerperium (6-week period after delivery) is lar patient determines the risk of thrombosis or bleeding. There
associated with a higher risk of thrombosis than pregnancy. may be a prolongation of PT, APTT, and thrombin time. These
The presence of coexistent inherited thrombophilia during transient abnormalities resolve within | to 2 weeks after ces-
pregnancy represents a significant added risk. In a Swedish sation of the drug. Recent studies indicate that, in children
population, approximately 50% of women with venous with ALL, L-asparaginase is a risk factor for thrombosis, but
thrombosis had APC-R, with 65% to 75% of the thrombotic those who develop thrombi have additional risk factors. Some
events occurring during pregnancy.'*>'*° Thrombosis during consideration should be given to the evaluation of other risk
pregnancy is attributable to the following conditions: factors for thrombophilia prior to beginning the medication. If
other risk factors are present, prophylaxis may be indicated.
1. Venous stasis in the lower extremities, caused by the gravid An increased risk of thrombosis has also been described
uterus. in women with breast cancer who are receiving chemother-
2. Trauma to pelvic veins during delivery. apy.'” The basis of thrombogenicity among these patients is
3. The placenta is rich in tissue factor and also releases plas- not well understood, and possible causes include low grade
minogen activator-2, which reduces fibrinolytic activity. DIC, impairment of vitamin K metabolism, and altered
4. Increased levels of most coagulation proteins (fibrinogen hepatic protein synthesis.
can be increased to 600 mg/dL).
5. A significant decline in plasma levels of free and total PS.
6. Elevated D-dimer levels during the third trimester. Thrombosis in Cancers
and Other Conditions
Although PC and AT levels remain normal during pregnancy,
both are decreased in preeclampsia. Patients with cancer are at high risk of VTE when compared to
The risk of venous or arterial thrombosis secondary noncancerous patients. The occurrence of thrombosis in patients
to oral contraceptives is related to estrogen dose. Most with malignancy can be caused by cancer itself or occur
682 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

secondary to chemotherapy (mitomycin D, L-asparaginase),

“Table28-11LD
surgery, or radiation. The most common sites of cancer predis-
posing to thrombosis are the gastrointestinal and genitourinary
tracts and lungs. The possible causes for thrombosis include
activation and production of procoagulants by tumor cells, and
Hereditary Conditions
low-grade DIC. None of the clotting tests currently available are
specific for cancer. The morbidity and mortality is substantial as . Factor V Leiden mutation
VTE in this group is often underdiagnosed and undertreated. . Prothrombin gene mutation
. AT deficiency
Recurrent thrombosis in the presence of adequate anticoagula- . Protein C deficiency
tion raises the possibility of underlying occult malignancy. . Protein S deficiency
Recent data suggest LMWH to be not only as effective as war- . Heparin cofactor II deficiency
farin in the secondary prevention of VTE in patients with cancer . TFPI deficiency
but also have a lower incidence of bleeding complications, sim- . Elevated lipoprotein a
. Homocysteinemia
MHWNr
OMAN
pler dosing, and more predictable anticoagulant activity.'*
. Factor XII deficiency
Microangiopathic hemolytic anemia (MAHA), dissemi- . Elevated factor VIII levels
nated intravascular coagulation (DIC), thrombotic thrombo- . Plasminogen deficiency
cytopenic purpura (TTP), and hemolytic uremic syndrome . Increased plasminogen activator inhibitor
(HUS) could lead to the formation of platelet and fibrin Acquired Conditions
microthrombi that occlude the capillaries and arterioles.
1. Antiphospholipid antibodies
These microthrombi are found most frequently in the kidneys, 2. Heparin-induced thrombocytopenia
heart, brain, spleen, and adrenal glands. 3. Malignancy
Myeloproliferative disorders predispose patients to venous 4, Autoimmune disorders
or arterial thrombosis, especially when the platelet count and 5. Nephrotic syndrome
6. Myeloproliferative disorders/PNH
hematocrit are not controlled by therapy. Increased blood vis-
7. Behget’s syndrome
cosity and activation of platelets may be the cause for thrombo- 8.Microangiopathic hemolytic aianemia a (DIC, TTP, HUS)
sis. In patients with paroxysmal nocturnal hemoglobinuria
Note: The list is not complete as many ones rare causes of
(PNH), there is a predilection for thrombosis in intra-abdominal
thrombophilia are not listed.
and cerebral vessels. A diagnosis of PNH should be suspected in
patients with pancytopenia, elevated reticulocyte count, low
iron studies, and a negative family history of thrombosis.
On the other hand, the hypercoagulable state in a 70-year-old
bedridden person is most likely to be acquired. Therefore, the
Thrombosis and Major Trauma knowledge of the coagulant, anticoagulant, and fibrinolytic
systems; complete history and physical examination; ethnic
DVT and PE are common complications after major trauma. background (factor V Leiden is very rare in African Americans
Five independent risk factors for DVT following major and Asians); and identification of the risk factors are the basic
trauma have been defined: older age, blood transfusion, but very important steps that need to be considered before one
surgery, fracture of the femur or tibia, and spinal cord injury. orders assays for inherited disorders.
In one study in which prophylaxis was not used, 58% of
patients admitted to the trauma unit developed lower extrem-
ity DVTs.'* Therefore, effective prophylaxis is necessary in
patients with major trauma.

Diagnostic Approach and Issues Pregnancy

Oral contraceptives
in Laboratory Testing in Patients Obesity
with Thrombosis Surgery
Trauma
The purpose of detecting hypercoagulable states is to predict General anesthesia
and prevent thromboembolic disease and its complications, Malignancy
Immobility
identify risk factors, modify treatment regimens, and discover
Varicose veins
underlying diseases that might be amenable to treatment Infection
(Tables 28-11 through 28-15). The diagnostic evaluation of Congestive heart failure
patients with thrombotic diathesis initially involves consider- Advancing age
ation of two factors: (1) whether the disorder is inherited or Infusion of prothrombin complex concentrates
acquired and (2) whether the thrombosis involves primarily Diabetes mellitus and hyperlipidemia
Treatment related:
the veins or arteries (Table 28—16). For example, a 35-year- L-Asparaginase
old person with recurrent DVT is most likely to have an Mitomycin
inherited defect of one or more of the anticoagulant proteins.
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 683

§ % tavie 28-16 Coagulation Defects

ate _and Sites of Thrombosis

Toes Venous Arterial

Factor V dee = se ae
1. Age at onset
Prothrombin gene mutation sheets
. Any underlying associated risk factors
PC deficiency** ap ae
. Number of events (initial or recurrent)
PS deficiency ++
. Site of thrombosis
AT deficiency aimets
. Any documented evidence of pulmonary embolism or DVT
Hyperhomocysteinemia af
. Whether recurrent thromboembolism occurred despite
Lipoprotein a =
therapeutic anticoagulation
Antiphospholipid syndrome 3Par
. Any history of recurrent fetal loss, MI, or stroke
Heparin-induced sear
. Any medications that can lead to thrombosis (oral contra-
thrombocytopenia
ceptives, chemotherapy)
. Family history of venous thromboembolism *Women who smoke and take oral contraceptives have an increased
risk of myocardial infarction.
**A slight increased risk of stroke has been reported.

4 Table 28-14
1 Suggested Evaluation — Specific laboratory tests of interest in patients with
-.__. Criteria for. Inherited thrombophilia have been addressed in other sections of this
_ Thrombophilia chapter. There are, however, some issues that deserve consid-
eration when approaching the laboratory evaluation of a
1. Thrombosis at any age especially in younger patients* patient who has suffered from thrombosis. They are summa-
2. Recurrent thrombosis rized in Table 28-17 and addressed briefly below.
3. Significant family history of thrombosis
4.Thrombosis atunusual sites (other that ee veins of legs)

*Studies have Onset that the yield in feeultnherlied risk Complete History and Physical
factors for thrombophilia in the aged occurs at the same rate and
would have the same value in guiding clinical management.
Examination
A complete personal, family, and drug history is extremely
important in evaluating patients with acute, recurrent, or
remote thrombosis. The patient should be carefully questioned
about all the possible risk factors for thrombosis. A significant
Table 28-15 Testing for Inherited | family history of thrombosis strongly suggests the possibility
: _ Thrombophilia- coe of inherited thrombophilia. If the patient is female, history of
oral contraceptive use and obstetric history is important. Pres-
Plasma Gaaeatgion Screening ence of constitutional symptoms, hemoptysis, melena, or hema-
Pal turia may suggest underlying occult malignancy. Recurrent
APTT
Thrombin and reptilase times thrombosis despite anticoagulation therapy may suggest under-
lying malignancy. Atherosclerotic vascular disease and renal
Anticoagulant System
APC resistance assay (APTT-based assay)
Protein C (functional and antigenic)
Protein S (functional and antigenic)
AT (functional and antigenic)
| Table 28-17 Issues in Laboratory |
- Testing in Patients —
Fibrinolytic System
Fibrinogen (functional and antigenic)
(an Thrombosis
Factor XII activity
¢ Ensure a complete history and physical examination 1s
Plasminogen (functional)
obtained.
Genetic Tests * Careful selection of tests during the acute event.
Factor V (FV:Q°°°) Leiden mutation * Consider conditions that can interfere with test results.
Prothrombin gene mutation (nucleotide G20210A) ¢ Test in the appropriate clinical setting.
MTHFR gene mutation ¢ Use functional assays when possible.
¢ In arterial thrombosis, consider the additional evaluation of
Additional Tests hyperhomocysteinemia and lipoprotein a.
Homocysteine level ¢ Repeat testing prior to diagnosis.
Lipoprotein a level ¢ Ensure that the yield in performing testing is high, making
Factor VIII activity evaluation worthwhile.
684 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

disease (nephrotic syndrome) can lead to arterial or venous TESTING DURING THE ACUTE EVENT
thromboembolism. Thrombotic events, whether on the arterial or venous side of the
Ethnic background is also important in evaluating patients circulation, are capable of consuming naturally occurring coag-
with inherited thrombophilia. The factor V Leiden mutation is ulation inhibitors (PC, PS, AT) that are of interest for testing. In
common in people of European descent and extremely rare in addition, a number of these components are either positive or
African American, Native American, or Asian populations. negative acute phase reactants. Anticoagulant therapy also
Physical examination should be specifically directed to exam- affects these concentrations variably. For these reasons, testing
ining vascular system, skin, extremities, heart, chest, and during the acute event and during anticoagulant therapy will
abdomen. produce both false-positive and false-negative results.

USE OF D-DIMER ASSAY IN THE DIAGNOSIS

Conditions That Can Interfere
OF THROMBOEMBOLISM
Owing to the nonspecific symptoms associated with vascular with Test Results
thromboemboli, a common clinical question is whether a A number of physiologic and pathologic states, as well as,
potentially dangerous clot is present. The risk of fatality or medications may affect plasma levels of PC, PS, and AT. One
morbidity requires the use of anticoagulation if an aberrant of the most common problems is the interpretation of these
hypercoagulable state is present and these medications are not assays under inappropriate conditions. Levels of PC, PS, and
without risk and indeed can have a great impact on lifestyle AT are physiologically low in newborns, during pregnancy
(such as with contact sport athletes). Establishing a diagnosis (PC may be high), and in the early postpartum states. Patho-
of venous thromboembolism is well studied and is typically logic decreases in levels of PC, PS, and AT can be seen in
assessed using a combination of clinical history and symp- postthrombotic states, the postoperative states, severe liver
toms along with clinical testing and various vascular analysis disease, and DIC. In nephrotic syndrome, PS and AT levels
techniques. D-Dimers are the specific cross-linked fibrin
are decreased while PC levels may be increased.
breakdown products formed in the dissolution phase of clot
PC and PS are vitamin K—dependent proteins, and both
formation, and therefore elevated levels are almost always
functional and antigenic levels will be reduced during oral
seen in patients with VTE. A negative D-dimer test result has
anticoagulation therapy. Warfarin has on rare occasion ele-
a negative predictive value of approximately 90%, which is
vated AT levels in patients with a hereditary defect of this
useful to exclude VTE. However, the power of the negative
coagulation inhibitor. Heparin therapy may falsely lower
predictive value is reduced in patients with a high prevalence
AT activity. Heparin therapy will not affect plasma levels of
of VTE (such as in malignancy or ICU patients). In these
PC and PS but will interfere with APTT-based activity assays.
patient populations, a high index of suspicion warrants further
For these reasons, it is a good practice to test for these deficiency
investigation. The addition of the more specific impedance
states at least 2 weeks after completing the initial 3- to 6-month
plethysmography or venous compression ultrasound yields a
course of oral anticoagulant therapy (PC and PS assays can be
more accurate diagnosis. On the other hand, elevated D-dimer
performed while the patient is anticoagulated with heparin).
levels are insufficient to establish the diagnosis of VTE, as
Deficiency states for PC, PS, and AT may be excluded if the lev-
elevated levels can be seen in hospitalized patients, infection,
els for these proteins are normal during the acute thrombotic
inflammation, surgery, pregnancy as well as other conditions.
episode. But finding a low level of these proteins during this
A confusing aspect of the D-dimer assay is the use of many dif-
period requires confirmation by repeat testing after anticoagula-
ferent concentration units in which the result is reported, which
tion is discontinued and sufficient time has elapsed for natural
makes interlaboratory comparisons difficult. D-dimers are
concentrations of factors to equilibrate (approximately 2 to
reported as either fibrinogen equivalent units (FEU) or D-dimer
4 weeks). Investigation of first-degree relatives can be useful to
units in ng/mL or mg/L or pg/mL or ug/L. In our coagulation
document the inherited nature of a deficiency.
lab, the threshold for exclusion of VTE is 230 ng/mL of D-dimer
Because most patients hospitalized for thrombosis are in
units. The ideal approach is that every hospital and lab should
the acute postthrombotic state and are receiving anticoagulant
establish their own cut-off values to rule out VTE. Because the
therapy or have illnesses that may interfere with testing, the
threshold values used in the literature may differ depending on
tests for PC, PS, and AT levels are generally better suited to
the methodology and reporting units used, the units can be con-
the outpatient setting and have a limited role in the diagnosis
verted based on the manufacture’s recommendation. The fol-
of thrombophilia in hospitalized patients.
lowing points regarding D-dimer are important to understand in
The genetically based tests, including factor V Leiden
the initial diagnosis of a thromboembolic episode:
mutation, prothrombin gene mutation, and MTHFR mutation,
1. D-dimer testing can be a sensitive test to screen for throm- are not affected by physiologic or pathologic states and anti-
boembolic disease, but is very non-specific, so a negative coagulation therapy. Therefore, the genetic tests can be per-
test is much more helpful than a positive result. formed in these settings.
2. ELISA testing methods have the best sensitivities (95%),
with a negative predictive value of 90%.
Testing in the Appropriate Clinical Setting
3. In patient populations where the likelihood of thromboem-
bolism is high, D-dimer alone may not be sufficient to This is a complex issue that extends beyond the scope of this
exclude a clotting episode.'°°! chapter; however, there are a few points to remember. The
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 685

development of a thrombus, regardless of the clinical setting, strategies will also evolve that will be directed by one or more
requires the accumulation of more than one inherited or of the thrombophilic risk factors.
acquired risk factor. Venous thrombosis in almost any clinical
setting would merit a comprehensive evaluation. For some
time, it was thought that evaluation for inherited throm- Anticoagulant Therapy
bophilia should be limited to the “young” patient with throm-
Patients with venous thrombosis are most frequently treated
bosis. Studies have demonstrated that the yield in detecting
with unfractionated heparin acutely, followed by long-term
inherited risk factors for thrombophilia in the aged occurs at
oral anticoagulants.'*"'*° This is done to prevent propagation
the same rate and would have the same value in guiding clin-
of the thrombus, to reduce the risk of embolus, and to allow
ical management.
for natural resolution. Because of difficulties with appropriate
monitoring, the use of LMWH is becoming more popular;
Functional Assays however, these medications are still considerably more
expensive than unfractionated heparin. Patients in a clinical
Many of the molecules of interest in thrombophilia can be setting (i.e., perioperative) with a high risk of thrombosis are
assayed using functional or antigenic assays. Whenever qual- also treated with low doses of anticoagulants to prevent
ity functional assays are available, they should be the first thrombus formation.
assays performed. In general, if the assay is within the refer-
ence range. further testing is not indicated. In contrast, if the
functional assay is below the reference range, evaluation by Unfractionated Heparin Therapy
antigenic assay is needed to determine if a normal amount of
abnormally functioning molecule is being produced. Heparin is an unbranched polysaccharide that is heavily sul-
fated, making it anionic. Commercial preparations are made
by extracting these molecules from bovine lungs or porcine
In Arterial Thrombosis, Consider intestines. Therefore, the heparins that are extracted are vari-
the Additional Evaluation of able in size (4 to 35 kD, averaging 12 to 13 kD). The nature
and degree of sulfation and the size of the molecule influence
Hyperhomocysteinemia and Lipoprotein a
the biologic activity.
The usefulness of testing for thrombophilia in patients with The anticoagulant activity of heparin is to function as a
arterial disease, particularly MI and stroke, continues to be dis- cofactor. When heparin is added to purified activated coagu-
cussed in the literature. A consensus has not evolved regarding lation factors (in the absence of AT), no anticoagulation
the usefulness of laboratory testing in these settings. Risks in occurs. Heparin acts by accelerating the rate at which AT is
relation to hyperhomocysteinemia and Lpa have been well capable of binding to the activated serine protease, irre-
demonstrated. LA and HIT can cause either venous or arterial versibly inhibiting its activity. In the absence of heparin, neu-
thrombosis. The absence of exposure to heparin or related tralization of thrombin or factor Xa will occur in a time frame
compounds eliminates the need to consider HIT. of approximately 15 minutes. In the presence of heparin, the
same reaction occurs more rapidly than it is possible to make
the first measurement.
Repeat Testing Prior to Diagnosis
Virtually all of the assays involved in the evaluation of throm- Administration and Monitoring
bophilia are influenced by a variety of factors in both the prean-
alytic and analytic phases. Because of this, false-positive testing Unfractionated heparin must be administered parenterally,
is acommon problem. Therefore, positive tests should be con- most frequently intravenously but it can also be used subcu-
firmed before the patient is labeled as having a deficiency state. taneously.'*” Clearance of heparin is primarily cellular (retic-
uloendothelial cells) and renal filtration. At usual doses, the
clearance is by the reticuloendothelial cells and provides an
Performing Testing and Making Evaluation average half-life of approximately 60 to 90 minutes. At higher
doses, renal filtration becomes a factor when the reticuloen-
Worthwhile
dothelial system is saturated. This may be an explanation for
These assays are not indicated as a routine preoperative eval- the varying half-life of heparin with increasing dose.
uation if the aim is to detect the risk of thrombosis in the The dose of heparin is determined by the weight of the
absence of a known personal or family history of thrombosis. patient. It is given as a bolus dose followed by continuous IV
Also, there is no valid indication for these tests in an elderly infusion. Owing to the length of the half-life of the drug,
patient who has a risk factor for venous thromboembolism. monitoring should not occur until equilibrium is reached at
Patients who present with thrombosis and who are compre- about 6 hours after beginning or changing therapy. Once
hensively evaluated will have more than a 50% probability of within the therapeutic interval, daily monitoring is sufficient.
having a thrombophilic risk factor identified. The usefulness When used for therapeutic purposes, heparin has a very
of determining thrombophilic risk factors continues to evolve. narrow therapeutic interval. The goal, of course, is to prevent
It is very likely that, as clinical studies evolve, treatment thrombosis without causing hemorrhage. If the drug is given in
686 — Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

inadequate doses and serum concentrations are below effective disadvantage is the wide variability resulting from the vari-
levels, the consequences can be thrombosis and embolism, ables described earlier.
causing significant morbidity and even mortality. When con- The heparin assay has the advantage of being repro-
centrations are elevated, the anticoagulant properties will cause ducible with more precise therapeutic intervals, making clin-
hemorrhage. It is the obligation of the laboratory to inform clin- ical management less problematic. Disadvantages include the
icians of the method that the laboratory recommends for mon- cost and a more technically demanding assay technique than
itoring heparin and the therapeutic interval. the APTT. Some clinicians are also concerned that a target
Heparin therapy can be monitored by one of two strate- concentration approach may not accurately reflect the state of
gies. The first involves monitoring the effect of heparin on the anticoagulation due to the numerous variables influencing in-
patient’s coagulation system, most frequently via the APTT. dividual patient response.
The second is the target concentration strategy in which the However, with the advent of better defined heparin prod-
concentration of heparin in the blood is determined. Both of ucts with more predictable patient responses, it is likely that
these strategies have been demonstrated to be effective in assays of heparin will move toward the target concentration
monitoring populations of patients receiving unfractionated strategy.
heparin. Unfortunately, when the two are compared directly,
there is very poor correlation between the concentration of
heparin and the resulting APTT. There is no dose-response Low-Molecular-Weight Heparin
relationship between the amount of heparin and the APTT.
Low-molecular-weight heparin (LMWH) is prepared from
The APTT has been used for the monitoring of heparin
unfractionated heparin by fractionation or depolymerization,
since the 1950s. The general “rule of thumb” is a therapeutic
which produces heparins of a more uniform molecular mass
interval of one-and-a-half to two-and-a-half times the upper
(ranging from | to 12 kD; averaging 5 kD) and function. They
limits of the reference range for the APTT. Problems in the
tend to have a greater impact on the inhibition of factor Xa than
monitoring of heparin are based on:
on other enzymes. A significant advantage of LMWH is that it
¢ Variability of heparin is administered subcutaneously and, in many patients, it does
¢ Patient response not require therapeutic monitoring. Dosing is generally once or
e Reagent twice daily and can be administered by patients, similar to the
¢ Instrument'** way diabetic patients can administer their own insulin.
It appears that LMWH will replace unfractionated heparin
It is necessary for each laboratory to determine the respon-
in many clinical circumstances where heparin is required. These
siveness of the instrument/reagent method for the APTT to
products are still not satisfactory for use in extracorporeal circu-
heparin in the patients’ specimens. This process needs to be
lation (cardiopulmonary bypass, extracorporeal membrane oxy-
done each time there is a change in the reagent or instrument
genation). In addition, despite the fact that many patients do not
used in the laboratory.
require monitoring, those of unusually low or high body weight,
The concentration of heparin in the blood can be reflected
patients with renal failure, pediatric patients, and patients with
by assays that take advantage of the neutralization of the activ-
other complicating medical conditions that may influence
ity of thrombin or factor Xa. These assays can use either a clot
heparin metabolism require careful ongoing evaluation. Moni-
endpoint or chromogenic substrate with spectrophotometric
toring of LMWH requires the target concentration strategy
endpoint. The assays provide the enzyme (thrombin or acti-
because the therapeutic concentrations do not influence the
vated factor X), the inhibitor (antithrombin), and a marker
APTT or other coagulation tests in a dose-dependent way. These
(fibrinogen in the plasma or chromogenic substrate). The rate
tests may, however, be prolonged during therapy and this should
or amount of conversion of the substrate is inversely propor-
be noted in testing situations before assigning a diagnosis of a
tional to the concentration of heparin present in the specimen.
hemostatic disorder.
Another assay for quantifying the amount of heparin in
A final comment is warranted regarding some preanalytic
the blood uses protamine sulfate, an antagonist to heparin.
issues in monitoring heparin therapy. A natural inhibitor of
Protamine sulfate binds to heparin in a stoichiometric fashion,
heparin, called PF4, is released from platelets. The presence of
neutralizing its activity. Assays, based on the thrombin time,
platelets in the specimen or the activation of platelets during col-
can be constructed using known concentrations of protamine
lection can thus lead to a false lowering of heparin assayed in
sulfate, thereby judging the amount of heparin by the amount
the specimen. Therefore, care should be taken to collect a high-
of protamine sulfate required to neutralize heparin.
quality specimen, and its preparation should include assuring
All of these approaches to monitoring heparin therapy
that the specimen has a very low platelet count. In addition, if
have their advantages and disadvantages. The dominating
plasma from a heparinized sample sits on cells, the PF4 can neu-
methods being used at the present time are the APTT and the
tralize the heparin, resulting in a falsely decreased APTT.
chromogenic assay, which uses the inhibition of factor Xa.
The APTT has the advantage of being inexpensive (one-tenth
the cost of the Xa inhibitory assay), it can be rapidly and eas-
Oral Anticoagulation
ily performed by many different technologists in the labora-
tory, is available at all hours, and reflects the anticoagulant As described in the synthesis of coagulation factors in the pre-
activity of heparin in the patient’s specimen. Its significant vious chapter, vitamin K plays a key role in the postribosomal
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 687

modification of many of the serine proteases. Vitamin K is a When monitoring patients who are taking oral anticoag-
cofactor for a carboxylase that inserts an additional carboxyl ulant therapy, the INR is maintained between 2 and 3. The
group on the glutamic acid residues in the amino terminal end exceptions to this therapeutic interval include patients who
of coagulation factors II, VII, [X, and X in addition to PC and have a complicating lupus anticoagulant in whom a higher
PS. This process is a kinetic one in which vitamin K, through INR may be indicated and patients who have cardiac valves
two enzymatic steps, is recycled and can be reused for the car- in whom the INR is recommended to be 2.5 to 3.5.
boxylation of multiple molecules. The greatest difficulty in managing oral anticoagulation
A coumarin compound, ultimately named warfarin, in patients with thrombosis is the large number of medica-
capable of inhibiting the recycling of vitamin K was identified, tions and other factors that can increase or decrease the
isolated, synthesized, and first administered to patients in 1941. metabolism of warfarin. These drug interactions are important
Warfarin acts by interfering with the recycling of vitamin K considerations for clinicians who are treating patients.
after it has performed its carboxylation function. This makes Recently, it has been thought that warfarin dose-response
warfarin interesting in two respects: (1) Warfarin is not truly an may be regulated at the transcriptional level. The variants in
anticoagulant; it leads to the production of coagulation factors the gene encoding vitamin K epoxide reductase complex
that have reduced anticoagulant function (adding warfarin to a | (VKORC1) has been described that may explain differences
tube of blood does not inhibit coagulation). (2) Warfarin func- in dose requirements among patients of different ethnici-
tions by changing the action of vitamin K in the synthesis of ties.'°! Genetic polymorphisms (i.e., CYP2C9) have been
coagulation factors from a kinetic one to a stoichiometric demonstrated in patients with resistance to warfarin.
one; that is, one vitamin K molecule produces one change in
the glutamic acid molecule rather than a single vitamin K
molecule being able to modify several through a cycling
Alternative Anticoagulants
process. DIRECT THROMBIN INHIBITORS
Because warfarin functions by altering the synthesis of Because some patients cannot tolerate heparin, especially
coagulation factors, the synthetic rate of coagulation factors is those with HIT, additional anticoagulants are currently being
variable. In most patients it takes 5 to 7 days of warfarin ther- used in a limited way. Direct thrombin inhibitors (DTIs)
apy before a stable anticoagulant effect can be achieved. inhibit thrombin through the binding of exosite | and the
Anticoagulant therapy with warfarin has the same prob- active binding site of thrombin.'”
lem that was described earlier with heparin. The drug has a Hirudin, an anticoagulant produced in the salivary
narrow therapeutic interval and less than optimal tests to glands of leeches, is a direct thrombin inhibitor that binds
monitor the drug. Since 1935, when Armand Quick described irreversibly in a 1:1 stoichiometric relationship. It has a
the prothrombin time, and in 1941, when the first patient was plasma half-life of approximately | hour. It has no structural
treated with warfarin, the PT has been used to monitor ther- similarity to heparin and does not cross-react with heparin-
apy. As with the problem of heparin and the APTT, it was dependent antibodies. A hirudin derivative (lepirudin) and
discovered in the late 1970s that there was a wide degree of recombinant hirudin are available for treatment of HIT.
variability in the response of thromboplastin reagents and Hirudin therapy is commonly monitored using the APTT,
instruments that were used for the PT when evaluating with a target value of 1.5 to 2.0 times the median of the nor-
patients who were receiving warfarin therapy. It has been pos- mal range. However, variable responses of APTT reagents to
sible to overcome some of the difficulties with this variability hirudin can be problematic. Early data show promising results
by normalizing the responses of thromboplastin reagents for the Ecarin clotting time (a snake venom—derived assay);
against an international standard. This process is referred to however, clinical outcome data are still being gathered. Daily
as the international normalized ratio (INR). The INR is monitoring is recommended because hirudin is predomi-
described by the following formula: nantly cleared by the kidneys. Therefore, monitoring is espe-
Pi
ISI cially important in patients with impaired renal function.'°*'™
Antibodies to lepirudin develop in one-third of patients on
INR =(=—=
a

PT initial exposure and in two-thirds patients on repeated expo-

n
sure. In addition, fatal anaphylactic reactions seen after sensi-
in which the INR is the international normalized ratio, tization to lepirudin preclude the use of this drug more
PT,,., is the patient’s prothrombin time, PT,, is the prothrombin than once.-?°
time that reflects the geometric mean of the reference range, Argatroban is a synthetic thrombin L-arginine derivative
and ISI is the international sensitivity index. The ISI needs to inhibitor that functions similarly to hirudin but is cleared even
be developed for each thromboplastin reagent and instrument in patients with moderate to severe renal failure. It shows
combination used in performing prothrombin times and cal- reversible inhibition and has a half life of approximately
culation of the INR. It is now understood that ideal reagents 45 minutes. It is a drug of choice in HIT patients, especially
for performing the INR are those that have ISIs below in patients in whom hirudin cannot be used (renal failure or
1.7.'#2!59 Some reagents are exquisitely sensitive, having an risk of anaphylaxis) with a target value of 1.5 to 3.0 times the
ISI of 1 or less, leading to other problems with accuracy and median of the normal range.'*’'°* The drug should be used
precision in specimens with high INR. The optimal reagent cautiously in patients with liver disease. Antibodies to arga-
would have an ISI of 1.3 to 1.5. troban have not been reported.
688 — Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

Bivalirudin is a reversible synthetic thrombin inhibitor ximelagatran shows oral bioavailability and has shown simi-
that can be used as an alternative drug in patients with HIT. It lar or enhanced efficacy when compared to warfarin with a
contains a C-terminus similar to hirudin that binds thrombin’s similar complication profile; however, questions regarding
exosite | and an amino terminus that binds thrombin’s active the risk of hepatotoxicity have currently delayed FDA
site. It is administered intravenously and has a half-life of approval. Another candidate for an oral thrombin inhibitor is
25 minutes. The current use is in coronary angioplasty for dabigatran, which is in clinical trials. Others in development
patients who cannot use heparins. DTIs can be monitored via may try to use tissue factor, factor VII, factor V, and factor VIII
anti-[la chromogenic assays. as potential targets. The use of recombinant activated protein
C is being developed.
FACTOR Xa INHIBITORS
Fondaparinux is a synthesized selective factor Xa inhibitor. A
pentasaccharide, it binds AT and increases its affinity for Antiplatelet Agents
factor Xa more than 300-fold. The molecule effects a confor- Used alone or in combination with other anticoagulants, a
mational change in AT and is released to inhibit another mol- number of antiplatelet function agents have been developed.
ecule. Because the inhibition is more specific for factor Xa, By far the most commonly used agent is aspirin. It functions
bleeding complications are expected to be decreased due to a by inhibiting prostaglandin synthesis. Its action has been well
small amount of thrombin generated by other uninhibited recognized for over 20 years, and its use (in low doses) for
coagulation factors. It is administered subcutaneously and has prophylaxis to prevent stroke and MI has been well character-
a half-life of 17 hours. A long acting variation, idraparinux, ized. Aspirin has been used in combination with heparin or
requires once a week dosing and is currently in clinical trials. LMWH in some patients who have arterial thrombotic events.
Oral factor Xa inhibitors are also in development and reliable Vascular surgeons and cardiologists are becoming
data are anxiously awaited. increasingly adept at using thrombolytic agents to lyse clots
formed in coronary arteries and other vessels. The site of
OTHER ANTICOAGULANTS
these thrombi can then be stented with devices that hold the
Danaparoid, a heparinoid, is a mixture of the glycosaminogly-
lumen of the artery open and allow recirculation beyond the
cans heparan sulfate, dermatan sulfate, and chondroitin sul-
previous occlusion. The difficulty that has occurred with these
fate. It is not available in the United States, but is used in
therapies has been the prevention of rethrombosis of the
Europe and Canada. Its anticoagulant effect is by the inactiva-
vessel. The most promising of the agents used in a postthrom-
tion of factor Xa. It also has some antithrombin (anti-IIa)
bolytic setting or with stents have been inhibitors of platelet
effects and interacts with heparin cofactor II. It can be admin-
function with or without heparin. Those used prevent platelet
istered subcutaneously or intravenously and has a half-life of
aggregation by binding to the GPIIb/IIla receptor on the sur-
18 hours. Because of a somewhat predictable distribution and
face of the platelet. In addition, new agents bind specifically
metabolism, dosing can be based on the patient’s weight.
to the ADP receptor on the surface of the platelet, thereby pre-
When used for the purpose of prophylaxis to prevent throm-
venting activation of the platelet. Three intravenous GP
bosis, it does not require monitoring. However, when needed
I[b/Ila inhibitors are in clinical use. Abciximab is a mono-
to therapeutically treat patients with thrombosis, the drug
clonal antibody directed against the receptor protein, and
requires monitoring using the Xa inhibitory assay. Thrombin-
tirofiban and eptifibatide are non-antibody receptor inhibitors.
based assays are not effective. Although recent publications
Of note, profound thrombocytopenia can develop within
suggest monitoring is not required during treatment unless the
hours of use of these drugs and should be suspected in the cor-
patient is very large (more than 90 kg) or very small (less than
rect clinical setting.
55 kg) or has some degree of renal failure, it seems prudent to
Oral GPIIb/IIIa inhibitors have been in development but
monitor danaparoid therapy. This is performed using the anti-Xa
have been associated with increased mortality and increased
activity assay (target range is 0.5 to 0.8 anti-Xa U/mL) and
bleeding risk as compared to placebo. The underlying mech-
requires the use of a standard curve prepared with danaparoid.
anism is not well understood.
H-PF4 antibodies may cross-react with danaparoid in vitro
Other antiplatelet agents are likely to be developed in the
but do not appear to have a negative effect on patient out-
near future. Their effectiveness is gradually increasing, as
come.'**'** However, new thromboembolic episodes occur
clinicians become more accustomed to their application. In
more frequently in patients treated with danaparoid than those
general, these antiplatelet agents have not required laboratory
using lepirudin.'* In addition, the rate of cross-reactivity of
monitoring.
danaparoid with heparin is approximately 3%. Another disad-
vantage is the absence of an antidote (protamine sulfate, the
antidote for unfractionated heparin and LMWH, has no effect
Thrombolytic Therapy
on danaparoid).
Anticoagulants currently in clinical trials are striving for If a thrombus has been formed and has been in place in situ
ease of administration and efficacy with low risk of bleeding for a short period of time, precluding organization, logic
complications. Melagatran and Ximelagatran are dipeptide would dictate that accelerating the lysis of the clot may help
mimics of fibrinopeptide A’s binding region to thrombin’s preserve function in the vascular bed served by the vessel.
active site. Melagatran is administered subcutaneously but Thrombolytic therapies using urokinase and streptokinase
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 689

have been available for more than two decades. More

recently, other thrombolytic agents have become available. PERTINENT PHYSICAL FINDINGS
The medications that convert plasminogen to plasmin Vital Signs: Blood pressure, 110/75 mm Hg; respirations,
without fibrin as a cofactor may be classified in two categories: 20/min; pulse, 72/min and regular; temperature, 38°C;
(1) agents that convert plasminogen to plasmin directly and weight, 130 kg.
(2) agents that are fibrin specific. In the former group are strep- Chest: Distant breath sounds on the left with dullness to
tokinase or urokinase. The latter group includes t-PA. percussion. Heart examination is not remarkable.
Because the goal is the lysis of fibrin clots, agents that are Bones, Joints, and Muscle: Right leg, ankle, and foot are
specific for fibrin would logically be preferred. Streptokinase slightly edematous and tender to palpation.
and urokinase cause a hypofibrinogenemia secondary to fib-
rinogenolysis in virtually all patients, even when the medica- LABORATORY FINDINGS
tion is given by a catheter into the specific artery containing Hgb, 14.5 g/dL; WBC, 12,000/uL (1); differential WBC:
the clot. In practice, the magnitude of this hypofibrinogenemia Neutrophils, 9100/uL (1); lymphocytes, 2200/uL; monocytes
tends not to be clinically significant with regard to hemor- 500/uL; eosinophils, 200/uL; platelet count; 220,000/uL
rhage, although hemorrhagic complications can occur. The PT, 12 seconds; INR, 1.0; APTT, 22 seconds; fibrinogen,
advantage of the fibrin-specific agents is that it is feasible to 269 mg/dL; thrombin time, 18 seconds; D-dimer,
administer these agent systemically rather into the vessel con- 500 ng/mL (7)
taining the thrombus in a catheter-directed fashion. It would be Cholesterol, 230 mg/dL (1), HDL, 25 mg/dL (J)
incorrect to assume that generalized fibrinogenolysis is pre-
cluded by fibrin specificity. In fact, t-PA can bind to circulating QUESTIONS
fibrin degradation products, activate plasmin, and cleave fibrino- 1. What is the differential diagnosis?
gen. Therefore, although less frequent and less severe, fibrin- 2. What diagnostic procedures may be of value?
specific agents can be complicated by hypofibrinogenemia. 3. What laboratory tests may be helpful in determining the
The thrombolytic activity in thrombolytic therapy, in gen- etiology of the condition (a) in the acute setting?
eral, is not monitored. However, prior hemostatic evaluation is (b) after cessation of anticoagulation?
of value. First, evaluation of hemostasis can be helpful in pre- . What risk factors are present in this patient?
dicting which patients are at the greatest risk for bleeding when . What therapy is indicated?
thrombolytic therapy is used. Second, patients who have had . How is the therapy monitored?
thrombolytic therapy that needs to be followed by a surgical
procedure, such as coronary artery bypass, need to have their he- ANSWERS
mostatic mechanism evaluated before the surgical procedure. 1. Swelling in the leg could be lymphatic obstruction from
Evaluation in this case would include the usual initial tests of infection or tumor. Chest pain has several possibilities in
hemostatic evaluation: PT, APTT, fibrinogen assay, thrombin the cardiac and pulmonary areas. However, collectively,
time, and platelet count. Frequently these patients are receiving in this sequence is suggestive of a venous thromboem-
therapeutic heparin, complicating the evaluation. Should evalu- bolism (VTE) or DVT with PE.
ation still be considered necessary, heparinase can be added to . Ventilation perfusion scan of the lungs, chest x-ray
the specimen to neutralize the heparin effect. exam, high-resolution CT scan, Doppler imaging of the
deep veins of the upper legs and, possibly, pulmonary
angiogram. V/Q and Doppler scans were positive in
this case. Chest x-ray exam showed an effusion on the
right.
Case Study 1 . (a) Activated protein C resistance, factor V Leiden by
PCR, prothrombin gene mutation 20210 by PCR,
MTHER by PCR, possibly anticardiolipin. (b) After
A 40-year-old man presented to the emergency department recovery and conclusion of anticoagulation, protein C,
with a swollen, painful right leg of 3 days’ duration and protein S, AT. All of these were performed, and the
shortness of breath for the past 12 hours. He also had sharp patient was found to be heterozygous for factor V
pain in the left side of his chest when he took a deep breath. Leiden only.
. Obesity, factor V Leiden, occupation. Cigarette smoking
PERTINENT HISTORY and abnormal lipid profiles are associated with arterial,
but not venous, thrombosis.
Past Illnesses: Had pneumonia 8 years ago with full
Nn. Parenteral anticoagulation (unfractionated heparin or
recovery.
LMWH) followed by oral anticoagulation.
Occupation: Cross-country truck driver.
. APTT and PT/INR.
Social Habits: Drinks alcohol occasionally on a social
basis. Has smoked a pack of cigarettes a day for 22 years.
Family History: Mother died of “clots in her lungs.” Father
and siblings are alive and well. One sister had a “clot in her
leg” after she delivered her only child.
continued
690 ~~ Chapter28 Introduction to Thrombosis
and Anticoagulant Therapy

QUESTIONS
Case Study 2 1. What is the initial test that should be ordered to evaluate
the patient’s prolonged APTT? What can this test tell
A 28-year-old gravida 2, para 0 woman desires to have chil-
you about the clotting abnormality?
dren. She seeks advice because she has miscarried two prior
2. An equal mix of the patient’s plasma with normal
pregnancies and is concerned about her ability to have a suc-
plasma demonstrates an APTT of 62 seconds. What can
cessful pregnancy.
you conclude regarding the cause of the prolongation of
Current Medications: Patient reports taking aspirin for
her APTT?
headaches but otherwise takes no medications.
3. Further laboratory testing reveals a prolonged dilute
Family History: Parents and siblings are alive and well.
Russell’s viper venom time, which normalizes when
excess phospholipid is added to the plasma. Based on
PERTINENT HISTORY the results of these tests, what is the probable cause of
Hematopoietic: No history of anemia, bleeding disorder, her prolonged APTT?
jaundice, or easy bruising. Two years ago she had a clot in 4. What further testing should be performed to help con-
her left leg that was treated with anticoagulants for firm these results?
6 months. The leg has a small amount of swelling by the end
of the day. She has never received a blood transfusion. ANSWERS
Menstrual History: Unremarkable menstrual cycle,
1. The 1:1 mixing study with normal plasma; the test will
although somewhat irregular. Last menstrual period:
differentiate between a coagulation inhibitor or factor
39 days ago.
deficiency.
2. The 1:1 mixing study fails to completely correct the
PERTINENT PHYSICAL FINDINGS APTT, indicating the presence of an inhibitor.
Vital Signs: Blood pressure, 125/80 mm Hg; respirations, 3. She has a lupus anticoagulant and history consistent
16/min; pulse, 72/min, and regular. with the antiphospholipid syndrome (recurrent sponta-
Skin: Mild livedo reticularis is noted on distal arms and legs. neous abortion, mild thrombocytopenia, and deep
Pelvic and Rectal Examination: The cervix is parous and venous thrombosis).
soft. 4. Repeat testing for lupus anticoagulant in 6 to 8 weeks to
Bones, Joints, and Muscle: No edema is apparent in the left show persistence and correlate with clinical history.
leg at the time of the examination.

LABORATORY FINDINGS
Hgb, 13.5 g/dL; WBC, 8000/uL; differential WBC:
Neutrophils, 5100/uL; lymphocytes, 2200/uL; monocytes,
500/uL; eosinophils, 200/uL; platelet count, 100,000/uL.
PT, 12 seconds; INR, 1.0; APTT, 70 seconds (1): fibrinogen,
269 mg/dL; thrombin time, 18 seconds.
continued

a oi one
1. Which characteristic of endothelial cells suppresses te. Fibroblast
fibrinolysis? d. Eosinophil
a. Platelet adhesion e. Erythrocyte
b. Production of t-PA inhibitor
4. A 28~year-old man suffered from recurrent deep venous
c. Secretion of von Willebrand’s factor thrombosis and pulmonary embolus. Studies in the labo-
d. Release of tissue factors
ratory show that his plasma is unable to inhibit factors
2. Which of the following statements concerning warfarin Villa and Va, indicating the plasma is deficient in which
is true? naturally occurring inhibitor/anticoagulant?
a. It inhibits production of contact coagulation factors. jot). Protein C

b. It decreases the function of vitamin K—dependent factors. b. Antithrombin

c. It interferes with cyclic vitamin K before carboxylation. c. High-molecular-weight kininogen
d. It will inhibit coagulation in a standard tube of blood. d. Ferrochelatase
3. Which cell plays a key role in the regulation of hemostasis? e. Fibronectin
a. Endothelial cell 5. A 28-year-old woman with known systemic lupus ery-
b. Neutrophil thematosus presents with a swollen painful left leg. She
 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy 691

had had one prior deep venous thrombosis. She has no c. Factor V Leiden
children but has had two spontaneous abortions. Which d Mutated forms of factors V and VII
laboratory test do you expect to be often abnormal?
8. What is the most significant inhibitor of tissue plasmino-
a. Thrombin time
gen activator?
oa . Activated partial thromboplastin time
a. Plasminogen activator inhibitor- |
. Assay for factor V
b. Plasminogen activator inhibitor-2
. Assay for plasminogen
c. Plasminogen activator inhibitor-3
CO
ee. Assay for antithrombin
d. Plasminogen activator inhibitor-4
6. A 25-year-old woman presents with a thrombus in her
9. What is the major inhibitor of thrombin and factor Xa?
right leg. She had a similar event 2 years ago in the left
a. Protein C
Jeg. Her mother, a paternal uncle, and her sister have all
b. Antithrombin
had episodes of thrombosis, and one of these was a pul-
c. Protein S
monary thromboembolus. Of the following potential eti- d. Heparin cofactor II
ologies; which is the most likely?
a. Protein S deficiency 10. Which of the following is not a thrombolytic agent?
b. Antithrombin deficiency a. Tissue plasminogen activator
c. Hyperhomocysteinemia b. Urokinase
d. Factor V Leiden ‘ c. Streptokinase
e. Factor XI deficiency d. a,-Antiplasmin

7. What is the most common cause of activated protein C See answers at the back of this book.
resistance?
a. Functional PS deficiency
b. Inhibitors of activated protein C

@ Inherited thrombophilia is a group of congenital hemato-

logic disorders that includes a variety of hypercoagulable
states that usually present as venous or arterial thrombo-
a Endothelial cells function to facilitate platelet adhesion sis, or both.
and platelet activation at the site of injury, secrete von
Willebrand’s factor, and secrete an inhibitor of tissue plas- w Activated protein C resistance (APC-R) can be defined as
minogen, which suppresses fibrinolysis. deficient anticoagulant response of plasma to the addition
of APC. Causes of APC-R include inhibitors to APC,
m Natural anticoagulants in plasma include antithrombin functional PS deficiency, and mutated forms of factor V
(AT), heparin cofactor II (HC-II), protein C (PC),
and VIII molecules; factor V Leiden is the most common
protein S (PS), and tissue factor pathway inhibitor (TFPI). cause of APC-R.
mwAntithrombin is a major inhibitor of thrombin and g@The prothrombin nucleotide G20210A mutation can be
factor Xa. described as a single guanine to adenine mutation at
m Protein C is a vitamin K—dependent zymogen that, nucleotide position 20210 and is associated with
once activated by thrombin, proteolytically degrades increased risk of deep vein thrombosis and elevated pro-
factors VIIla and Va, two of the major cofactors thrombin levels.
involved in thrombin generation. w Antiphospholipid (aPL) syndrome is an acquired throm-
a TFPI inhibits plasma coagulation by (1) binding the acti- botic disorder. Laboratory evidence of aPL antibodies can
vated form of either factor X or factor LX, and (2) inhibit- be in the form of either immunologically demonstrated
ing further binding of factor X or factor IX via the aPL antibodies; enzyme-linked immunosorbent assay
TFPI-X-IX membrane complex. (ELISA) for anticardiolipin antibody, and/or lupus
@ Inhibitors of plasmin include a,-antiplasmin, @,-antitrypsin, anticoagulant.
a-macroglobulin, AT, and C1 esterase inhibitor. mw The APTT may be prolonged in heparin therapy or conta-
mination, insufficient volume of sample, disseminated
w Inhibitors of plasminogen include plasminogen activator
intravascular coagulation (DIC), factor deficiencies, and
inhibitor-1 (PAI-1), PAI-2, and PAI-3, with PAI-1 being
the most significant inhibitor of tissue plasminogen antibodies directed against factors in intrinsic or common
pathways.
activator (t-PA).
continued
692 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

They inhibit factor Xa more efficiently and do not require

therapeutic monitoring.

w Heparin-induced thrombocytopenia is described as a sud- w Vitamin K is a cofactor for a carboxylase that inserts an
den decrease in the platelet count 5 days after heparin additional carboxyl group on the glutamic acid residues in
therapy is initiated and is caused by the interaction of the amino terminal end of coagulation factors I, VII, IX,
platelet factor 4 (PF4) on the platelet membrane. and X.

w Unfractionated heparin is an unbranched polysaccharide mw Warfarin therapy or oral anticoagulation therapy acts by
that is prepared from bovine lungs or porcine intestines with interfering with the recycling of vitamin K and depressing
a range of molecular mass from 4000 to 35,000 daltons. It the activities of factors II, VII, LX, and X; PC; and PS.
acts by accelerating the rate at which AT is capable of w Warfarin therapy is monitored by the prothrombin time
binding thrombin. Heparin therapy is monitored by the (PT) and international normalized ratio (INR); the INR is
APTT, with a target value of one-and-a-half times the maintained between 2 and 3 for patients on anticoagulant
upper limits of the reference range for the APTT. therapy.
@ Low-molecular-weight heparin (LMWH) is_ prepared wg Thrombolytic agents include t-PA, urokinase, and strep-
from unfractionated heparin by fractionation or depoly- tokinase. Hemostatic evaluation includes PT, APTT,
merization, producing heparins of 1000 to 12,000 daltons. fibrinogen assay, thrombin time, and platelet count.

Acknowledgment
The authors wish to express their deep gratitude to
John D. Olson, MD, PhD, for his assistance in preparing this
chapter. His knowledge of and commitment to the subject
were inspiring and invaluable. Without his guidance this work
would not have been possible.

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nolysis 3(6):749, 1992. elevation is found twice as frequent as in Haematol 116(1):173, 2002.
694 Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy

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PART 5
LABORATORY METHODS

Chapter ')

Quality Management,
Quality Assurance,
and Quality Control
Kim A. Przekop, BS, MT(ASCP)

introduction OBJECTIVES
Quality Management At the end of this chapter the learner should be able to:
Definition of Quality
Management and Quality . Define a quality laboratory test result.
Testing . List four divisions of quality management and their role in laboratory quality.
Legal Implications
“Quality Management . Explain three quality approaches.
Improves Bottom Lines” . Describe two general quality control activities.
Quality Management Plans
Quality Approaches . Recall quality questions to ask when choosing a new method.
Divisions of Quality . Illustrate the difference between accuracy and precision.
Management
Quality Software . Calculate the mean, standard deviation, and coefficient of variation from a set of data.

Quality Assurance and . Characterize random and systematic error.

Quality Control
. Identify the experiments that should be performed in validation of a new method.
Definitions of Quality
Assurance, Quality . Suggest an experiment in which a correlation coefficient would be useful.
Control, and Quality
. Explain the importance of the CLIA-88 guidelines in laboratory quality.
Assessment
General Quality Assurance . Understand the minimum quality required by CLIA-88.
Control Activity . Describe a Gaussian distribution and how it applies to quality control.
Guidelines
Preanalytical, Analytical, . Show examples of a trend and shift from a Levy—Jennings graph.
Postanalytical, and . List three Westgard Rules and describe how to use them in quality control.
Nonanalytical Factors in
Testing . Summarize QC troubleshooting paths depending on whether the violated Westgard
Accuracy, Precision, and Rule is random or systematic.
Error . Illustrate the Six Sigma scale and how it can enumerate the quality of a test.
Method Validation
Quality Control Definitions
Minimum Quality Control
Requirements, CLIA-88 CFR
Levy—-Jennings Graphs
Westgard MultiRule Quality
Control
697
698 — Chapter 29 Quality Management, Quality Assurance, and Quality Control

Sigma-metrics
Troubleshooting Quality
Control Problems
Peer Group Quality Control
Hematology Laboratory
Applications
Quality Plan Example
Method Validation Studies
Quality Control
Laboratory Quality Updates
Quality Meeting Launches
New Organization
Equivalent Quality Control
Option 4
Case Study 1
Case Study 2

Introduction The birth of total quality in the United States came as

a direct response to the quality revolution in Japan follow-
In the last few years, the health-care industry has adopted suc- ing World War II. The Japanese welcomed the input of
cessful quality control (QC) practices already in use in the man- Americans Joseph M. Juran and W. Edwards Deming, and
ufacturing and other industries. News of preventable deaths and rather than concentrating on inspection, focused on
incorrect diagnoses and treatments caused a search for better improving all organizational processes through the people
ways to define quality and monitoring systems. Every part of who used them. By the 1970s, U.S. industrial sectors such
every process is now under scrutiny. Sophisticated statistical as automobiles and electronics had been broadsided
approaches and error-free goals are now becoming mainstream by Japan’s high-quality competition. The U.S.
response,
in hospitals and laboratories. Regulations usually require emphasizing not only statistics but also approaches that
the minimum QC, but is this enough? Probably not. In this embraced the entire organization, became known as Total
chapter some of the top minds in the QC field will help the lab- Quality Management (TQM).
oratorian navigate through the alphabet soup of regulatory By the last decade of the 20th century, TQM was con-
agencies and standards, and learn how to uncover the real sidered a fad by many business leaders. But while the use of
meanings of and methods to obtain a quality result. the term TQM has faded somewhat, particularly in the
United States, its practices continue. In the few years since
The quality movement can trace its roots back to medieval
the turn of the century, the quality movement seems to have
Europe, where craftsmen began organizing into unions called
matured beyond Total Quality (Fig. 29-1). New quality sys-
guilds in the late 13th century. Until the early 19th century,
tems have evolved from the foundations of Deming, Juran
manufacturing in the industrialized world tended to follow
and the early Japanese practitioners of quality, and quality
this craftsmanship model. The factory system, with its empha-
has moved beyond manufacturing into service, healthcare,
sis on product inspection, started in Great Britain in the mid-
education and government sectors. (Reprinted with permis-
1750s and grew into the Industrial Revolution in the early
sion from www.asq.org © 2006 American Society for
1800s.
Quality).
In the early 20th century, manufacturers began to include
quality processes in quality practices. After the United States
entered World War II, quality became a critical component of Guilds Sampling USA TQM
the war effort: bullets manufactured in one state, for example,
had to work consistently in rifles made in another. The armed
forces initially inspected virtually every unit of product; then
to simplify and speed up this process without compromising
safety, the military began to use sampling techniques for
inspection, aided by the publication of military-specification Factories Japan TQM we
standards and training courses in Walter Shewhart’s statisti-
Figure 29-] m The history of quality.
cal process control techniques.
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 699

Quality Management technical personnel can be held liable for harm if the patient
can show that the laboratory person acted negligently. '
It is most important that top management be quality-minded.
In the absence of sincere manifestation of interest at the top,
little will happen below. “Quality Management Improves
Bottom Lines”
—Joseph M. Juran
Companies that were reluctant to include Total Quality
Definition of Quality Management Management (TQM) in their policies in the 1970s and 1980s
and Quality Testing were reassured of the positive financial impact on their
bottom lines with several studies. One study looked at the
Quality Management (QM) comprises all of the processes winners of quality awards versus control companies.
that affect the quality of a product (laboratory test result) from The extent to which award-winning companies signifi-
beginning to end. What is quality (Fig. 29-2)? A quality test cantly outperformed the controls during the period following
result is free of defects (accurate and precise), gives the clin- TQM implementation is shown in Figure 29-3. Award-winning
ician the information he or she needs to make a medical deci- companies experienced an average 91% growth in operating
sion (customer service), and is within a reasonable cost (cost income compared to 43% for the controls; 69% increase in
efficient). How does a laboratorian ensure quality results? sales versus 32%; 79% increase in total assets versus 37%;
Processes are instituted at every step of the way to comply 23% increase in number of employees versus 7%; 8% rise in
with regulatory guidelines and best practice models. Quality return on sales versus no improvement for the controls; and 9%
tools are used correctly to find all types of errors and apply improvement in return on assets versus 6%. They sum up their
that knowledge to the control systems. Everyone is involved findings with three guidelines for companies implementing
in decision-making, including the physicians who will be quality practices:
ordering the test. From the patient’s posture (sitting vs. lying
1. TQM is a good investment. (“Don’t give up on TQM.
down) and manner of collection, to the analysis of a tube of
When implemented effectively, it improves financial per-
blood on an instrument and the acceptance of results in a
formance dramatically.”’)
computer; there are a myriad of variables to consider and to
2. Be patient. (“The benefits of TQM are achieved over a long
control.
period.... Even after effective implementation, it still takes
a couple of years before financial performance starts to
Legal Implications improve.)
3. Be realistic. (“Set realistic expectations on the potential
There are possible legal costs to laboratories and to personnel impact of TQM. Organizational characteristics such as
when quality management procedures are not instituted or size, capital intensity, extent of diversification, and the
followed. When a laboratory professional fails to adhere to maturity of the TQM implementations influence the gains
“that which is acceptable,” errors may occur that lead to from TQM. These and other factors should be considered
adverse consequences for patients. In general, laboratories are in setting expectations.”)*
more likely to be liable for regulatory violations than to be
sued for negligence (medical malpractice) or other legal rea-
sons. However, litigation can occur and individual laboratory Quality Management Plans
The Centers for Medicare and Medicaid Services (CMS) regu-
late all laboratory testing (except research) performed on
Accurate &
humans in the United States through the Clinical Laboratory
precise
Improvement Amendments of 1988 (CLIA-88). These amend-
ments have been updated five times and were finalized in 2003.
According to CLIA’s “Survey Procedures and Interpretive
Guidelines for Laboratories and Laboratory Services (Appen-
dix C) Part 493, Laboratory Requirements,” all laboratories
that perform patient testing need a quality management plan.’
Quality
laboratory This chapter is mostly concerned with non-waived testing
test result (moderate and highly complex tests). To assist laboratories, the
College of American Pathologists (CAP) has incorporated reg-
ulations from CLIA into their checklist questions on quality.
‘Customer Cost Even if a lab is not registered with CAP, the checklists are
service efficient
available (www.cap.org). There are examples of key indicators

Figure 29-2 m The elements of a quality laboratory result. *Reprinted with permission from www.asq.org © 2004 American Society for
Quality.
700 Chapter 29 Quality Management, Quality Assurance, and Quality Control

100
@ Quality award winners
B Control firms

Oo©

40
30
performance
in
change
%
20
10
-
Operating Sales Total Employees Return on Return on
income assets sales assets

Figure 29-3 ® Quality award-winning companies outperform control firms. (Reprinted with permission from Hendricks and Singhal, “Don’t
Count TQM Out.” Quality Progress, April, 1999, p. 38.)

to monitor, how to interpret statistical data, how to measure the world-class company (not necessarily another laboratory) to
effectiveness of a plan, etc. The following are a few of the per- learn what can be done better. For example, if a laboratory wants
tinent questions: to raise the standard of its courier service to excellent, it might
compare its current service to UPS or FedEx, both considered
* Does the Quality Management (QM) plan cover all aspects
best in class, and implement its quality practices. °
of the laboratory service?
¢ Are appropriate corrective and/or preventive actions taken
ROOT CAUSE ANALYSIS
when opportunities for improvement are identified?
¢ Has the QM plan been implemented as designed? This approach is used after a problem occurs to investigate
* Does the laboratory summarize and review its records of errors what happened, how it happened, and why it happened. There
and incident reports at defined intervals to identify trends and are four steps to a Root Cause Analysis:
initiate corrective/preventive actions as appropriate? 1. Data collection
* Does the laboratory have a procedure for reporting device- 2. Charting of each sequence of events for each contributing
related adverse patient events, as required by FDA (Food factor
and Drug Administration)? * 3. Root Cause Mapping to uncover how a factor contributed
A laboratory can follow any currently accepted quality-related to the problem
plan, or establish its own plan. A quality management plan is not 4. Recommendations and Implementation
designed or expected to eliminate all errors in a laboratory. The
primary objective should be patient safety based on quality test INTERNATIONAL STANDARDS ORGANIZATION (ISO)
results. Every plan should contain the following elements: The ISO organization has members from more than 156 coun-
tries. Their common goal is to standardize quality products
* Climate of patient safety and quality
around the globe and provide a common technological lan-
¢ Risk assessment
guage to transfer knowledge. ISO 9000:2000 is an industry
¢ Control measures to monitor high-risk processes
standard for QM systems. 7
¢ Data management; customer service
¢ Continuous improvement.*?
SIX SIGMA
This approach focuses on reducing waste by reducing varia-
Quality Approaches tion. The term “Six Sigma” refers to a calculation for the
Sigma scale; a quality process would have a Sigma score
There are numerous books on approaches to laboratory qual- between 3 and 6 Sigma. To achieve Six Sigma (world-class
ity management plans. However, to simplify the process, sev- quality), a process must not produce more than 3.4 defects per
eral popular quality approaches are defined below. million opportunities. Many organizations, from the govern-
ment to private and public companies, are now using Six
BENCHMARKING Sigma practices. New York-Presbyterian Hospital began their
World-class quality cannot be obtained by simply comparing Six Sigma program in 2003. Today it is ranked 7th in U.S.
one laboratory’s process to another laboratory’s process. Bench- News & World Report's Best Hospitals Honor Roll (before
marking requires that performance be measured against another implementing Six Sigma they were ranked 14th.) *!°
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 701

RISK MANAGEMENT AND FAILURE MODE and results. The president of the United States gives the award
AND EFFECTS ANALYSIS annually to companies in varying sectors who apply. Since
Risk Management came from the insurance and financial sec- 1988, 65 companies have been honored with this award. The
tors, and basically asks, “What can go wrong, and what can first Healthcare Award was given in 2002 to the Franciscan
we do about it?” Failure Mode and Effects Analysis (FMEA) Sisters of Mary Healthcare System in the Midwest, which
began in the aerospace industry in the 1960s and is a proac- includes 21 hospitals and 3 nursing homes in 4 states.'°
tive statistical approach used to identify possible failures of a
product or service and then determine the frequency and
impact of the failure. For example, a test result would be the Divisions of Quality Management
product, and a failure could be an incorrect test result that
In a clinical laboratory, there are 12 major divisions of Qual-
could potentially harm the patient. A Hazard score can be
ity Management, also called Quality System Essentials. They
obtained by combining a rating of the severity of harm with
are Organization, Facilities and Safety, Information Manage-
the frequency that the harm might occur. The relationship
ment, Occurrence Management, Customer Service, Person-
between cause and effect is at the heart of FMEA. Six Sigma
nel, Equipment, Documents and Records, Assessments,
includes the discipline of FMEA." ?
Purchasing and Inventory, Process Control, and Process
Improvement (Fig. 29-4). There are many steps that can
LEAN
affect the quality of a patient test result, and all steps need to
Maximizing customer value is the main goal of Lean. The
be subjected to quality standards.
ideas of Lean were pioneered by the Toyota car maker after
WWII, and some expressions of Lean still have Japanese
ORGANIZATION
names today. The Lean method identifies seven types of
All personnel should commit to quality at every step of every
waste. Examples of laboratory waste include:
process.
1. Defects: Errors in laboratory results or reports, missing
reports, late reports. FACILITIES AND SAFETY
2. Waiting: Time periods where laboratory personnel are The layout of buildings, elevators, walkways, and storage
waiting for the patient or the next batch of samples at rooms should facilitate easy access and short time frames for
receiving or testing. transporting specimens and supplies. Work areas should
3. Extra processing: Rework, such as redraws, retesting, incorporate a workflow approach to reduce turnaround times
rehandling, or resending that occur because of defects in and wasted movement. Temperatures are critical in a labora-
the original effort. tory and should be stable. The most up-to-date protective
4. Transport: Unnecessary movement of samples, supplies, equipment and procedures should be in use to protect work-
paper, or staff, and retrieval of lost, moved, or misplaced ers from injury and illness; unscheduled time off from work
items needed in the work. affects quality. Nonworkers are also considered in any safety
5. Motion: Extra steps taken by staff to accommodate and infection control plan.
“workarounds” arising from inefficient layout of sample
paths, instrumentation, supplies, storage, or information. INFORMATION MANAGEMENT
6. Inventory: Extra inventory of laboratory reagents, supplies, There are multiple concerns for the laboratory concerning the
paper, and other materials that is not directly required for control of information. Patient data is readily available, in
the current work. paper and electronic form, and must be protected from inter-
7. Overproduction: Performing unnecessary laboratory test- nal and external threats. Statistical reports and company
ing for any reason. information on employee computers is vulnerable. Regardless
of the cost, it is imperative that every healthcare facility
A person can be certified as a Lean Green Belt. The Mayo review the risks and implement procedures to protect vital
Clinic recently adopted Lean principles in its operations.'>"* information from attack or abuse.

TOTAL QUALITY MANAGEMENT OCCURRENCE MANAGEMENT

Total Quality Management (TQM) is a holistic approach that Adverse events such as accidents, errors, and customer com-
involves all individuals in an organization. Together, they plaints should be detected, documented, and analyzed. The
improve processes and ultimately increase the quality of their processes involved should then be reviewed in detail to prevent
products to meet customer expectations. The tools used can future occurrences. Root Cause Analysis would be appropriate
be any of those listed in this chapter, or others that the orga- here.
nization decides are useful to improve quality.
CUSTOMER SERVICE
MALCOLM BALDRIGE NATIONAL QUALITY AWARD A crucial piece of quality, but sometimes overlooked. Who is
Any organization can apply the seven criteria used for this the customer? The customer is the recipient of a product or
prestigious award: leadership; strategic planning; customer service. Technologists should ask who the very next person is
and market focus; measurement/analysis and knowledge in the chain of information.'® Every laboratory test and the for-
management; human resource focus; process management; mat of that result should have meaning to a nurse/physician,
702 Chapter 29 Quality Management, Quality Assurance, and Quality Control

Customer
Service

Purchasing
Documents
and Inventory
and Records
Information
Management

Personnel

Process Facilities/Safety
Improvement
Process Control

Figure 29-4 ™ The 12 divisions of Quality Management (QM), also called Quality Systems Essentials (QSE).

who will then translate the result into an action for a Procedures should follow current guidelines and need to be
patient. The best quality test result that tells a physician noth- written for a newly hired employee who would read the pro-
ing useful is useless. Answering the phone properly, coopera- cedure and then be able to perform the test with minimal
tion among laboratory departments, and calling a critical result supervision. An excellent source for writing laboratory
promptly are other examples of excellent customer service. procedures can be found in the Clinical and Laboratory
Standards Institute’s (CLSI) “New Laboratory Documents:
PERSONNEL Development and Control; Approved Guideline-Fifth
Employees should be hired for attitude and competence; qual- Edition” (GP2-A5), updated May 2006. (In 2005, CLSI
ity results start with quality people. All employees deserve to changed its name from National Committee for Clinical
be trained correctly and consistently. Performance appraisals Laboratory Standards, i.e. NCCLS). Records show what has
and competency assessments are tools for praise, corrective been done; instrument records serve as a history of the
action, and learning.'’ Licensure of all laboratory personnel is instrument. Appropriate records are stored according to
a hot topic; currently 11 states require licensure.'* Because rules of several agencies and state and federal regulations
technology is swiftly moving forward, continuing education and change frequently. The storage time to use is the longest
must be a priority, whether a state is licensed or not. time of all the regulations that affect a department.

EQUIPMENT ASSESSMENTS
When purchasing new equipment, a careful review of all There are internal assessments (1.e., correlations between
currently available instruments and their specifications is similar instruments) and external assessments (1.e., surveys,
warranted: cost, accuracy, precision, risk, usefulness, ease- inspections, correlations between two labs). CLIA, JCAHO
of-operation, maintenance schedule, consumables required, (Joint Commission on Accreditation for Healthcare Organiza-
test menu, etc.'? Consider future needs and technological tions), and CAP requires correlations every 6 months on
advances because most equipment is replaced every 5 years instruments reporting the same result; a laboratory must
or so. Method validation studies must be done correctly to determine the limits of correlation.*°' If there are multiple lab
uncover any hidden biases so that the foundation for a qual- sites within a health-care system, one site is deemed the
ity monitoring system is solid. “core” site, and the others must perform correlations every
6 months with the core site if they are reporting the same
DOCUMENTS AND RECORDS automated results as the core lab. Physicians and patients may
Documenting any variations in normal operations is essen- be using more than one lab in the system; the test results need to
tial to understanding what processes need to be monitored. be comparable. There may be several agencies that can inspect
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 703

a laboratory, i.e., AABB, CAP, JCAHO, CMS, FDA, plus state complaints, and errors. Any QC program can be used to
agencies; the laboratory management team must be knowledge- improve a process (see “Quality Approaches’’).°
able of all regulatory requirements that pertain to them.

PURCHASING AND INVENTORY Quality Software

Products are purchased with quality and cost in mind. Inven-
Several software companies distribute software for managing
tory is monitored for usage and expiration dates, and rotated
quality information. Which one is best for a particular lab
for maximum usage without waste. Extra inventory that will
depends on many factors; a thorough needs analysis may be a
not be used for months consumes storage space, technologist’s good way to start.
time for rotating expiration dates, and dollars that could be
used elsewhere. Purchasing sequestered lot numbers of ¢ Harrington Group (www.harrington-group.com)
reagents is desirable to reduce parallel testing time and money, ¢ Paradigm Software (www.paradigmsoftware.com.au)
and to ensure a more stable testing environment. ° Quality Link (www.qmsonline.com)
¢ Beckman Coulter Lab Accelerator (www.beckmancoulter.
PROCESS CONTROL com/labquality)
This is where most laboratories concentrate their quality prac- * Q-Pulse (www.Qpulse.com) **
tices. There are preanalytical, analytical, and postanalytical
variables to monitor for every laboratory test or procedure. QC Quality Assurance and Quality Control
programs are devised to make sure that test systems work the
way they were designed and to produce quality results. Control- Everything that can be counted does not necessarily count;
ling processes can also be achieved by flowcharting a process everything that counts cannot necessarily be counted.
and writing corresponding procedures that identify each task
—A\lbert Einstein
and who is responsible for each task. A simplified flowchart for
performing a sedimentation rate procedure is shown in Figure
29-5. When procedures are written in a clear, concise manner Definitions of Quality Assurance, Quality
with diagrams, the tasks can be accomplished consistently and Control, and Quality Assessment
successfully. :
The terms “quality assurance” and “quality control” are often
PROCESS IMPROVEMENT used interchangeably to refer to ways of ensuring the quality
Improvement is possible by monitoring a process, then finding of a service or product. However, these two terms have differ-
ways to improve the quality of that process. Areas to monitor ent meanings:
can be found in the results of assessments, benchmarking,
* Quality assurance: The planned and systematic activities
implemented in a quality system so that quality require-
ments for a product or service will be fulfilled. Quality
assurance is a direct result of performing quality assess-
Determine if
the
eis # ments. If the proper method validation was performed,
quality requirements met, and assessments completed, the
clinician is assured that the test results are of the highest
quality possible.
Quality control: The observation techniques and activities
Remove cap from ESR plastic used to fulfill requirements for quality. (Reprinted with per-
vial. Using a disposable pipet,
add enough blood to the fill line- mission from www.asq.org © 2004 American Society for
on the vial. Replace cap and Quality). Quality control is a tool to control and monitor the
gently mix. — inherent variables that lead to error.
Quality Assessment is the new term for QA per CLIA-88 final
rule (CFR 2003). Quality efforts are concentrated on assess-
ments of the testing process, procedures, and corrective action
to prevent future problems. Assessment of accuracy 1s required
under Subpart K, section §493.1236 and is accomplished
either through proficiency testing or by some other system for
Place the ESR assembly into verifying the accuracy and reliability of its test results at least
a rack. Note the position number twice a year.
and time (write both down).
Set timer for 60 minutes. At
60 minutes, read the ESR at the
interface between the blood General Quality Assurance Control Activity
and plasma. Guidelines
The CFR 2003 ruling provides a broad outline of quality
Figure 29-5 m Flowchart of a sedimentation rate procedure. assurance activities based on a quality systems approach that
704 Chapter 29 Quality Management, Quality Assurance, and Quality Control

follows the route of a specimen through the laboratory. The method’s errors, probability of error detection and false rejec-
standards for quality systems are under the Analytic Systems tion. control rules, and number of control measurements. For
section of subpart K: more information on QC statistics, go to www.westgard.com.

¢ Procedure manual ($493.1251)

POSTANALYTICAL FACTORS
* Test systems, equipment, instruments, reagents, materials, Postanalytical factors are errors that occur after the patient
and supplies (§493.1252) result is obtained, but before the result is posted to a chart or
¢ Establishment and verification of method performance
sent to a physician. Almost every patient result is entered into
specifications (§493.1253) a computer, and computer entry errors are a major source of
¢ Equipment maintenance and function checks (§493.1254)
postanalytical errors. Calculation errors for a manual method
* Calibration and calibration verification procedures also contribute to incorrect patient results. Technologists
($493.1255) should review patient test results carefully before accepting
* Control procedures ($493.1256)
them in the computer to prevent incorrect results from reach-
* Comparison of test results (§$493.1281)
ing a physician, and possibly causing harm to the patient.
¢ Corrective actions (§493.1282)
¢ Test records (§493.1283) NONANALYTICAL FACTORS
¢ Analytic systems assessment ($493.1289) Nonanalytical factors are not directly connected to the actual
specimen testing; they are more supportive of the entire test-
Preanalytical, Analytical, Postanalytical, ing process. Examples include choosing an appropriate
method for testing an analyte; hiring and training qualified
and Nonanalytical Factors in Testing personnel; developing policies and procedures that meet reg-
PREANALYTICAL FACTORS ulatory guidelines and are useful for a bench technologist;
Preanalytical factors are the most common source of labora- instrument maintenance; utilizing quality assurance at every
tory errors. Proper identification of the patient continues to be step; suitable QC materials and procedures for the method;
a concern, as is collection and labeling of the specimens at the reporting critical values in a timely manner to a health profes-
bedside. Bar-coded labels have improved this process over sional in charge of the patient; and proper collection and stor-
the years. Timing of specimen collection for specific tests is a age of specimens (Table 29-1).
challenge, and often the specimen is not collected at the exact
time necessary for optimum quality results. Deviations from Accuracy, Precision, and Error
the specimen transport policies reduce the quality of the spec-
imen, and ultimately the quality of the result (ammonia and Accuracy describes a test result that is close to its true value,
blood gases are examples of specimens that must be trans- while precision refers to reproducibility, 1.e., whether the
ported on ice). Specimens that need to be analyzed within a same test can be run on the same sample and produce the
short time frame, such as coagulation tests, should be tested same result. The target analogy is used in Figure 29-6 to
as soon as possible or stored appropriately. Most samples col- visually describe accuracy and precision.
lected at off-site locations can be transported in coolers with All analytical methods have some error. Error is the dif-
ice packs and thermometers to reduce temperature-related ference between the true value and the obtained value. How
changes to the specimens. much accuracy and precision is necessary or desirable? What
is the allowable error and bias for the method? These ques-
ANALYTICAL FACTORS tions should be answered before purchasing a new instrument
Analytical factors are errors that occur during the testing or performing a new analytical method.
phase of a specimen. For an automated instrument, the analyt-
ical factors in testing include imprecision and inaccuracy of HOW TO MEASURE ACCURACY
the test method, and the sensitivity of the QC procedures to Accuracy is measured in bias, the difference between the
detect significant errors. Imprecision and inaccuracy can be mean of a set of replicate measurements and the true value.
caused by many factors including calibration drift, reagent Accuracy may be expressed as a percentage or as a number
issues, and instrument malfunction. The QC _ procedures with units. The mean (average) of a set of numbers shows sys-
chosen should be the result of a logical evaluation of the tematic error, or error inherent in the data.

Preanalytical Analytical Postanalytical Nonanalytical

Patient identification Calibration Result entry Method choice

Sample identification Malfunctions Calculations Personnel
Sample transport New lots of reagent/QC Policies/Procedures
Sample handling Pipetting Maintenance
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 705

A. Accuracy B. Precision

Figure 29-6 m Accuracy and precision can be compared to a

target. A. Example of accuracy and precision (the shooter hit the
“target” value, i.e., the bulls-eye, and also reproduced the shot
several times. B. Precise, but not accurate.

Mean = the arithmetic average of a set of values =

where n = number of observations (data points) and

Figure 29-7 mi Accuracy: constant and proportional systematic
x = sum of all data error in relation to the mean.
For example, if a sample were run 10 times for hemoglo-
bin with the following results:
where s represents the standard deviation, 2 means sum-
6107 8:509:0)7.8. 8-5, 9.1558.9,,9.1.8.6,.8.6, — the mean
mation of all the (x; — x)? values, x; is an individual result, ¥ is
is the average, which is 8.7. (To obtain the mean, the numbers
the mean of the results, and n is the total number of results
are added together and then divided by 10, the number of
included in the group.** For example, to calculate the SD, first
observations). How accurate are the data? The “true” value
observe a set of results and then calculate the mean of those
would need to be known. In this case the true hemoglobin
results. For the hemoglobin study, the number of observations
value was really 8.8. Calculate the accuracy two ways:
was 10, and the mean was 8.7. The calculated SD is 0.45.
Accuracy (bias) = 8.8 — 8.7 = 0.1 g/dL or The standard deviation is related to the spread or distribu-
Accuracy (bias) = 0.1/8.8 x 100 = 1.14% tion of results about the mean. Whereas the mean is an
indicator of accuracy or systematic error, the standard deviation
Accuracy studies show constant systematic error or pro-
is a measure of the width of the distribution and is related to
portional systematic error (both are biases). Accuracy studies
imprecision or random error. The bigger the standard deviation,
should be performed with a patient sample repeated at least
the wider the distribution, the greater the random error, and the
10 times within a short period of time. How the constant and
poorer the precision of the method; the smaller the standard
proportional systematic error relates to the mean is shown in
deviation, the narrower and sharper the distribution, the smaller
Figure 29-7.
the random error, and the better the precision of the method.”
When using a measurement procedure, it is generally
HOW TO MEASURE PRECISION
expected that the distribution of results will be normal or
Precision is measured by the standard deviation (SD) or coef-
Gaussian, named after the mathematician Carl Friedrich
ficient of variation (CV) of a set of replicate measurements
Gauss (1777-1855), as shown above. In a Gaussian distribu-
(i.e., where did the five shots land in relation to the target’).
tion, the percentage of results that are expected within certain
Precision studies will show random error (Table 29-2).
limits (1, 2, and 3 SD) can be predicted. Approximately 68%
of the values will fall within 1 SD, 96% of the values will fall
WHAT IS THE EQUATION FOR THE STANDARD
within 2 SD, and 99% will fall within 3 SD (Fig. 29-8).
DEVIATION (SD)? The standard deviation is determined by
first calculating the mean, then taking the difference of each
result from the mean, squaring that difference, dividing by
n—1, then taking the square root. All these operations are out-
lined in the following equation:
Standard deviation (SD or s): Accuracy _ Precision

Measured in oe Measured in1 SD orCV

S= > (x,—x)? Measures systematic error Measures random error
(n-1)
706 Chapter 29 Quality Management, Quality Assurance, and Quality Control

= Sra
i - a)

Sy eee
ne dh
10d
IE
as
a Vtats
¥ “ aed :
Seger
Se) > ee

Hematology Test or Analyte Acceptable Performance

Cell identification 90% or greater

consensus on
identification
WBC differentiation Target + 3 SD based on
percentage of different
types of white blood
-3SD -2SD —1SD x +1SD +2SD +3SD cells
RBC count Target + 6%
Figure 29-8 @ Example of Gaussian distribution curve for data of Hematocrit Target + 6%
normal populations. Hemoglobin Target + 7%
WBC count Target + 15%
Platelet count Target + 25%
Fibrinogen Target + 20%
WHAT IS A CV? The coefficient of variation (CV) describes APTT Target + 15%
the standard deviation as a percentage of the mean, as shown Pay Target + 15%
in the following equation:
CV = (s/X) 100
Method Validation
where s is the standard deviation, ¥ is the mean, and
the multiplier of 100 is used to convert the s/¥ ratio to a How does a laboratory select a new method? There are three
percentage. general categories to consider:
For example, in the hemoglobin study the standard devi-
¢ Application characteristics should include volume of
ation (0.45) is divided by the mean (8.7) and multiplied by
specimen, type of specimen, workload appropriate for the
100. The CV for the hemoglobin study is 5.2%.
testing situation (high volume centralized lab vs. low vol-
ume point-of-care), operator skills, cost per test, time for
WHY IS A CV USEFUL?
analysis, and operator training requirements.
The standard deviation of a method often changes with con-
¢ Methodology characteristics should include type of stan-
centration, 1.e., the larger the concentration, the larger the
dards or calibrators, reagents and reaction conditions, traceabil-
standard deviation; therefore it is usually necessary to esti-
ity of standard or calibrator assigned values, method principle,
mate the standard deviation at the concentration level of
measurement principle, and measurement capabilities.
interest. Because the CV reflects a ratio of the standard
* Performance characteristics should include working
deviation to the concentration, it often provides a better
range, imprecision, interference, bias vs. the reference
estimate of method performance over a range of concentra-
method, and total errors less than 10%.
tions (Box 29-1).
When you are ready to begin the method validation process,
TOTAL ERROR a plan of action is very helpful. James O. Westgard, PhD rec-
Total Error (TE) estimates have been established by CLIA ommends following a logical plan (Box 29-2).”’
in the 1992 Federal Register for many tests and are used for
WHAT STUDIES NEED TO BE RUN ON A NEW METHOD
allowable differences on proficiency surveys (Table 29-3).
OR INSTRUMENT?
Because most proficiency samples are analyzed only once
When a new instrument or method is introduced to the labo-
for a survey (like a patient), the maximum error will be
the total error possible due to both inaccuracy and impreci-
ratory, a series of experiments and calculations must be per-
formed to validate each parameter. The responsibility has
sion in the same direction. The TE is also considered an
been shifted to the laboratory to run the studies necessary to
estimate of analytical quality for a particular test because
show that the method is accurate and precise. Even if the
it is the largest cushion of error allowable. CLIA lists
the TE in three ways: an absolute concentration limit manufacturer has already performed some studies and has the
(i.e. 5mg/dl), a percentage (i.e. 10%), and in SDs (i.e., + printed data to support their claims, the laboratory must vali-
DES)7° date the studies required by CLIA:
* Accuracy (bias)
¢ Precision (SD or CV)
| Box 29-1 Coefficient of Variation 2 * Reportable range of test results for the test system (linearity).
* Manufacturer’s reference intervals (normal values); Are
/ © Why is a CV useful? The coefficient of variation (CV) they appropriate for the laboratory’s patient population?
shows random error across a wide range of concentrations * Other studies that are usually performed: Comparison of
Methods and Recovery experiments
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 707

valid (within a predetermined percentage of the original nor-

Box 29-2 Method Validatio
|
mal result). Hemolysis can be induced by freezing and thaw-
¢ Familiarization period ing aliquots of patient CBCs. Test samples are prepared as in
* Establish working procedure. the lipemia experiment. The average result of each duplicate
¢ Check working range. | pair is calculated, and then the difference between the total
¢ Check calibration. average normal values and the total average altered values is
¢ Check detection limit. determined (similar to paired f-test statistics). This difference
* Preliminary MV experiments is compared to the CLIA standards for total allowable error
* Perform within-run replication study. (TE). If the experiments revealed an interference difference
* Perform interference study. greater than the CLIA standards, then the method or instru-
| * Perform recovery study. ment would not be acceptable.
| ¢ Judge analytical acceptability.
¢ Final MV experiments i REPLICATION EXPERIMENT
__ ¢ Perform total replication study. A replication experiment provides information about random
¢ Perform comparison of methods study. i error (precision, SD or CV) and is performed by making repli-
¢ Judge analytical acceptability. cate measurements on a series of patient samples and/or QC
° Verify reference interval(s). material within a specified period of time, usually within an
¢ Document studies. analytical run, within a day, or over a period of a month. The
¢ Implementation preliminary experiment usually involves determining within-
¢ Select QC procedure. run imprecision by running a sample 10 or more times within
¢ Write operating protocol or procedure. one run. The final experiment generally requires at least
¢ Train analysts. 20 working days to provide a good estimate of the total impre-
¢ Introduce method for service. | cision, which includes within and between run components.
¢ Monitor routine performance. |
¢ Introduce method for service. REPORTABLE RANGE (LINEARITY) EXPERIMENT
A reportable rage (linearity) experiment is useful to assess
the lowest and highest values that can be reliably reported.
it is good laboratory practice to only report values that can
A brief description of the different method validation experi- be proven accurate and not rely on manufacturer’s claims.
ments a laboratorian will need to run is provided in Box 29-3. Linearity kits can be purchased for most hematology ana-
lytes that contain five or more levels for testing. Each level
INTERFERENCE EXPERIMENT is tested at least three times. The statistical charts and
An interference experiment provides information about the graphs needed to make decisions are usually provided by
constant systematic error (accuracy, bias) caused by interfer- the manufacturer of the linearity kit. The laboratory estab-
ing substances. Examples of interfering substances in hema- lishes expected limits for each parameter at each level. A
tology and coagulation are lipemia and hemolysis. For best best-fit linear line is drawn through all the points on a plot
results, obtain fresh lipemic patient samples; alternately, a of the means of the measured values (y-axis) vs. known val-
commercial lipid emulsion can be purchased such as ues (x-axis). The low and high ends of the reportable range
Intralipid (Pharmacic and Upjohn) or Liposyn (Abbott Labo- are the values just before the laboratory-established limits
ratories). Several concentrations of lipemia are created in a are exceeded.
linear fashion by adding different amounts of the lipid to a
normal whole blood specimen. At least three different speci- REFERENCE RANGE (NORMAL RANGE) EXPERIMENT
mens should be used for the experiment to decrease the ran- A reference rage (normal range) experiment is essential to
dom error. The test samples are analyzed in duplicate by the establish a new reference range or to verify the manufac-
test method to determine the point at which the results are turer’s normal range claims. An experiment to establish a new
range could include more than 120 specimens per age and
sex, if the ranges changed significantly across these cate-
gories. The patients are usually screened via a health ques-
tionnaire according to CLSI recommendations. If the new
;;
* Replication experiment: within-run and between-run for method has well documented studies available either from the
i {
;;
HH random error manufacturer, in literature, or in-house, then a smaller study
i;
¢ Interference experiment: make dilutions with the interfering of twenty samples may be useful to validate the range: if two
substance for constant systematic error samples or fewer fall outside the stated ranges, then the
* Recovery experiment: make dilutions of patient samples for | ranges are statistically verified. If there is any question as to
proportional systematic error the validity of the original study to be transferred, or if the
* Comparison of Methods experiment: run patient samples on new method is significantly different from the old method,
two test methods for average systematic error then a larger validation study of at least 60 samples should be
prc
remrewnranenencannmatonnnrereiancnnin
Rt
performed.”*
708 Chapter 29 Quality Management, Quality Assurance, and Quality Control

COMPARISON OF METHODS EXPERIMENT reject the method or to identify and eliminate the causes of the
A comparison of methods experiment is primarily used to errors. The laboratory director should be consulted when the
estimate the average systematic error observed between two data are difficult to interpret.
similar methods, but can also reveal the constant or propor-
tional nature of that error. The results of patient specimens are RECOVERY EXPERIMENT
compared to determine the analytical errors between the
A recovery experiment can be used when there is no compar-
methods. A minimum of 40 well chosen patient samples
ison method. The purpose is to uncover proportional system-
should be tested over a minimum of 5 working days. These
atic error. Commercial solutions of high concentrations of the
samples should be distributed one-third in the low to low-
analyte to be measured are obtained and added in a linear
normal range, one-third in the normal range, and one-third in
fashion to patient samples, and the samples are run two or
the high abnormal range. The correlation coefficient (r) is a
more times to reduce the random error. The results are aver-
useful statistical tool for comparing two sets of data. A perfect
aged and compared, and a percent difference is calculated.
linear relationship between the data would result in a correla-
The total error defined by CLIA is compared to the percent
tion coefficient of 1.0. Figure 29-9 is an example of a corre- difference: if the difference is greater than the total error al-
lation study between identical hematology analyzers. Each lowed, the method or test is not acceptable.
lab must establish its own correlation acceptable limits for CLSI also provides a series of documents that provide
each assay. Statistical software, such as Microsoft Excel, can
extensive information about individual experiments:
produce a graph along with the correlation coefficient.
Regression statistics are another mathematical comparison of * EP5-A. Evaluation of precision performance of clinical
two sets of data; systematic differences can be detected more chemistry devices.
readily with this method. (For a complete discussion of labo- ¢ EP6-P. Evaluation of the linearity of quantitative analytical
ratory statistics for method validation log on to http:// methods.
westgard.com). Once the data have been collected, method ¢ EP9-A. Method comparison and bias estimation using
acceptability should be judged on the basis of the sizes of the patient samples.
random, systematic, and total analytical errors. If these errors ¢ EP10-A. Preliminary evaluation of quantitative clinical lab-
are small compared to the amount of error that would invali- oratory methods.
date the use and interpretation of a test result, the method is ¢ EP14-P. Evaluation of matrix effects.
acceptable. If the errors are too large, it may be necessary to * NRSCL12-P. Source book of reference methods.

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124 ei22
125 12.4
127 12:8
12ST
12 lige st3.0
182 ~~«18.1
13.6 13.5
187 1) 126 Sample number
13.8 13.6 Correlation Coefficient = 0.9978
13.9 13.8
14 an
Lav AA
15.0 15.0
Figure 29-9 @ Correlation study of hematology analyzers (hemoglobin).
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 709

* H20-A. Reference leukocyte differential count and evalua-

tion of instrumental methods. : Box 29-4 Judging a Method’s Perforr
* EPI5-P. User demonstration of performance for precision | ¢ Find the TE (Total Error) from CLIA list. |
and accuracy.
_ * Select experiments to reveal expected types of errors. |
* C24-A3. Statistical Quality control for Quantitative Mea- | ¢ Perform experiments, collect data. |
surement Procedures: Principles and Definitions. _ © Calculate the size of the errors. |
* C28-A. How to define and determine reference intervals in | ¢ Compare observed error with TE. |
the clinical laboratory.” * Judge the method performance.
How will the test be QC’d? What control rules will be used?
i ee Ee ee
How many control measurements are needed and how often?
These questions need to be answered before implementing
STATISTICAL PROCESS CONTROL
any new method in a laboratory (Box 29-4).
Statistical process control is a general term for the parts of a
control system in which statistics are used, such as a Levy—
Quality Control Definitions Jennings graph.

QUALITY CONTROL MATERIAL RUN

The samples that will be used for QC procedures. The pur- Arun is a finite time period or number of patient samples. QC
pose of a QC material is to monitor analytical quality of a is designed to be analyzed at the beginning and end of a run.
method so as to alert the operator when there is a problem; CLIA changed the definition of a run for QC purposes to
however, the QC material should not alert the operator if 24 hours for most analytes. However, coagulation and a few
everything is working as designed. Most laboratories pur- other tests are still required to have a QC material analyzed
chase commercially prepared materials due to ease of prepa- every 8 hours.
ration, stability, and long shelf life. At least two levels of
controls are usually utilized per method. CONTROL LIMITS
Control limits are statistical criteria of acceptability for a par-
QUALITY CONTROE STATISTICS ticular control material, usually derived from the mean and
All of the many mathematical calculations available to help SD; the limits are placed on a control chart for ease of use.
determine if a method is accurate and precise. Examples
include: Levy—Jennings plots, Youdin plots, OPSpecs charts, CONTROL RULE
Critical-Error graphs, Power Function graphs, correlation A control rule is a decision criterion for judging a control
coefficient (7), linear regression, and ¢ and F tests. material as acceptable or not. Westgard MultiRules are exam-
ples of control rules.
MATRIX
Matrix is a term describing the substance of the QC material. MOVING AVERAGES
Regulations require that the control material be as close as pos- Dr. Barr discovered that red blood cell (RBC) indices (mean
sible to real human specimens. For example, urine tests should corpuscular volume [MCV], mean corpuscular hemoglobin
be controlled by a urine-like control material, etc. Sometimes [MCH], and mean corpuscular hemoglobin concentration
animal materials are used because they are very close in nature [MCHC]) were fairly stable in a normal person over a long
to the human materials and easier to manufacture. period of time. He translated this into a QC program called Xz.
The mean of the RBC indices in the population of a laboratory
ASSAYED CONTROL MATERIAL is monitored for significant drifts or shifts in calibration, which
A commercially prepared control material that has expected may mean method quality problems. This QC procedure
mean and SD values from the manufacturer. The laboratory should not be the only source of QC of RBC indices, and lab-
will need to validate the values, usually by running a new lot oratories running fewer than 100 CBCs a day should not uti-
of QC before an old lot has been exhausted or expired (paral- lize this procedure due to lack of sufficient data.”
lel testing), before running the new lot of control material to
monitor patient samples. CONTROL CHART
A control chart is graphical method to display control results
UNASSAYED CONTROL MATERIAL so that it is easier to determine if a control run is in or out-
Unassayed control material is a commercially prepared control of-control. Shifts and trends can be visually assessed using
that does not have specific ranges verified by the manufacturer. control charts.
The laboratory must run the control material in replicate over
several days or weeks to establish an initial mean and SD. SHIFT
Shifts are consecutive observations on a QC chart (usually
QUALITY CONTROL LEVELS Levy—Jennings) above or below the mean. Recommendations
Each control level should be carefully chosen to monitor a are that six or more values above or below the mean consti-
critical medical decision level whenever possible. tute a shift.
710 Chapter 29 Quality Management, Quality Assurance, and Quality Control

TREND
A trend is a slow change in QC values on a QC chart, either
Box 29-5 Minimum QC Requirements
rising or falling steadily across several days or weeks. ((@BF...)
¢ Run two controls each day of testing for quantitative tests.
ELECTRONIC QC * Run controls after changing all reagents, preventive mainte-
Some analyzers and test systems have internal electronic nance, or replacement of a critical part or component.
checks such as clot detectors, abnormal plasma/serum detec- ¢ Rotate QC among the staff.
tors (lipemia, hemolysis), or a mechanism to detect insuffi- * Test QC the same as patients.
cient samples. There could be monitors for temperatures, light e Establish or verify the QC ranges.
source outputs, moving parts sensors, etc. For calibration, ¢ Establish a mean and SD for new unassayed QC before
there might be verification rules in the software for acceptable going live.
runs, or statistical control rules for QC. Electronic QC usually * Do not report patients unless QC is within limits.
monitors variables during the analytical phase only. The dif- ¢ Document all QC procedures performed.
ferent types of QC procedures in the laboratory and the errors haere renee nintninaneievnenttn nA NAST CNRS ARIN LARS RRA

they monitor are shown in Figure 29-10.

All new QC lots must be run in “parallel” with current control

Minimum Quality Control material to establish or verify the acceptable range before
Requirements, CLIA-88 CFR using the new control lot to monitor patient results. The num-
ber of data points needed is dependent on a couple of factors,
The government requirements are listed:
such as the stability of the test system, the stability of the
¢ Run two levels of controls for quantitative tests each day control material, laboratory policy, etc. For automated hema-
when, patient specimens are assayed or examined (note that tology analyzers, a parallel experiment of a few runs per day
this general requirement may be modified by specific for several days on all shifts should be sufficient because most
requirements in specialty areas as described in sections systems and the commercially prepared control material are
493.1261 through 493.1278, e.g., hematology manual cell very stable. For new lots of coagulation controls, a few runs
counts require one control every 8 hours, nonmanual coag- per day for a week or more may be necessary depending on
ulation requires two levels of control every 8 hours, but whether the controls are assayed (fewer runs to validate
manual coagulation tests require two levels of control each mean) or unassayed (more runs to establish mean).
time specimens are analyzed, etc.).
¢ Test controls after a complete change of reagents, pre- ESTABLISHING IN-HOUSE QC MEAN AND SD
ventive maintenance, or replacement of a critical part or Almost all current regulations require a laboratorian to estab-
component. lish an in-house mean and SD for any QC material to be used,
* Rotate controls among all operators performing the test. before the material is placed in service to monitor a method.
¢ Test control materials the same as patient materials. Why is this necessary? Even if the manufacturer supplies
¢ Establish or verify criteria for acceptability for all control ranges and SDs, the data are usually combined from many
materials. instruments and possibly multiple methods to arrive at the
* Determine the statistical parameters (mean and SD) over acceptable mean and SD listed in their package insert, which
time through concurrent testing for all unassayed control usually makes the ranges wider than is useful for daily
materials. method monitoring. In addition, the manufacturer’s data can-
¢ Make sure controls meet criteria before reporting patient test not predict how the controi material will behave on a particu-
results. lar lab’s instrument, handled and run by the staff. Coagulation
¢ Document all control procedures performed (Box 29-5). ”° laboratories have been establishing control means and ranges
for years with unassayed materials and for special tests that
require frequent calibration.
Hematology is somewhat trickier: the commercial QC
lot number changes every 6 weeks or so. Calculating new SDs
every 6 weeks is not very useful or productive. To establish
in-house ranges, a technologist could average the SDs for
each CBC and differential parameter from all control levels
from the instrument peer group reports for the past 6 months.
<— Elect OC — (Note: Be careful when reviewing peer group data; some SDs
listed are actually 1 SD, and the SDs are probably very low).
mr acitlonalG en
The average SDs are used as the basis for calculating in-house
—m
——Patient sample replicates>
ranges. The controls are run and means established for each
—
—— Patient sample comparisons ————————__> parameter. The instrument software should allow the user to
choose these new means as the target means. New ranges are
Figure 29-10 @ Different types of errors and QC actions.
then calculated: be sure that the new ranges are not wider than
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 711

the manufacturer’s package insert ranges. The SDs can be in-house and calculations for quality (accuracy and precision).
entered into the instrument for future use with each new lot of Figure 29-1] is an example of a L-J graph for WBC QC
controls. To test the new ranges, run the controls a few times normal level.
every day for a week. If the control values fall outside the new
ranges, then the new ranges may be too “tight” and need to be SHIFTS AND TRENDS
widened a bit. Multiply the SD by 2, then 3, then 4, and so on The L—J graph is a visual view of how the QC is performing,
until the controls are within the new range. Document the and therefore, how the method is performing. According to
process adequately for inspection purposes. Note: some dif- the Gaussian distribution, data points should occur equally
ferential parameters, such as basophils and eosinophils, nor- around the mean, and this is true for most analytes. A shift in
mally have a high SD due to relatively low actual values. the values represents a sudden and consistent bias from the
mean, 1.e., a group of data points are clustered on one side of
Levy-Jennings Graphs the mean. A shift is representative of systematic error. It is
commonly accepted that six points on one side of the mean
Levy and Jennings introduced the idea of control graphs in constitutes a shift; however, every lab should determine how
1950.°*° The Levy—Jennings (L-J) graph used today consists of many data points constitute a shift. Causes of a shift include
individual data points plotted on a graph of the mean and the improperly prepared reagents or controls, reagent deteriora-
control limits of the particular control level. The L-J graph tion, change in lot numbers, and failure of an instrument com-
historically has consisted of 1, 2, or 3 SD ranges. The roots of ponent (Fig. 29-12).
the 2 SD phenomenon can be traced back to Carl Frederick A trend is a gradual change in the same direction, either
Gauss (1777-1855), who introduced the concept of the normal increasing or decreasing (Fig. 29-13). A trend is also system-
curve. Because 95% of the points in a normal data set for a atic error. Again, each lab must determine how many data
particular parameter are expected to be within 2 SD of the points constitute a trend. Trends can be caused by the same
mean, it seemed appropriate to use + 2 SD limits for labora- items as a shift, but because the data are slowly changing the
tory QC. Therefore, when Levy and Jennings started using a root cause may be more difficult to identify. Look for items
chart for QC, the 2 SD range was implanted and has remained that can change over time, such as reagent or lamp deteriora-
ever since. *! However, laboratories must establish their own tion, temperature fluctuation, calibration shift, and so forth.
mean and limits for each assay based on experiments run Use a systematic logical troubleshooting approach in isolating

WBC QC NORMAL LEVEL

WBC
1000
x

(eZ Sea Se CP e/e Ge Oe Omani

Sat) tom) Omli/jel OnlomeO

Day of the month

Figure 29-11 mA typical Levy-Jennings graph. Each data point is plotted so an overall view of the data is possible.

WBC QC NORMAL LEVEL

+2SD

Mean

& a
WBC
1000
x —2SD

) 2 Boal Be ty 7h te Sy AO Sa ae ake ake 1) 7A ait IS} 28)

Day of the month

Figure 29-12 m™ Example of a shift in QC values on a Levy-Jennings graph. Notice that between day 8 and day 20 the data has abruptly
shifted above the mean.
712. Chapter 29 Quality Management, Quality Assurance, and Quality Control

WBC QC NORMAL LEVEL

WBC
1000
x

128 456 7 & & 10 1 12 18 1 Ws 16 U7 is 1 Ae

Day of the month

Figure 29-13 m Example of a trend in QC values on a Levy-Jennings graph. Notice that between day 8 and day 20 there is a slow trend
above the mean.

the cause, making only one change at a time and document- ¢ 1,, This rule refers to one data point exceeding the + 3 SD
ing each action taken. limit.
Another type of change commonly seen on an L-J graph ¢ 2,, This rule refers to two consecutive data points exceeding
is an increase in imprecision. The data points become further the + 2 SD limit.
away from the mean, and the SD becomes larger. If there is ¢ R,, This rule refers to one data point exceeding the +2 SD
contamination of the diluent water used in coagulation recon- limit, and another close data point exceeds the —2 SD limit
stitution, the QC material may display such a dispersion of (opposite directions). An example of this rule is illustrated
results.*Other causes of imprecision include poor mixing of in Figure 29-14.
the control material, pipetting errors, and so forth. * 2of3,, This rule refers to two out of three control points exceed-
ing either the +2 SD or the —2 SD limit on the same side.
Westgard MultiRule Quality Control ¢ 7, This rule refers to seven consecutive data points trending
in the same direction, either progressively lower or higher.
When QC data points have been run and are entered onto a ¢ 4,, This rule refers to four consecutive control measurements
L-J QC chart, the results must be reviewed. When is a data exceeding the +1 SD or —1 SD limit on the same side of the
point too far out? What if all the points are above the mean? graph.
Dr. James O. Westgard, the originator of “Westgard Rules,” ¢ 10, This rule refers to 10 consecutive control measurements
has answered these questions and more. Westgard Rules is a falling on one side of the mean.
series of decision criteria (control rules) to decide whether an
analytical run is in-control or out-of-control. There are rules CHOOSING WESTGARD RULES
for two levels or three levels of control material. To improve Consider these factors when choosing Westgard Rules for a
sensitivity, it is suggested that at least two rules be used method:
together for every control material level. Here is a list of the
* Is the method highly automated, a new method, or a manual
most commonly used Westgard Rules:
method? The type of method, automation, and reliability
¢ 1,, This rule refers to one data point exceeding the +2 SD will have an effect on the rules chosen.
limit. * Select a combination of rules having at least one rule that
¢ 1,;, This rule refers to one data point exceeding the + 2.5 SD responds to random error and one rule that responds to sys-
limit. tematic error (Table 29-4)

WBC QC NORMAL LEVEL

WBC
1000
x

i) 2 SPE ey oy 7h eek SP AKO I aie Ih SS IG. I aS} ie) BO

Day of the month

Figure 29-14 mA Levy-Jennings graph showing a violation of the Westgard Rule R,..
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 713

* Assess the probabilities of rejecting a control value for that method is performing, which in turn can assist in choosing
combination of rules. how to control that method (Box 29-6). A Sigma refers to pre-
* Automatically eliminate the rules that normally have a high cision expressed as a standard deviation of the measurement
probability of rejection procedure. In laboratory testing, a measurement procedure is
the sequence of events from obtaining a sample to reporting a
Rejection refers to false rejection. A technologist does not
result. A method can be any number between 2 and 6 Sigma.
want to be notified by the controls when there is nothing
For Six Sigma quality, the ideal is to have the smallest method
wrong (false rejection). However, she does want to be notified
SD so that six times the SD will fit into a process’s perfor-
if there is a problem with the method. The information in the
mance and produce a high-quality result. The process should
table below was interpolated from a critical-error graph,
be judged, not just the QC material.
which shows whether or not a rule would detect a medically
To provide some perspective on the observed sigma-
important error (see Table 29-4). From the table, it is obvious
metrics for production processes, a number of benchmarks
that the 1,, rule has the highest false rejection rate of any of
are often cited:
the rules—around 9% for a two-control system. This means
that 9 times out of 100 runs a technologist would be respond- * “World class quality” is represented by 3.4 DPM (defects
ing to a false alarm. If three control levels are being analyzed, per million opportunities in a process) or 6 Sigma
the false alarms go even higher. Yet it is the most commonly performance.
used rule in laboratories today. For highly automated systems, * Airline baggage handling shows a 0.4% or 4000 DPM mis-
a better rule would be 1, ;, or 1,,. handling rate, which corresponds to a 4.15 Sigma process.
Each method has a different accuracy and precision; the * Airline travel has a very low fatality rate: 0.43 deaths per
control rules are chosen to match them. If a method is perform- million passenger flights, which is better than a 6 Sigma
ing well (low CV and SD), fewer rules are needed. If the method process.
is a problem method (moderate to high CV and SD), more rules * Typical process performance in business and industry is
are needed to maintain a higher quality of patient results. The often suggested to be about 4 Sigma.
accuracy (bias) and precision (CV) of a method are known from ¢ Firestone production of tires for Ford Explorers appears to
the method validation studies. To review an existing method, the be near the 5 Sigma level on the basis of available informa-
control material CVs for precision (in percent) and CLIA profi- tion on accident rates in the popular press.
ciency testing results for that method can be used to estimate
To find out how well a method is performing, use the follow-
accuracy (bias in percent). The CV and bias can be used in a
ing basic formula (percentages for all):
metric (calculation) which makes it easier to determine how
much and which type of control rules are needed, based on how Sigma-metric = (TE — bias)/SD
well the method is performing. The method’s performance can
To obtain a Sigma-metric, subtract the method bias from
be plotted on the graph in Figure 29-15 to find your Six Sigma
the TE (use the CLIA TE for the method) and divide the prod-
Scores
uct by the method SD. This number shows statistically if the
method is high quality or not as long as the method remains sta-
Sigma-metrics ble. That is why it is crucial to run quality control materials—
to detect instances of instability in the method that could
Six Sigma is not new; it has been around since the 1980s and produce an erroneous result.
has revolutionized many other industries and saved compa- The minimum acceptable quality for a Six Sigma
nies billions of dollars. The principle is based on the Gauss- process is 3 Sigma. If a method is below 3 Sigma, the method
ian distribution and has been updated to correspond to a is not a quality method. You first need to make changes in the
process. Now it is time to start applying this proven quality process and repeat the experiments until your method has
tool in the laboratories. Six Sigma can quantify how well a attained a 3 Sigma level. For a 3 Sigma method, the maxi-
mum number of QCs and rules must be applied; a poorer
quality method requires more controls to maintain the maxi-
mum quality possible, and the opposite is true of a higher
Table20-4 Westgard MultiRules:
quality method. It is a win-win situation when a method is a
Bhilai aed of Detecting —
Errors, False’ Rejections
High Probability High Probability of
D sox 25-6SirSees
Error False Rejection of Error Detection
(atGa 20%) ¢ Well-documented principles
Condition ‘Gt feastOe)
* Useful way to gauge method quality
No errors I. ¢ Easy to calculate
Random Ly 55, I36,13 55,Ras
e Easy to understand
error
¢ Based on Gaussian curve
Systematic 296, 41s, 20f39,, 31,, 10,
error ¢ Use for further quality tests
714 ~— Chapter 29 Quality Management, Quality Assurance, and Quality Control

12.0

6.0

%)
in
(bias
inaccuracy
Observed
0.0
0.0 2.0 3.0 4.0 6.0

Observed imprecision (SD in %)

Figure 29-15 @ Imprecision and the Six Sigma scale.

quality method. Not only are the patient results more precise Operator suspects a problem when the control was run: short
and accurate, QC material can be kept to a minimum. sampling, mix up of control levels, etc. Do not run patients
For a 3 Sigma or less method, the maximum error detec- until you are satisfied that the problem has been solved or a sys-
tion with low false rejection rate would be a combination of tem is in place to monitor the method for future reoccurrence.
these rules: 1,;, with six controls and the 1,/2,/ R,/3,/6, The first step of troubleshooting QC is to determine whether the
MultiRules with six controls. If you are using a trilevel mate- error is systematic or random. How can the operator distinguish
rial (3 levels) with a 4 Sigma process, you could choose a rule between them? Review Figure 29-14. Which rule was violated?
such as 2o0f3,, for systematic error, and another rule for ran- Does that rule do a better job of detecting random or systematic
dom error, such as 1;,. For higher Sigma methods, possibly error? There is also a third kind of error, “flyers,” that are diffi-
only a single rule needs to be used (choose a rule for detect- cult to catch because they are caused by a bubble or a one-time
ing systematic error).** problem. The type of error should be related to the potential
causes for that error. Remember: Matching the type of error to
CALCULATING QUALITY the potential causes makes good troubleshooting sense.
To support the interest in easy calculations for quality proce-
dures, computer programs are available to automatically SYSTEMATIC ERRORS
select the QC rule(s), number of control levels, and frequency If a multitest system is being run (i.e., automated CBC), how
needed to maintain a particular level of quality desired. many of the parameters were out of control? If more than one
EZ Rules3° by Westgard QC, Inc. (http://www.westgard. parameter was affected, then the troubleshooting should be
con/ezrules.html) is the latest version of the popular Validator® logically aimed at processes that the parameters have in com-
software. A demo of the program can be downloaded for mon. For example, the WBC counting chamber also deter-
free. A SigmaMetric score can also be calculated with this mines the Hgb parameter. Another route to investigate is any
program. The data gathered from the method validation recent changes to the system, reagents, calibration, and main-
experiments are entered into the forms along with the tenance, as they cause many systematic errors.
desired number of control levels and rejection rate.
Several other QC planning tools are available on www.
RANDOM ERRORS
westgard.com: OPSpecs Charts, Critical-Error Graphs, and
The first and easiest way to troubleshoot random errors is to in-
Power Function Charts. These programs use new grids and
spect the system while it is operating. Watch every process that
charts for calculating the necessary QC procedures for individ-
can be visually accessed. Look for loose seals, bubbles, leaks,
ual analytes using imprecision and inaccuracy of the method.
faulty delivery volumes, clogs, and any mechanical parts and
reagents. If there is nothing visually wrong, consult the opera-
Troubleshooting Quality Control Problems tor’s manual and troubleshooting guide for assistance. Ask key
operators to take a look; review the error log for clues. Call
The method validation is performed, the Sigma-metric is calcu- Technical Service for suggestions or to have an engineer inspect
lated, and the Westgard Rules have been chosen for the method. the instrument. Run a precision check of at least 10 replicates of
If all of these procedures were performed correctly, there is a a fresh patient sample to rule out imprecision. If no problems
low false rejection rate for the control values. Thus if a control can be detected, add another random error Westgard Rule to
value falls outside the QC rules, there is a high probability that help detect a future error and monitor the system (tell all staff
the control is doing its job: detecting random or systematic about the problem also). Always document all investigations
error. Rerunning the control should be considered only if the and findings in a problem log for the method or instrument.
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 715

Peer Group Quality Control and less if possible. If the ranges were adjusted every month to
match the peer mean, the QC process would be negated in
Most of the larger companies that manufacture instruments favor of looking good on paper.
and/or QC products have a peer group QC program. The daily As with any quality review, written documentation must
or monthly data are sent to the company to compare it to other be noted on the peer group report that it was reviewed and no
labs running the same instrument or QC material for the same problems were seen with the data. If any assays require inves-
assays. This serves two purposes: to satisfy one of the CLIA tigation, the details should be written as if an inspector were
requirements for outside assessments and to provide feedback reading them. Always date any reviews and list any corrective
about a laboratory’s methods versus peers. Review the group- or preventive measures. Writing “will monitor” 1s unaccept-
ing criteria to be sure that the instrument is in the correct able these days.
group according to reagent lot number or control lot number.
Knowing how a method compares with peers gives laborato-
rians One more tool in their quality toolbox. Hematology Laboratory Applications
STATISTICS
Quality Plan Example
The manufacturer will probably provide peer statistics in their Laboratory Alpha decides to combine selections from several
report containing one or more of the following: Quality Management approaches to form their own unique
SDI (Standard Deviation Index): This calculation describes Quality Plan. From the Lean approach, the staff follows a
the systematic error or bias of a method as a multiplier of the CBC, a PT, and a urinalysis sample from collection through
standard deviation for the group: result reporting to the physician. Samples are chosen from
SDI = (Lab mean — Group mean)/ Group SD different areas, 1.e., outpatients, inpatients, clinics, to cover as
many scenarios as possible. Staff observe the motions of the
An SDI of 0.0 indicates that the lab mean is exactly the sample and the employees to document extra waiting, extra
same as the group mean. A SDI of 2.0 or higher requires steps, and unnecessary movements. If a defect or extra pro-
investigation: the method is in danger of systematic inaccu- cessing step occurs, these will be noted. The laboratory must
racy. An advantage to using SDI is a person can scan a peer define how often the study will be performed, how the data
group report and view the SDI of all the assays fairly quickly. will be collected and presented, and ongoing review and cor-
CVI (CV Index): The CV of the lab and the CV of the group rective actions. Many quality entities are covered with this
compared: plan, making it comprehensive. However, this would be only
CVI = Lab CV/Group CV a portion of a total Quality Management Plan. The staff also
decide to use Six Sigma for automated processes; the Sigma
A CVI of 0.0 indicates the laboratory’s CV is the same score is calculated for all assays and changes are instituted to
as the group CV. Any CVI greater than 0.0 suggests that the raise the Sigma score to at least a 3 Sigma. Once they are con-
laboratory’s imprecision is less than that observed for the fident that most of the processes in the laboratory are at peak
group. A CVI greater than 1.0 suggests the lab’s imprecision efficiency, the staff decide to apply for the Malcolm Baldrige
is higher than that of the group (Box 29-7). National Quality Award, which will help focus improvements
Z-score. Measures how many standard deviations the in several customer service areas. There are regulatory
mean of an analyte is from the mean of the peer group. A requirements from several agencies that should be included in
Z-score has no units; however, the score can be positive or the plan: turnaround time for critical tests, participation in a
negative. proficiency testing program, continuous competency assess-
Z-Score = Lab mean — Peer mean/SD
ments, appropriate quality control procedures for each assay,
reviewing errors and correcting the causes, and others. A
Hematology Department must also have its own plan,
REVIEW OF PEER GROUP DATA
although the general Laboratory plan may cover most of the
While reviewing peer data, the laboratory’s assay mean should
elements.
be compared to the peer’s mean: is the lab’s mean consistently
above or below the peer mean every month, but the lab’s SDI
and CVI are acceptable? Is it time to consider an adjustment in Method Validation Studies
the ranges? QC ranges need a little adjustment now and then
Once a new method or new instrument has been chosen and
as part of the normal instrument and QC material process. A
is installed, the method validation studies must be run. The
good rule of thumb is to adjust QC ranges only every 90 days,
focus is to introduce about the same amount of error that
would be present on a daily basis in the normal running of the
test or instrument. If the test will be performed on three shifts,
Box 29-7 SDI and CY
validation samples should be performed on three shifts. Allow
e sDI (SD Index), compares your mean to the group mean | each technologist who will be performing the test to also
* CVI (CV Index), compares your CV in the group’s CV | analyze some of the validation samples. For normal patient
}
i CSS SNe
| ener ein oe oop parent ne ae studies, the goal is to duplicate the same types of patients
716 Chapter 29 Quality Management, Quality Assurance, and Quality Control

tested every day. A plan is necessary to organize the samples Atlanta and created a new organization, The Institute for
needed for each study, the time required for each study, and Quality in Laboratory Medicine (IQLM). This consortium
the personnel who will be conducting the studies. will bring together more than 70 groups from payers, govern-
ment agencies, accrediting organizations, laboratory profes-
EXAMPLE OF METHOD VALIDATION STUDIES sionals, health systems, device manufacturers, clinicians, and
Hematology Laboratory Alpha has just installed a new auto- patients to discuss how to improve patient safety through lab-
mated hematology instrument. After the basic functions of the oratory testing.** **
instrument are verified by the manufacturer, the validation
studies begin. A replication experiment is planned in two
phases, preliminary and comprehensive. The preliminary
Equivalent Quality Control Option 4
phase is completed in one day with one run of 20 replicates of This term was penned by CMS in their Surveyor’s Guidelines
the same sample (within-run random error). The comprehen- in the final CLIA-88 rule. Equivalent Quality Control (QC)
sive phase includes analyzing quality control material of dif- describes situations in which a laboratory could run QC
ferent levels on all shifts by several technologists for at least materials Jess often than the previous CLIA rules of two levels
20 days (between-run random error). The mean, SD, and CV per run. There was much discussion of EQC in laboratory
are calculated for all parameters for both phases and are organizations, and most of them agreed that the level of quality
acceptable. A reportable range experiment is performed with is not yet high enough to allow QC to be run less often than cur-
a commercial CBC and reticulocyte linearity kit that covers as rent practice. In 2004, AdvaMed went as far as to propose a
much of the analytical range as possible (proportional system- new Option 4 where the manufacturer performs a risk assess-
atic error and linearity). The manufacturer of the kit receives ment, recommends a quality plan to monitor and detect the
the data and calculates the statistics; the laboratory personnel risks, and produces data to prove that the quality plan really
review the data and decide on a reportable range. Because the works. This information would be reviewed and approved by
new automated analyzer is similar to the current one, a small the FDA. Laboratories could then implement the quality plan in
normal range study is performed with 20 carefully screened place of one of the current CLIA options. Since the final CLIA-
adult patient samples from both sexes, and the previous range 88 rule has eliminated FDA clearance of manufacturer’s QC
is verified. For pediatric normal values, a decision is made to instructions, the laboratory now must validate all quality con-
transfer the ranges by calculation from the pediatric sample trol procedures, even manufacturer’s guidelines.*°
portion of the comparison of methods study. The comparison
of methods experiment is performed with 40 adult and
40 pediatric patient samples, using normal and abnormal
patients across the entire analytical range. The results of all
the studies are acceptable. Case Study 1
A technologist has just run the three levels of controls on a
Quality Control hematology analyzer. The low level for WBC has been
within 1SD of the mean for the past 3 weeks; however,
Now that the instrument performance has been investigated today the value is greater than 4 SD of the mean. The lab
and deemed acceptable, the monitoring of the quality required uses the following Westgard Rules: R,., 20f3>,, 77, 10,.
needs to be planned and executed. The first step is to calculate
the Sigma-metric score for the instrument. For a hematology QUESTIONS
analyzer with multiple parameters, a critical parameter might
be chosen, or a parameter with the highest SD; the laboratory 1. What should be done next?
2. Is this a random or systematic error?
must decide this. Suppose the Sigma-metric score was 4.0 for
3. Do all the patients need to be rerun since the last low
hemoglobin. With this information, the supervisor uses the
level was within limits?
EZRules3 software to determine which MultiRules to use
with the analyzer. After entering in the information into the
ANSWERS
calculator, the MultiRules given are 1,;, and R,, with two
levels of controls (example only). 1. Most control material for hematology analyzers is pack-
aged as a set of three levels: low, normal, and high. The
Westgard Rule of R,, has been violated; action is required.
Laboratory Quality Updates Errors from improper mixing of the control material
should be ruled out first; the control is then rerun once.
Quality Meeting Launches New If the WBC low level is still outside 4 SD, a new control
Organization vial is opened, mixed, and rerun. A background check is
also appropriate for troubleshooting problems with low
In recent years, many health-care organizations have con- levels of controls. If the background check is acceptable
and the control is still outside 4 SD, observe the instru-
vened to discuss improving the health of the public with
ment during all phases of testing: are there any leaks,
better use of laboratory tests. In April 2003, the Centers for
continued
Disease Control and Prevention (CDC) hosted a conference in
 Chapter 29 Quality Management, Quality Assurance, and Quality Control 717

partial aspiration errors, interference flags, bubbles in QUESTIONS

any of the lines, buildup on the sampling valve?
Zapping or bleaching the apertures may resolve the 1. Is this a systematic or random error?
problem. Run a 10-sample precision check of a fresh 2. What are the next steps?
patient or control. Review all of the quality control on 3. Is the problem more likely to be instrument/reagent-
the instrument for the past few weeks for shifts and related or quality control material-related (manufactur-
trends. Call Technical Service or check the operator’s ing, handling)?
manual for more suggestions. If no source of the prob-
lem is found, add another Westgard Rule to detect future ANSWERS
errors. At some point in the future, the additional rule
may seem unnecessary and can be removed. 1. This is a systematic error, affecting more than one para-
2. This is a random error. Only one parameter is affected. meter.
No shifts or trends were found when the QC was
2. Handling of the control material should be ruled out
reviewed. first. All the QC records on this analyzer for the past few
3. If the normal and high levels controls are within 1 SD, weeks should be reviewed for shifts and trends. Is there
another identical or very similar instrument that uses the
there may be an issue with the low range of reportable
same controls? Compare how the controls are running
results. After the instrument is given a clean bill of
on both instruments. Since the WBC and platelets are
health by an engineer, calibration and linearity studies
counted in the same chamber, look for instrument prob-
may be appropriate to verify the reportable range.
lems that affect this pathway. Check the background
counts, check reagents and tubing, clean the sampling
valve, bleach the apertures, etc. Observe the instrument
as it processes a sample for any clues: bubbles, leaks,
etc. The lower levels of controls may pick up a blockage

Case Study2 or small leak first.

3. This is more likely to be an instrument problem. A high
CV is hardly seen with mishandled control material; the
A technologist is running 10 runs of a new lot of hematol- cells are stabilized and are resistant to most mishandling.
ogy control material with three levels: low, normal, and
high. She notices that the low control has a much higher CV
for the platelet count and the WBC than the previous lot
number.
continued

Di cctiont x

1. A quality test result would: . ¢, Performed only by outside agencies

a. pe accurate Performed internally as well as externally
b. Be meaningless to a physician 6. Which of the following are not types of quality manage-
c. Cost a lot of money ment activities?
d. Take a very long time to process a. Equipment maintenance
2. All of the following are part of Root Cause Analysis, b, Procedure manuals
except: (¢.Taking a coffee break
a. Sequence of events charting d. Calibration
b. Data collection 7. Trends can be seen on:
> Recommendations a. Gaussian graph
(a Wastedefinitions b. Youden plot
5 Cie : ) |
lr (1 Clans Ag t
3. Lean’s main focus is: VMUL 30 CUpOWre c. Sigma-metric
Levy—Jennings graph
4, All of the following are examples of good customer ser-
vice except: 8. Westgard Rules help to identity:
a. Calling critical results promptly a. Random error
b. Answering the phone politely b. Systematic error
Asking a nurse to call back later c. Trends
d. Giving a physician information he/she asked for . Shifts
es andb only
5. Assessments are:
~f. c and d only
a. Only important for passing an inspection
g. all of the above
b. Something you do only once a year
718 Chapter 29 Quality Management, Quality Assurance, and Quality Control

9. Which of the following is a high quality process? c. Performing a precision check

a. 3 Sigma @ Rechecking recent reagent changes, calibrations
b. 4 Sigma 12. Explain the types of statistics commonly included in
(c. 6 Sigma peer group data.
d. 2 Sigma
10. List four method validation experiments required by See answers at the back of this book.
CLIA.
11. Troubleshooting steps for a systematic error do not
include:
a. Investigating all parameters for errors
b. Running control material

m There are many factors to consider when choosing a

new method or instrument. Instead of simply looking at
cost, other equally important issues should be addressed:
= Quality should be a priority for every activity in a risk, bias, working range, sample requirements, interfer-
laboratory. ence, methodology, and so forth.
= The focus on quality has been around for centuries. The # CLIA-88 requires that a laboratory validate any new
Industrial Revolution started the quality process move- method or instrument. This means that experiments must
ment. Recent studies of medical errors causing many be run to prove the manufacturer’s claims: replication,
deaths refocused the quality microscope on the laboratory. interference, recovery, and method comparison. This
@ Quality management involves all processes in a labora- is the minimum amount that should be run; a particular
tory. A quality management plan is required of every method may require more studies. Before beginning
laboratory. a validation, a plan is helpful to reduce time and
m There are several successful quality approaches: Bench- wasted steps.
marking, Root Cause Analysis, ISO, Six Sigma, FMEA, a Different quality control procedures monitor different
Lean, TQM, Malcolm Baldrige, and others. A laboratory parts of the analytic process. A laboratory should maxi-
can choose any or all of the quality approaches to mize quality control at critical points in the process to
implement. reduce the chances of a medically important errors.
w Quality Management involves twelve divisions, or Qual- mw CLIA-88 details all the quality control activities a labora-
ity System Essentials (QSE). Each of the divisions is tory should be performing: establishing or verifying all
equally important and none should be ignored. control ranges, testing controls after reagent changes,
m Quality Assurance is doing the quality activities necessary rotating the running of controls among the staff, docu-
to produce a quality test result. Quality Control is an mentation of activities, and so forth.
observation technique or activity of a quality process. m Levy—Jennings graphs are useful for determining how a
mwCLIA-88 regulations outline all quality requirements method is performing. Trends, shifts, and controls out of
from the government. These guidelines are minimum range can be easily seen.
requirements for quality. w Westgard Rules give a laboratorian a set of rules to judge
m Accuracy is hitting the bulls-eye; the target has been the control results by. Running controls can be custom fit
achieved. Precision is reproducibility: hitting the same to the method and less time can be spent rerunning con-
part of the target over and over. trols. Troubleshooting is more focused when you know
the types of errors the method is experiencing.
m Accuracy is measured in bias from a mean or target;
precision is measured in variation from a mean or target. m Sigma-metrics can demonstrate the quality of an entire
process, not just a control result. Now a method’s quality
w Standard deviation (SD) relates to the spread of the data
can be easily calculated; this information is important for
and indicates random error. Coefficient of variation (CV)
process improvement.
also indicates random error, and uses the SD to monitor a
method along varying concentrations. = The commonly used 1,, Westgard Rule has a high false
rejection rate. In other words, there is a high probability
@ Proficiency testing results are used for Total Error (TE)
of false alarms—control values that are not indicating a
estimates because only one sample is run once, increasing
method problem. It would be wise to adopt other
the possible errors to the maximum.
Westgard Rules instead.
 Chapter 29 Quality Management, Quality Assurance, and Quality Control TAQ

# Troubleshooting control value problems is easier when a instrument, and a report is sent with useful statistical
specific rule(s) is matched to a specific method. information, such as SDI, CVI, and the means and SD’s
m Systematic errors affect an entire method (proportional or from the past few months.
constant); random errors affect a part of the process. m The next few years should be exciting as laboratory
m Peer group data is a great tool for quality management. A groups discuss where quality is going.
method is compared to others with the same method or

REFERENCES . Powers, D: Laboratory quality control . Westgard, JO: Basic QC Practices, ed 2.

requirements should be based on risk Madison, WI: Westgard QC, Inc, 2002.
l . Sazama, K: Legal Implications of Labo-
management principles. Lab Med . Westgard, QC: Method Validation—
ratory Errors. Lab Med 36:213, 2005.
36:633, 2005. Selecting a method to validate .
2 . Centers for Disease Control and Preven-
. Lean Enterprise Institute (1997-2006): Retrieved April 28, 2006 from
tion: Current CLIA regulations, July 2004.
Principles of lean. April 20, 2006, from http://www.westgard.com
Retrieved May 2, 2006, from http://www.
http://www.lean.org. Pile Westgard, QC: Final? CLIA Rule. Part I:
phppo.cdc.gov/clia/regs/toc.asp.
. Berte, L: Don’t waste your Laboratory Key Changes (2006). Retrieved April 28,
. College of American Pathologists: Labo-
away. Lab Med 37:224, 2000. 2006 from http://www.westgard.com.
ratory Accreditation Program Laboratory
. Baldrige National Quality Program 28 . Clinical and Laboratory Standards Insti-
General Checklist, April 2006. Retrieved
(2001-2006): History of the Malcolm tute: How to define and determine refer-
April 11, 2006, from http://www.cap.org.
Baldrige National Quality Award. ence intervals in the clinical laboratory.
. The Clinical and Laboratory Standards
Retrieved May 20, 2006, from Retrieved May 10, 2006, from http://
Institute: A quality management system
http://www.quality.nist.gov. www.nccls.org.
model for health care; Approved guide-
. Berte, L: Who is the laboratory’s cus- 29! College of American Pathologists: Labo-
line HS1-A2. Wayne, PA: NCCLS, 2004.
tomer? Lab Med 36:287, 2005. ratory Accreditation Program Laboratory
Cn. The Clinical and Laboratory Standards
. Halstead, D, and Oblack, D: Executing General Checklist, April 2006. Retrieved
Institute: Application of a quality man-
successful performance appraisals and April 11, 2006, from http://www.cap.org.
agement system model for laboratory
competency assessments. Lab Med . Levey, S, and Jennings, ER: The use of
services; Approved guideline GP26-A3.
36:145, 2005. control charts in the clinical laboratory.
Wayne, PA: NCCLS, 2004. . Steward, C, and Schulze, M: ASCP sur- Am J Clin Pathol 20:1059, 1950.
. Berte, L: The right word. Lab Med 36: . Westgard, JO: QC- The “Westgard
vey on laboratory personnel state licen-
416, 2005. sure. Lab Med 36:524, 2005. Rules.” In: Westgard, JO (ed): Basic QC
— . International Organization for Standard-
. McDowell, J: Using activity-based cost- Practices, ed 2. Westgard QC, Inc.,
ization (n.d.): ISO 9000: 2000. Retrieved
ing to aid in the selection of laboratory Madison, WI, 2002, pp. 77-89
May 21, 2006, from http://www.iso.org. . Westgard, JO: Six Sigma Quality Design
equipment. Lab Med 36:278, 2005. Ooi)
oo. Six Sigma (2000-2006): Six sigma, 20. Lasky, F: Technology variations: Strate- & Control, ed. ed 2. Westgard QC, Inc.,
quality resources for achieving six sigma gies for assuring quality results. Lab Med Madison, WI, 2006.
results. Retrieved May 25, 2006, from 36:617, 2005. 33. Institute for Quality in Laboratory Medi-
http://www.isixsigma.com 2G Joint Commission: Joint Commission cine: Retrieved May 16, 2006, from
\o. South, S: Achieving breakthrough
Requirements. Retrieved April 17, 2006, http://www.iqim.org.
improvements with the application of from http://www.jointcommission.org. 34, ASCP Press: What is IQLM? Creating
Lean Six Sigma tools and principles . Berte, L: Managing laboratory quality— the Institute for Quality in Laboratory
within process excellence. Lab Med 36: A systematic approach. Lab Med 35:621, Medicine. Special Report. Lab Med
240, 2005. 2004. 36:605, 2005.
. Westgard, JO: Six Sigma Quality Design . Woodcock, S: Quality management soft- . Yost, J, and Mattingly, P: CLIA and
& Control, ed 2. Westgard QC, Inc., ware. Lab Med 36:333, 2005. equivalent quality control: Options for
Madison, WI, 2006. . Westgard, QC: QC—The calculations the future. Lab Med 36:614, 2005S.
. Woodhouse, S: Engineering for safety: (2006). Retrieved April 26, 2006 from
Use of failure mode and effects analysis http://www.westgard.com.
in the laboratory. Lab Med 36:16, 2004.
 Body Fluid Examination
The Qualitative, Quantitative,
and Morphologic Analysis of Serous,
Cerebrospinal, and Synovial Fluids
Sharon L. Schwartz, MS, MT(ASCP)SH, CLS(NCA)

Introduction OBJECTIVES
Types of Body Fluids At the end of this chapter, the reader should be able to:
and Anatomy
Pericardial, Pleural, L List the types of body fluids from closed body cavities studied in the hematology
and Peritoneal (Serous) laboratory.
Fluids . Identify the type of procedure used to obtain each type of fluid.
Nw
Cerebrospinal Fluid
Synovial Fluid . Describe the formation of serous (peritoneal, pericardial, and pleural), cerebrospinal,
and synovial fluids.
Specimen Collection
and Preparation . Identify the cell types normally found in serous, cerebrospinal fluid, and synovial fluids.
Collection
. Define effusion, transudate, and exudate and describe their characteristics.
Preparation
. Describe the laboratory methods for body fluid analysis.
Cellular Components
of Body Fluids eS
eC. Describe
SCN the principle, advantages, and disadvantages of the cytocentrifuge method
Neutrophils for body fluid morphological examination.
Lymphocytes
. Describe the common cellular artifacts introduced by cytocentrifugation.
Monocytes
Tissue Cells . Differentiate normal from reactive or malignant cells in body fluids.
Eosinophils, Basophils, Mast
Cells 10. Associate morphologic abnormalities in body fluids with their reactive and malignant
causes.
Pleural, Pericardial, and
Peritoneal (Serous) Fluids lidbs Describe the importance of distinguishing a traumatic lumbar puncture from a true
Effusions: Transudates and CNS hemorrhage.
Exudates . Explain the clinical importance of detecting leukemia, lymphoma, and metastatic
Cellular Responses, cancer cells in body fluids.
Microorganisms, and
Malignant Cells in Serous . Describe the purpose of synovial fluid analysis.
Fluids . Describe the types of crystals found in synovial fluids.
Types of Effusions,
. Associate the types of crystals found in synovial fluid with the types of joint
Laboratory Analysis, and
diseases.
Clinical Correlations
Pleural and Pericardial . Define birefringence and relate it to crystal analysis.
Effusions
Peritoneal Effusions
Cerebrospinal Fluid
Specimen Collection and
Processing

720
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis WaN

Laboratory Analysis and

Clinical Correlations
Cellular Responses,
Malignant Cells, and
Microorganisms in CSF
Synovial Fluid
Specimen Collection and
Processing
Laboratory Analysis and
Clinical Correlations
Quantitative Analysis:
Biochemical Analysis and
Microscopic Examination
Crystal Examination
Summary
Case Study 1
Case Study 2
Case Study 3
Case Study 4

Introduction found in closed body cavities: the thoracic and abdominal

cavities, central nervous system, and joint spaces. Fluids
The analysis of fluids from normally sterile body compart- from the thoracic and abdominal cavities are referred to as
ments is an important element of patient care and provides serous fluids, which mean that they are related to or resem-
clinicians with valuable information in the diagnosis and ble serum in their composition. In reality, they are ultrafil-
treatment of disease. Under the abnormal conditions of dis- trates of plasma.
ease processes, these fluids may significantly increase in
volume and contain infectious and/or inflammatory sub-
stances and cellular elements. This chapter describes the
Pericardial, Pleural, and Peritoneal (Serous)
hematological analysis of the most commonly encountered Fluids
body fluids in detail, while discussing the chemical, micro-
The thoracic or chest cavity contains the heart and lungs.
biological, serological, and cytologic analyses, when
The heart is enclosed within a fibrous structure, known as
appropriate, as performed by those respective laboratory
the pericardial sac. The pericardial sac is lined by a single
disciplines.
layer of mesodermal cells called mesothelial cells. These
The hematology laboratory is responsible for cell enu-
cells form a continuous membrane, known as the peri-
meration (or cell count) and morphologic evaluation (or
cardium, which covers the inner or parietal surface of the
differential) of the body fluid specimen. The types of cells
pericardial sac, and the outer or visceral surface of the
and their concentrations are useful in disease diagnosis. The
heart. The potential space between the two membranous
cells that can be found include white blood cells (WBCs),
surfaces is called the pericardial cavity. It contains a small
red blood cells (RBCs), tissue cells, and in some cases
amount of serous fluid that is secreted by the parietal peri-
tumor cells. It is important for the laboratory professional
cardium and absorbed by the visceral pericardium. It func-
to be able to distinguish benign from reactive or malignant
tions as a lubricant between the membranes as the heart
cells and appropriately refer these findings to the patholo-
relaxes and contracts.
gist. Crystal formation in body fluids is also diagnostically
The lungs are encased by a thin membrane composed of
significant. a single layer of mesothelial cells known as the pleura. The
lung surface is covered by the visceral pleura. The pleura also
lines the inside surface of the chest wall, forming the parietal
Types of Body Fluids and Anatomy pleura. The potential space between these surfaces forms the
Body fluids, an all-inclusive term, may be used to describe pleural cavity that contains a thin film of serous fluid pro-
a diverse group of non-blood, non-urine fluids that are duced by the parietal pleura and absorbed by the visceral
722 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

pleura. This fluid provides lubrication for the membranes dur- reabsorbed at the same rate by the arachnoid villi of the
ing respiration while preventing the formation of any space arachnoid mater. The arachnoid mater penetrates the inner
between the lungs and chest wall. This maintains the lungs in dura and its venous sinuses. The choroid plexus epithelium
an expanded state. and the endothelium of the capillaries in contact with the
The abdominal cavity is lined by a membrane also CSF form the blood—CSF barrier.'! This barrier controls
composed of a single layer of mesothelial cells known as the the concentrations of solutes between the blood and CSF.
peritoneum. It is the largest and most complex serous mem- The normal total volume of CSF in adults is 90 to 150 mL,
brane in the body. The parietal peritoneum covers the inner and 10 to 60 mL in neonates.
surface of the abdominal wall. The visceral peritoneum cov-
ers the surfaces of the stomach, small and large intestines,
liver, and the superior aspect of the urinary bladder and
Synovial Fluid
uterus. The kidneys, pancreas, duodenum, some lymph Joints, also known as articulations, are the junctions between
nodes, and the abdominal aorta are posterior to (not two or more bones. The freely movable limb joints of the
enclosed by) the peritoneum, and are known as retroperi- body (e.g., shoulder, elbow, knee, hip, etc.) are enclosed by
toneal. The potential space created by the two layers of peri- strong fibrous tissue for support and alignment of the bones,
toneal membranes is known as the peritoneal cavity. The known as the joint capsule. It consists of an outer fibrous
parietal peritoneum secretes a serous fluid that prevents fric- layer and an inner membrane with a rich blood supply, called
tion between the membranes from the constant motion of the the synovium or synovial membrane, composed of a single
abdominal organs, and is absorbed by the visceral peri- layer of mononuclear synovial cells. Articular cartilage covers
toneum. Normal pleural, pericardial, and peritoneal cavities the bony surfaces of the joint that permits smooth motion dur-
do not contain appreciable amounts of fluid, and as such, are ing movement. The synovium also surrounds the tendons and
not true cavities until a disease state causes the accumula- free margins of the ligaments and articular cartilage, but does
tion of fluid. not extend over the surface of the intra-articular cartilage. An
ultrafiltrate of plasma, known as synovial fluid (or mucin; an
older terminology), is secreted by the synovial cells. This fluid
Cerebrospinal Fluid
fills the synovial (joint) cavity, the space that exists between the
The central nervous system (CNS) consists of the brain and bones and enclosed by the synovium and intra-articular carti-
spinal cord, enclosed by the bony structures of the skull and lage. The synovial cells also produce a mucopolysaccharide
vertebrae of the spine. These structures are lined by special known as hyaluronic acid. This gives the fluid a viscous
membranes known as the meningeal membranes or consistency providing lubrication and facilitation of movement.
meninges. The meninges consist of three layers of tissue: a Synovial fluid also transports nutrients to the articular cartilage.
relatively thick outer membrane known as the dura mater, Synovial cells are also phagocytic, removing cellular debris that
which provides the major protection for the brain and spinal exists at the surface of the membrane. The large joints may nor-
cord, a thinner middle membrane called the arachnoid mally contain approximately 1 mL of synovial fluid.
mater, and an inner membrane that lies directly on the sur-
face of the brain and spinal cord known as the pia mater.
The area between the arachnoid mater and pia mater is Specimen Collection and Preparation
known as the subarachnoid space. It contains a selective Collection
ultrafiltrate of plasma known as cerebrospinal fluid (CSF),
which protects and supports the brain and spinal cord, and A properly collected specimen is crucial to proper diagnosis.
maintains a constant ionic environment by circulating nutri- Body fluids are collected only by a physician or properly
ents and removal of waste products. These functions are trained medical personnel because these procedures are
controlled by the blood-brain barrier, which consists of the invasive and may potentially cause harm to the patient.
endothelial cells of brain capillaries wrapped by supporting Specimens are always obtained by aseptic technique for
cells of the brain known as astrocytes. These cells permit the diagnostic or therapeutic purposes. They are potentially
passage of essential substances such as water, oxygen, and infectious and must be handled with universal precautions.
carbon dioxide, and exclude the passage of large molecules They must also be handled with great care, as they
such as proteins, peptides, and many drugs. frequently are nonretrievable, especially in neonates. The
The CSF circulates through the ventricular system of aspirated fluid is placed into evacuated, anticoagulated,
the cerebrum, cerebellum, and brain stem. Masses of spe- and/or sterile tubes to be sent to the laboratory. Details about
cialized capillaries in the pia mater, known as the choroid the collection of each body fluid type are discussed in more
plexus, project into the ventricles and are the main source detail later in the chapter.
of production of CSF. The ventricles and central spinal
canal are lined by a layer of epithelial cells with villous
Preparation
projections and cilia known as ependymal cells, or the
ependyma. Together, the ependyma and choroid plexus On receipt in the laboratory, the body fluid specimen must be
produce approximately 20 mL of CSF per hour, which is processed immediately. The cells within a fluid are usually in
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 728,

a lower concentration than peripheral blood and more fragile; cells within a fluid suspension directly onto a glass slide as a
their viability deteriorates once they are removed from monolayer. The method utilizes a small funnel apparatus that is
the body. held against a glass slide by a clamp that fits into the cytocen-
First, the specimen is evaluated for the appropriate trifuge. Between the funnel and the slide is a fitted strip of
collection container and visually inspected for volume, filter paper with a hole under the funnel outlet. Cytocentrifuga-
color, clarity, viscosity, and/or the presence of fibrin clot. tion is based on the principle that cells are concentrated by slow
Next, the sample is set up to perform the cell count. The centrifugation with simultaneous absorption of the noncellular
automated methods currently in use for peripheral blood portion of the fluid into a filter paper. The filter preferentially
cell counts are not recommended for use on body fluids for absorbs the fluid portion leaving the cells in a concentrated but-
several reasons: ton on a microscope slide. This method markedly improves the
1. The peripheral blood cell-counting instruments are not quality of the cell morphology obtained as compared to the
standardized for a body fluid medium. direct smear technique. It is superior for differential analysis
2. Cell size variation in fluids is greater than in peripheral blood. over the performance of an older technique known as the hema-
3. Background debris and clots are often present in serous cytometer chamber differential count, which had the following
fluids. limitations:
4. Certain fluids (e.g., synovial) are quite viscous. ¢ Chamber differential counts only provided the clinician
Despite its imprecision, manual cell counting using a hema- with cell categorization limited to polymorphonuclear or
cytometer is the preferred method for total cell counts on mononuclear leukocytes; the mononuclear category included
body fluid specimens (see Chap. 31). Unlike peripheral many different cell types but was usually interpreted as
lymphocytic.
blood, and unless the specimen is obviously bloody or
cloudy, the fluid is counted undiluted. If it is necessary to * Cell differentials were done on a limited number of cells,
i.e., the number present in the chamber.
dilute the sample, the diluent used must be appropriate for
the type of specimen (i.e., synovial fluid diluent must not ¢ The cells were either unstained or minimally stained to
determine nuclear detail.
contain acetic acid; see discussion on Synovial Fluid.) The
¢ Malignant or reactive cells were impossible to differentiate
diluent must be filtered and free of any background debris
from other cells.
by microscopic verification. Separate dilutions may be
¢ No permanent preparation could be retained for future
required for the RBCs and WBCs to reduce error. The num-
review.
ber of squares (area) to be counted is determined by the
concentration of cells present:* The College of American Pathologists has recommended
¢ If more than 200 cells are seen in a square millimeter of the cytocentrifugation as the preferred method for hematologic
chamber (e.g., a WBC square), the cells in the five RBC analysis.* The obvious advantages include (Table 30-1):
squares of the center square should be counted. * Its relative ease and speed of preparation
¢ If more than 200 cells are seen in the entire ruled area (..e., * Cell differentiation is done on a concentrated preparation
all nine squares), the cells in each of the four corner WBC (good cell recovery).
squares should be counted. * Cell differentiation is determined via Romanowsky (Wright;
¢ If fewer than 200 cells are seen in the entire ruled area, the Wright-Giemsa) staining.
cells in all nine squares should be counted. ¢ Normal, reactive, and malignant cells can be identified with

The calculation of the cell count is based on the formula:

adequate training.
¢ The cytocentrifuge slide preparation becomes a permanent
number of cells counted X dilution factor record that can be retrieved for review.
Total cells/uL =
number of squares counted < volume/square

volume/RBC square: 0.004 uL

volume/WBC square: 0.1 uL
The same area on both sides of the hemacytometer
is counted and the percent difference of the count from
each side must be less than or equal to 10%; if greater, the
cellular distribution was poor and the counts must be
repeated. e Ease of preparation.
Finally, the sample must be prepared for morphological e Speed of preparation.
evaluation. This portion of the body fluid analysis is the most ¢ Concentrates specimen for good cell recovery.
important aspect of the process, because it becomes the * Cellular differentiation is determined by Wright or
Wright—Giemsa stain.
permanent record of the cellular constituents within the spec- ¢ Normal, reactive, and malignant cells can be identified.
imen, and provides the basis for diagnosis. Body fluid slides * Cytocentrifuge slide becomes retrievable permanent
are prepared by a technique known as cytocentrifugation, using record.
a cytocentrifuge: a device that concentrates and deposits the
724 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

There are, however, certain disadvantages of the technique.

Cytocentrifugation may create artifacts that the laboratory
professional should be aware of. These include (Table 30-2):

* Overconcentration of cells in fluids with high cell counts

* Cells in the interior of the cell button may be smaller in
overall size with a denser nucleus than cells at the periphery.
¢ Abnormal cells are more likely to be affected because of
their propensity to be more fragile.
¢ Nuclear-induced changes can include peripheralization; dis-
torted shape; and segmentation, fragmentation, prominent
nucleoli, or holes.
* Cytoplasmic artifacts can include irregular fragmentation,
projections, localization of granules, and peripheral vac-
uolization (Figs. 30-1, 30-2, and 30-3).
Figure 30-1 m@ Artifact of cytocentrifugation. Peripheralization,
However, even with these issues, the cytocentrifuge method vacuolization, and hyperlobulation of neutrophils. Wright stain,
1000 magnification. (From Best, M: Body Fluids Examination.
produces a much better quality representation of the cellular
ASCP Workshop 9294, Boston, 1990, with permission.)
elements present in the fluid. The best results are obtained
with fresh specimens, processed immediately or as soon as
possible after collection. A small amount of added protein,
such as a drop of bovine or human serum albumin, will pro-
tect the cells during cytocentrifugation. The cell count helps
to determine the volume of body fluid specimen necessary in
performing the cytocentrifuge preparation; high cell counts
require less sample or predilution. Slides should be prepared
regardless of whether any cells were seen in the hemacytome-
ter, as the potential to detect abnormal or malignant cells
increases with the cytocentrifugation process. The optimal
speed and duration is determined by the individual laboratory
based on its equipment design.
After cytocentrifugation, the slides are allowed to air-dry
and then stained with Wright or Wright—Giemsa stain; addi-
tional slides can be made for other cytochemical stains. These
slides are then used to perform the differential WBC count as
well as for detection of any reactive or malignant cells. Figure 50-2 m@ Artifact of cytocentrifugation. Prominence of nucle-
Laboratories without cytocentrifugation equipment would oli, vacuolization in lymphocytes. Wright stain, x1000 magnifica-
have to prepare body fluid slides either on resuspended tion. (From Best, M: Body Fluids Examination. ASCP Workshop
sediment after centrifugation, or with a drop of undiluted or 9294, Boston, 1990, with permission.)

RBC-lysed hypercellular fluid, using a standard push smear or

4 table 50-2 Disadvantages of the

Cytocentrifugation
Method for Body Fluids
Creation of artifacts:

* Overconcentration of cells in fluids with high cell counts.

° Cells in the interior of the cell button appear smaller with
denser nucleus than cells at the periphery.
¢ Abnormal cells are more likely to be affected because of
their fragility.
* Nuclear-induced changes can include peripheralization;
distorted shape; segmentation; fragmentation; holes; more
obvious nucleoli.
* Cytoplasmic artifacts can include irregular fragmentation and Figure 30-5 @ Artifact of cytocentrifugation. Cytoplasmic projec-
string-like processes; clustering of granules; peripheral vac- tions of lymphocytes. Wright stain, 1000 magnification. (From
uolization; perinuclear zone in lymphocytes and monocytes. Best, M: Body Fluids Examination. ASCP Workshop 9294, Boston,
1990, with permission.)
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis = 725

slide-on-slide method. The slide-on-slide technique involves Monocytes

the following’:
Monocytes present in body fluids may appear similar to those
1. Place a drop of the sample in the middle of a glass slide.
seen in peripheral blood smears, or may be larger with abun-
2. Place a second slide over the first, perpendicular or paral-
dant, vacuolated cytoplasm. These transformed monocytes
lel, and allow the sample to spread by capillary action. Do
are referred to as histiocytes: they have migrated into tissue
not apply any pressure.
spaces from the peripheral blood (Figs. 30-4 and 30-5).
3. The slides are then pulled straight apart laterally, in one
These histiocytes may contain phagocytosed material; they
smooth motion. Do not pull up or down as the slides are
are then referred to as macrophages or phagocytes. Classify-
being separated.
ing these cells between types is not clinically important. In
4. Allow the slides to air dry.
some cases, however, phagocytosed organisms or cells may
be of diagnostic importance. Some monocytic cells’ vacuoles
may fuse together into one large cytoplasmic vacuole, caus-
Cellular Components of Body Fluids ing the nucleus to become flattened against the cell mem-
Under normal conditions, the cells present in body fluids
brane; this gives the cell the appearance of a signet ring
are composed of peripheral blood leukocytes that are capa- (Fig. 30-6).
ble of migration through the vascular endothelium into Few monocytes are present in CSF; histiocytes or
extravascular spaces, and a small number of epithelial macrophages are usually seen only in pathologic states.
cells sloughed from the membrane that lines the compart-
ment. The most common types of cells encountered are
neutrophils, lymphocytes, monocytes, and tissue cells.
Other cells that are less frequently seen are eosinophils,
basophils, and mast cells.

Neutrophils e

Neutrophils are frequently seen in pleural, peritoneal, and

pericardial fluids, as well as synovial fluids, but normally con-
stitute fewer than 25% of the total cells in the differential
analysis. Very few, if any, should be observed in CSF. After
staining, the neutrophils would appear as in a peripheral blood
smear; however, the process of cytocentrifugation may cause
the nuclei to be pushed to the cell’s periphery, resulting in an
artifactual hypersegmented appearance (see Fig. 30-1). Some Figure 30-4 @ Histiocytes/macrophages (center). Also monocytes
cells can be degenerated, exhibiting cytoplasmic vacuoliza- and neutrophils. Wright stain, 1000 magnification. (From Best, M:
tion and nuclear pyknosis. Body Fluids Examination. ASCP Workshop 9294, Boston, 1990, with
Immature neutrophils (i.e., promyelocytes, myelocytes, permission.)
and metamyelocytes) are not commonly seen; if present, they
may represent a chronic inflammatory process or a bone mar-
row disorder (e.g., myeloproliferative, myelodysplastic, or
leukemic syndrome.) Blasts are found only in the presence of
bone marrow disorders.

Lymphocytes
Lymphocytes are often seen in all types of fluids in variable
numbers. They can vary in appearance from small to large in
size, and normal to reactive in their nuclear morphology. The
nuclei are artifactually more prominent in cytocentrifuge
preparations. The shape of the nucleus may be irregular
and the cytoplasm may have artifactual projections (see
Figs. 30-2 and 30-3).* Lymphocyte morphology in malig- a
nancyis determined by the type of neoplasm involved, el
i.e., leukemia or lymphoma. The lymphocytes seen in these
Figure 50-5 @ Macrophages. Also a lymphocyte (A) and a plasma
conditions will appear homogeneous, as they are clonal in cell (B). Wright stain, x1000 magnification. (Courtesy of Judith
origin. Plasma cells, if present, are usually seen only in Brody, M.D., North Shore-Long Island Jewish Health System Labora-
chronic inflammatory disorders. tories, Lake Success, New York.)
726 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

Figure 30-6 ® Signet-ring type macrophages. Wright stain,

1000 magnification. (From Best, M: Body Fluids Examination. Figure 30-8 ® Mesothelial cells; degenerated with peripheral
ASCP Workshop 9294, Boston, 1990, with permission.) vacuoles, resembling macrophages. Wright stain, x 1000 magnifica-
tion. (Courtesy of Judith Brody, M.D., North Shore-Long Island
Jewish Health System Laboratories, Lake Success, New York.)

Tissue Cells
Synovial cells have a similar appearance to mesothelial
Benign tissue cells are seen in all fluids and must be differen-
cells, although the cytoplasm appears denser than the
tiated from malignant cells. Pleural, pericardial, and peri-
mesothelial cell. Cartilage cells (chondrocytes) may be seen
toneal fluids all contain benign mesothelial cells, as they are
in a variety of arthritic conditions. These cells contain a cen-
normally sloughed from the membrane. These appear as large
tral, small, round, pyknotic nucleus surrounded by a clear
cells with moderate to abundant cytoplasm that may be
zone and a distinct burgundy colored cytoplasm.°
stained a light or dark blue and may contain phagocytosed
debris or granules. The nucleus is eccentric with a smooth
nuclear outline, and a fine, homogeneous chromatin pattern. Eosinophils, Basophils, Mast Cells
Nucleoli may be large and prominent, usually uniform in size
and shape (Figs. 30-7 and 30-8). The appearance of mesothe- Eosinophils, basophils, or mast cells may be present in
lial cells can vary within the same fluid specimen, which may small numbers in pericardial, pleural, peritoneal, or syn-
cause some difficulty in identification (Fig. 30-9). ovial fluids. Increased numbers can be seen in various dis-
CSF tissue cells (choroid plexus cells, ependymal cells) orders, and their presence may or may not be correlated
tend to form clusters. They may have cytoplasmic granules with their concentration in the peripheral blood. Mast cells
and slightly irregular nuclei. Arachnoid mater cells are fre- can be distinguished from basophils by having a round
quently seen as a syncytium: a mass of cytoplasm containing nucleus (not segmented) and a higher number of cytoplas-
several nuclei. These benign cells are usually only seen in the mic granules that are smaller in size than those usually seen
CSF from infants or adults who have had recent neurosurgery in basophils.*
or an implanted reservoir to deliver chemotherapeutic agents
or antimicrobials directly into the CNS.*

Figure 30-9 ™ Macrophages (right) and mesothelial cells (left).

Figure 50-7 ® Mesothelial cells. Normal, quiescent. Wright stain, Wright stain, X1000 magnification. (Courtesy of Judith Brody, M.D.
*1000 magnification. (From Best, M: Body Fluids Examination.
’
North Shore-Long Island Jewish Health System Laboratories, Lake
ASCP Workshop 9294, Boston, 1990, with permission.) Success, New York.)
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis VOM,

Pleural, Pericardial, and Peritoneal laboratory parameters most frequently characterize a fluid as a
(Serous) Fluids transudate:

¢ WBC count usually less than 1000/uL

Effusions: Transudates and Exudates
* Specific gravity of 1.015 or less
The serous fluids (pleural, pericardial, and peritoneal) are dis- ¢ Total protein (TP) of 3.0 g/dL or less
cussed together because they present similar findings in nor- ¢ Fluid TP/serum TP ratio less than 0.5
mal and pathologic states. Normally, these compartments Occasionally, a fluid specimen cannot be definitively identi-
contain a minimal amount of fluid, only enough to keep the fied as a transudate. In this case, the following parameters can
membranes moist and slippery. The fluid is produced by the assist in the identification:
parietal lining by plasma filtration through capillary endothe-
lial cells and absorbed by the visceral lining. Production is ¢ Fluid lactate dehydrogenase (LDH): less than 200 IU/L
dependent on four factors: capillary hydrostatic pressure, ¢ Fluid LDH/serum LDH ratio less than 0.6
plasma oncotic pressure, lymphatic resorption, and capillary ¢ Glucose level equivalent to serum
permeability. Pathology affecting any one or more of these ¢ Fluid cholesterol less than 60 mg/dL®
factors, as well as trauma, infection, or malignancy, may ¢ Fluid cholesterol/serum cholesterol ratio less than 0.3’
result in an abnormal fluid collection known as an effusion Transudates usually do not require further laboratory or diag-
(Table 30-3). Effusions that accumulate due to a systemic nostic evaluations.
disease state are categorized as transudates. Effusions that Exudates are caused by increased capillary permeability
accumulate due to a primary pathologic state within the com- and/or decreased lymphatic resorption. Exudative effusions can
partment are categorized as exudates. The purpose of identi- be caused by many different pathologic processes, such as bac-
fying the type of effusion as transudative or exudative is to terial infections, viral infections, neoplasms, trauma or infarc-
determine if further laboratory or other extensive diagnostic tion, noninfectious inflammatory conditions (e.g., rheumatoid
tests are required in order to discover the cause (Table 30-4). arthritis [RA]) and collagen vascular diseases (ee., systemic
Transudates are frequently the result of increased capillary lupus erythematosus [SLE]). The following laboratory parame-
hydrostatic pressure, as seen in congestive heart failure; or ters characterize a fluid as an exudate:
decreased plasma oncotic pressure, as seen in the hypoproteine-
mia of the nephrotic syndrome or liver failure. They are usually ¢ WBC count usually greater than 1000/uL
clear, pale yellow in color, and do not clot. The following ¢ Specific gravity greater than 1.015
* Total protein greater than 3.0 g/dL
¢ Fluid TP/serum TP ratio greater than 0.5

Fluids that cannot be characterized by the above criteria can

be further analyzed by the following to aid in classification:
(Factors «
¢ Fluid LDH >200 IU/L
1. Increased capillary hydrostatic pressure ¢ Fluid LDH/serum LDH ratio greater than 0.6
2. Increased capillary permeability * Glucose level possibly lower than serum level
3. Decreased lymphatic resorption
¢ Fluid cholesterol greater than 60 mg/dL®
4.Decreased ‘plasma oncotic pressure”
¢ Fluid cholesterol/serum cholesterol ratio greater than 0.3’
A fetect in
i | or more of 1!
these factors results in ieae, of an
effusion. Exudates tend to be cloudy, turbid, or purulent owing
to the presence of lipids or the increased WBC count, and

pu aooudates pu Aad ates

Form as a an ‘of7 Goillacy hydride pressure Form as a eae of ihee Secneapninesor : emipnare
or J plasma oncotic pressure resorption
Clear, pale yellow; do not clot Cloudy, turbid, purulent, bloody; clots on standing
WBC: <1000 cells/uL WBC: > 1000 cells/uL
Specific gravity: =1.015 Specific gravity: >1.015
Total protein: =3.0 g/dL Total protein: >3.0 g/dL
Fluid/serum TP ratio: <0.5 Fluid/serum TP ratio: >0.5
LDH: <200 IU/L LDH: >200 IU/L
Fluid/serum LDH ratio: <0.6 Fluid/serum LDH ratio: >0.6
Glucose: ~serum/plasma glucose Glucose: <serum/plasma glucose
Cholesterol: <60 mg/dL Cholesterol: >60 mg/dL
Fluid/serum cholesterol ratio: <0.3 Fluid/serum cholesterol ratio: >0.3
728 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

frequently clot on standing owing to the presence of fibrino- categorized as benign/reactive when the cellular abnormalities
gen. If a portion of the sample is centrifuged, a clear super- are caused by infection, inflammation, or other sterile reactive
natant indicates the presence of an abundance of leukocytes processes such as pulmonary infarction or cirrhosis of the liver.
or cellular debris; a white supernatant is caused by the pres- Serous fluids associated with bacterial infections will demon-
ence of lipids. strate an inflammatory cellular response that, on differential
If the fluid has a milky or opaque appearance that analysis, consists predominantly of segmented neutrophils.
remains in the supernatant after centrifugation, this is known These cells may reveal Dohle bodies, toxic granulation, and/or
as a chylous effusion, an exudate resulting from leakage or a vacuolization. Viral, fungal, or mycobacterial infections may
blockage of the lymphatic vessels. It is rich in chylomicrons, be associated with a predominance of lymphocytes or show
has an elevated triglyceride level (greater than 110 mg/dL), a mixed inflammatory response. These lymphocytes are
and contains predominantly lymphocytes. Chylous effusion frequently reactive and transformed, resembling immunoblasts.
most often results from a malignancy such as lymphoma or Benign/reactive lymphocytes consist of a heterogeneous
carcinoma, or from trauma. population of cells with varying nuclear shape, amount of
An exudate unrelated to the lymphatics, known as a cytoplasm, and degree of cytoplasmic basophilia, whereas
pseudochylous effusion, results from a persistent, chronic malignant lymphoma cells would appear as a homogeneous
effusion due to diseases such as tuberculosis (TB) and population, most with the same nuclear and cytoplasmic fea-
RA-associated inflammation of the pleura (i.e., rheumatoid tures. Eosinophilic responses can occur with parasitic or fungal
pleuritis). Unlike a chylous effusion, this exudate is caused infections, hypersensitivities, pneumothorax, some immune dis-
by the breakdown of cellular lipids, does not contain chy- orders, and peritoneal dialysis. Many will have an inflammatory
lomicrons, and usually has a low triglyceride level (less cell response that is not diagnostic for any specific disorder;
than 50 mg/dL). Its appearance is caused by cellular debris these would be referred to as nonspecific, reactive cellular
and cholesterol crystals, and contains a mixed reactive cell responses (Table 30-6).
population with many inflammatory and necrotic cells In rare cases, lupus erythematosus (LE) cells form spon-
(Table 30-5). taneously. An LE cell is a phagocyte, either neutrophil or
Trauma, aneurysm, malignancy, pancreatitis, or TB may monocyte, that has phagocytosed a naked nucleus showing a
also produce a bloody, or hemorrhagic effusion. homogeneous, smooth chromatin pattern. This finding is sus-
The aforementioned analyses are not always able to picious but not diagnostic of SLE; other autoimmune disor-
definitively distinguish transudates from exudates when the ders may also show this phenomenon.
results are equivocal; the patient’s clinical history and Mesothelial cells, as previously described, can have a
physical examination can provide further information. The wide variation in morphology. They may show nonspecific
finding of an exudate must always be followed by more reactive changes that include multinuclearity, the presence of
extensive tests to determine its cause. nucleoli, mitotic activity, and sometimes an increase in cell size
(Figs. 30-10 and 30-11). They have been classified into cate-
gories, based on their diverse morphology as quiescent; hyper-
Cellular Responses, Microorganisms, trophied; epithelioid; phagocytic; and senescent* (Table 30-7).
and Malignant Cells in Serous Fluids Quiescent mesothelial cells are the normal constituents of the
membrane that are shed into the fluid (see Fig. 30-7). Hyper-
Effusions can occur as a result of a wide variety of local or sys- trophied cells are larger, with more prominent nucleoli, and can
temic disease processes. They may be categorized as normal be seen in reactive processes that cause cellular hyperplasia
cellular, benign/reactive, or malignant, based on the cell types (Fig. 30-12). Epithelioid mesothelial cells are seen as clumps
present. Normal cellular effusions, as their name implies, are or sheets, owing to the disrupted membrane surfaces in reactive
neither reactive nor malignant; the cell morphology is normal processes (Fig. 30-13). Phagocytic mesothelial cells are indis-
regardless of the cause of the effusion. A serous fluid is tinguishable from macrophages or histiocytes (Fig. 30-14).

Feature Chylous Effusion Pseudochylous Effusion

Appearance Milky-white; opaque; supernatant Milky-white, yellow, green; opaque; supernatant

remains milky; forms creamy top remains milky
layer upon standing
Cause Leakage, damage or blockage of Chronic effusion; breakdown of cellular lipids
thoracic duct
Disease process Trauma, malignancy TB, Rheumatoid pleuritis
Presence of chylomicrons Rich in chylomicrons Chylomicrons absent
Triglycerides Increased (>110 mg/dL) Decreased (<50 mg/dL)
Cellular content Lymphocytosis Mixed reactive cell population; necrotic cells,
cholesterol crystals
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 729

Reactive Process Cell Types Present

Acute bacterial infection Greater than 50%

(e.g., pneumonia; neutrophils; many
peritonitis; pericarditis) macrophages
Chronic inflammation or Many reactive mesothelial
infection (e.g., RA; TB) cells, macrophages, and
lymphocytes; few
plasma cells and
neutrophils
Chronic sterile reactive Many reactive mesothelial
processes (e.g., cirrhosis; cells and macrophages; Figure 350-1 1 ® Mesothelial cell; multinucleated. Also
pulmonary embolism/ few neutrophils and macrophages (A) and lymphocytes (B). Wright stain, 1000 magni-
infarction) lymphocytes; signet fication. (Courtesy of Judith Brody, M.D., North Shore-Long Island
ring-type macrophages Jewish Health System Laboratories, Lake Success, New York.)
“may be present
Eosinophilic effusions Greater than the fine focus adjustment knob, the presence of infection is
(e.g., parasitic/fungal 10% eosinophils indicated as opposed to in vitro contamination of the slide
infections; hypersensi-
tivities; pneumothorax; or stain. Most pathogenic yeasts are found in CSF as opposed to
immune disorders; serous fluids. They may or may not be found intracellularly. The
peritoneal dialysis) most frequently encountered fungal organisms in fluids are:

° Cryptococcus spp.
¢ Histoplasma spp.
The senescent mesothelial cell is degenerated, with a single ¢ Candida albicans
large vacuole forming a signet ring configuration (Fig. 30-15). * Candida tropicalis
The nucleus may show pyknosis and karyorrhexis: one or more
spherical, densely staining nuclear fragments. All except quies- Malignancy is a major cause of serous effusion; therefore,
cent mesothelial cells can closely resemble malignant cells. pleural, pericardial, and peritoneal fluids may contain malig-
Extreme caution must be taken to avoid misinterpretation; nant cells from a variety of neoplasms. Their identification is
referral to pathology and/or cytology is required. critical for accurate diagnosis. A malignancy may already be
Most types of pathogenic bacterial and fungal organisms known to exist, but its presence in the fluid demonstrates
will stain with Wright and Wright-Giemsa stain, and are metastasis; it may also be an initial diagnosis. The laboratory
detectable on a routine cytocentrifuge preparation. Bacteria professional must look at the entire cellular area of the slide
will stain blue regardless of the Gram’s stain reaction under low power to detect suspicious clusters of cells. It is not
(Fig. 30-16). If the organisms are intracellular (Fig. 30-17), possible to distinguish malignant cells from benign reactive
discernible as being in the same plane of view as the cell via

Quiescent Normal constituents of the fluid;

Normally shed from mesothelial
lining
Hypertrophied Larger;
More prominent nucleoli;
Can be seen in reactive processes that
cause cellular hyperplasia
Epithelioid Seen as clumps/sheets, due to
disrupted membrane surfaces in
reactive processes
Phagocytic Indistinguishable from macrophages
or histiocytes
Senescent Degenerated cell;
Single large vacuole forming signet
Figure 30-10 @ Reactive mesothelial cell; binucleated. Also ring configuration;
macrophages, neutrophils, and lymphocytes. Wright stain, x 1000 Nucleus may show pyknosis and
magnification. (From Best, M: Body Fluids Examination. ASCP Work- karyorrhexis
shop 9294, Boston, 1990, with permission.)
730 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

Pigure 50-12 ® Hyperplastic mesothelial cells. Wright stain, Figure 30-15 @ Senescent mesothelial cell (right); becoming
400 magnification. (Courtesy of Judith Brody, M.D., North signet-ring configuration. Wright stain, *1000 magnification.
Shore-Long Island Jewish Health System Laboratories, Lake Success, (Courtesy of Judith Brody, M.D., North Shore-Long Island
New York.) Jewish Health System Laboratories, Lake Success, New York.)

Figure 350-13 @ Reactive mesothelial cells in sheets (epithelioid)

Figure 50-16 @ Bacteria in body fluid: Staphylococcus species;
with prominent nucleoli. Wright stain, 1000 magnification. (From
note the intra- and extracellular distribution. Wright stain, x 1000
Best, M: Body Fluids Examination. ASCP Workshop 9294, Boston,
magnification. (From Best, M: Body Fluids Examination. ASCP Work-
1990, with permission.)
shop 9294, Boston, 1990, with permission.)

Figure 50-14 @ Phagocytic macrophages/mesothelial cell. Note Figure 30-17 m Bacteria in body fluid: Neisseria species; note the
the ingested RBCs (Jeft). Wright stain, x1000 magnification. intracellular diplococci in neutrophils. Wright stain, 1000 magnifi-
(Courtesy of Judith Brody, M.D., North Shore-Long Island Jewish cation. (From Best, M: Body Fluids Examination. ASCP Workshop
Health System Laboratories, Lake Success, New York.) 9294, Boston, 1990, with permission.)
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 731

cells by a single cellular characteristic; the cells must be

judged based on collective criteria that include the gross
appearance of the fluid as well as the cellular features. Addi-
tional studies such as immunocytochemistry, flow cytometry,
tumor marker analyses, and biochemical and enzyme assays
may also be required.
The general features of malignant cells are:
¢ Multilayered formations; three-dimensional spheroidal ar-
rangements (ball-like formations (Fig. 30-18 and 30-19)
* Large cell size (often greater than 50 um diameter) or giantism
* An irregular nuclear membrane: jagged or showing multiple
folds
* Multinucleation; nuclear molding; Indian file arrangements
(Figs. 30-20 and 30-21)
* Nuclear hyperchromasia, unevenly distributed chromatin Figure 350-19 ® Cell-ball formation; malignant cell cluster. Wright
¢ Prominent nucleoli with irregular size and shape stain, x500 magnification. (Courtesy of Judith Brody, M.D., North
* High/variable N:C ratio Shore-Long Island Jewish Health System Laboratories, Lake Success,
* Increased/abnormal mitotic activity (Fig. 30-22) New York.)

* Bizarre vacuolation; abnormal inclusions (Fig. 30-23)

*Cannibalism (one cell engulfing/ingesting another)
(Fig. 30-24)
¢ Uneven staining of cytoplasm

Two very characteristic features of malignant cells in fluid

preparations are nuclear molding and paranuclear blue body
inclusions.
Nuclear molding describes the process whereby the
nucleus of one cell molds around the shape of an adjacent
cell. This occurs with the cohesive growth of tumor cells that
require the presence of tight junctions between the cytoplas-
mic membranes of the cells. It is most often seen in small cell
carcinoma, but may be seen in any type of carcinoma.
Paranuclear blue body inclusions occur in small cell carci-
noma or rarely in sarcoma, but have not yet been characterized.
They may represent early cell degeneration of phagocytosed
material. The inclusions may appear to be intranuclear, depend- Figure 30-20 © Indian file cellular arrangements: Breast carci-
ing on the orientation of the cell on the slide. They are only seen noma. Wright stain, 500 magnification. (From Best, M: Body
Fluids Examination. ASCP Workshop 9294, Boston, 1990, with
on Wright stained preparations. Cytology preparations and other
permission.)
laboratory studies are required to specifically identify the type of
malignancy (Table 30-8).

Figure 350-18 @ Clusters of malignant cells. Wright stain, x1000 Figure 50-21 ® Nuclear molding. Wright stain, 1000 magnifica-
magnification. (From Best, M: Body Fluids Examination. ASCP Work- tion. (From Best, M: Body Fluids Examination. ASCP Workshop
shop 9294, Boston, 1990, with permission.) 9294, Boston, 1990, with permission.)
 Large, frequently >50 um
Giantism
High/variable N:C ratio (21:1)
Nuclear Multinucleation; variable size
Pleomorphic, nonuniformity
Irregular nuclear membrane;
herniation into cytoplasm; clefts
Nuclear hyperchromasia
Unevenly distributed chromatin
Nuclear molding
Figure 350-22 ™ Abnormal mitotic activity; acinar (glandular) Prominent nucleoli with irregular
arrangement of malignant cells. Wright stain, x1000 magnification. size and shape
(From Best, M: Body Fluids Examination. ASCP Workshop 9294, Increased/abnormal mitotic
Boston, 1990, with permission.) activity
Cytoplasmic Uneven staining of cytoplasm
Bizarre vacuolization or large,
singular vacuole
Abnormal inclusions
Fused cytoplasmic membranes
Cannibalism
Cellular Arrangement Multilayered formations
3-D forms
Spherical aggregates
Cell clumps lacking, or having
- poorly defined margins
Wraparound, rosette or acinar
formations
Single cells

Hematopoietic malignancies that can most commonly be

found in body fluids are:

¢ The lymphocytic and nonlymphocytic leukemias

Figure 50-25 @ Foamy, bizarre vacuolization in cytoplasm of
malignant cells. Wright stain, 1000 magnification. (From Best, M:
¢ Non-Hodgkin lymphomas
Body Fluids Examination. ASCP Workshop 9294, Boston, 1990, ¢ Hodgkin’s lymphoma
with permission.) ¢ Plasma cell neoplasms

The abnormal cells found in a fluid in any of these disorders

have the same morphologic features as seen in the peripheral
blood and bone marrow.
The most common nonhematopoietic malignancies that
can be seen in body fluids are:

¢ Small cell carcinoma (oat cell)

¢ Metastatic adenocarcinoma (breast, ovarian, stomach, colon,
rectum)

Types of Effusions, Laboratory Analysis,

and Clinical Correlations
Pleural and Pericardial Effusions

A pleural effusion is defined as an abnormal accumulation of

fluid in the pleural cavity. It occurs when the rate of production
Figure 50-24 @ Nuclear molding; cannibalism of malignant cell
(right). Wright stain, 1000 magnification. (From Best, M: Body exceeds the rate of absorption. Conditions that affect capillary
Fluids Examination. ASCP Workshop 9294, Boston, 1990, with pressure or permeability, colloid osmotic pressure, lymphatic
permission.) drainage, or increased negative intrapleural pressure (which
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 7 WwWw

can occur with atelectasis [collapsed lung]), can lead to the for- The abnormal accumulation of fluid in the pericardial
mation of a pleural effusion. The presence of large amounts of space is known as a pericardial effusion. This is most fre-
fluid may restrict lung expansion, causing dyspnea and mild quently caused by damage to the lining of the cavity and
hypoxemia. Pleural effusions caused by congestive heart fail- increased capillary permeability. The function of the normal
ure, nephrotic syndrome, or cirrhosis tend to be bilateral, pericardium is to oppose dilatation of the heart; this is
whereas unilateral effusions are caused by diseases that reflected by the mean central venous pressure. Interference
develop below the diaphragm, such as cirrhosis with ascites, with pericardial venous and lymphatic drainage, which occurs
hepatic abscess, pancreatitis, and tumors.° in acute pericarditis, can lead to the formation of an effusion.
The volume of fluid, the rate of its formation, and the elastic-
SPECIMEN COLLECTION AND PROCESSING ity of the pericardial membrane will determine what effect the
Analysis of a pleural effusion begins with the removal of fluid effusion will have on cardiac function. A small effusion may
by a procedure known as thoracentesis (Table 30-9). Portions have no effect and not produce any symptoms; a large effu-
of the pleural or thoracentesis fluid, as it may also be classi- sion that develops very slowly may also not cause any symp-
fied, are placed into heparinized tubes for chemical analysis, toms provided that the pericardium has the ability to stretch.
sterile tubes for microbiological cultures and Gram and/or A severe complication of pericarditis or a traumatic injury can
acid-fast stains, and tubes containing ethylenediaminete- cause a condition known as cardiac tamponade. This occurs
traacetic acid (EDTA) for cell counts and differential analysis. when pericardial fluid or blood within the pericardial space
Additional nonadditive evacuated tubes may also be collected under increased pressure restricts the motion of the heart. This
for cytology and immunophenotyping studies. Thoracentesis produces a state of critical cardiovascular dysfunction, caus-
may also be used for therapeutic purposes. ing decreased cardiac output and hypotension, and if left
The gross appearance of the pleural fluid provides untreated will cause death. Treatment requires the aspiration
important diagnostic clues to the nature of the disease of the pericardial fluid in a procedure known as pericardio-
process. Pure blood in the pleural cavity, a hemothorax, can centesis (see Table 30-9). A portion of the aspirate is sent to
result from severe chest injuries, stab or gunshot wounds, or the laboratory for analysis as discussed earlier for thoracente-
surgical procedures. A bloody, or hemorrhagic, effusion in the sis. A wide variety of diseases and disorders can produce peri-
absence of trauma almost always suggests the presence of cardial effusions, such as neoplastic disease, infectious
malignancy, or occasionally a pulmonary infarction. Leakage agents, cardiovascular disease, renal disease, collagen vascu-
of the thoracic duct causes a chylothorax, most commonly lar diseases, and hemorrhagic events (e.g., trauma, aneurysm,
caused by a malignancy such as lymphoma or carcinoma.'° or anticoagulant therapy.)
This produces a milky-white, opaque pleural fluid that
remains opaque after centrifugation. Infection of the pleural LABORATORY ANALYSIS AND CLINICAL
space by bacterial pneumonia or a ruptured lung abscess will CORRELATIONS
cause empyema: the collection of pus in the pleural cavity. QUALITATIVE ANALYSIS The hematology laboratory will
This fluid will be turbid to opaque and have an extremely receive the body fluid in tubes containing EDTA, which pre-
elevated WBC count of 25,000/uL or higher. After centrifuga- vents clotting and preserves the cell morphology for analysis.
tion, the supernatant of this type of fluid will be clear due to If the fluid is completely clotted no analysis can be per-
the removal of the cellular debris. formed. If the specimen is partially clotted, which can occur
if the specimen is not properly mixed after collection, the cell
count would be inaccurate, but an attempt should be made at
performing the morphological examination for the presence
‘tabieso-0 Body Fluid = —t™S of reactive or malignant cells, with the required physician
a nd Collection notification and comment on the report.
_ Procedure Gross visual inspection of the fluid is initially per-
formed. A normal pleural fluid is pale yellow in color and
Nomenclature> transparent. Abnormal findings would be cloudy, turbid,
bloody, yellow, green, or chylous. If the fluid is grossly
Body Cavity/ Collection
Region Fluid Name Procedure bloody, and a microhematocrit performed on the specimen
has a packed cell volume (PCV) greater than 50% of the
Pleural cavity/lungs Pleural (serous) |Thoracentesis peripheral blood PCV, this would indicate the presence of a
Pericardial cavity/ Pericardial Pericardiocentesis hemothorax.
heart (serous)
Peritoneal cavity/ Peritoneal, Paracentesis
A normal pericardial fluid is pale yellow in color
abdomen ascites and transparent. Turbidity can be caused by infection or
(serous) malignancy. Grossly bloody fluid may result from trauma
CNS; subarachnoid — Cerebrospinal Lumbar puncture; or disease, or the possibility that intracardiac blood was
space/brain spinal tap
aspirated into the specimen during pericardiocentesis. The
and spinal cord
Arthrocentesis findings of a chylous fluid are rare, but can be caused by
Synovial cavity/ Synovial
joints the leakage of lymphatic vessels from trauma, lymphoma,
or carcinoma.
734 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC

ANALYSIS Next, the specimen is examined microscopically
for its cellular concentration. RBC and WBC counts are per-
formed on the undiluted fluid. Cloudy or turbid fluids may be
diluted with saline or buffered cell diluent, and the corrected
final cell count should be multiplied by the dilution factor.
Cell counts are of limited value in making a differential diag-
nosis because they are nonspecific. As few as 5000 RBCs/uL
will cause a blood-tinged appearance to the fluid. Red cell
counts greater than 100,000/uL in pleural fluid are highly sug-
gestive of malignant neoplasm, trauma, pulmonary embolism,
or infarction.’ The performance of a microhematocrit can be
useful in distinguishing a hemorrhagic effusion from aspi-
rated blood in the specimen. Generally, a total WBC count
less than 1000/uL is associated with a transudate, while Figure 30-25 m™ Neutrophils with vacuolization, fragmentation,
higher counts are associated with exudates and suggestive of decreased granulation; one pyknotic neutrophil (center). Wright
microbial infection or malignancy. These values alone should stain, 1000 magnification. (From Best, M: Body Fluids Examina-
tion. ASCP Workshop 9294, Boston, 1990, with permission.)
not be used to classify the fluid as transudative or exudative;
other biochemical analyses must be considered, as previously
discussed. Neutrophils are present in higher concentrations in exudates
Examination of the Wright-stained cytocentrifuge prepa- of a bacterial etiology, such as a pleural effusion of bacterial
ration is required regardless of the cell count. Concentration pneumonia or a ruptured lung abscess; in pericardial fluid a
of the fluid will demonstrate the presence of cellular material. predominance of neutrophils is seen in bacterial pericarditis
The entire circular field of stained sediment is first scanned (see Table 30-6).
under the low-power objective (10), searching for evidence Lymphocytes in pleural fluid resemble small peripheral
of cellular clumps or sheets, and the presence of large cells. A blood lymphocytes and are seen in variable numbers. They may
standard 100-cell differential is then performed under high- be variable in size and have an immature or reactive (trans-
power oil-immersion (50 or 100). If fewer than 100 cells formed) appearance in response to various stimuli. The nucleus
are present, all the cells in the preparation are counted, and can be cleaved and exhibit nucleoli that are often more promi-
the actual numbers of cells counted and the percentages of nent than those in peripheral blood lymphocytes. Some of these
each cell type are calculated and reported. The cells are cate- nuclear changes are artifactual effects of cytocentrifugation (see
gorized in the same manner as peripheral blood differential Fig. 30-2). Degenerative changes can occur in aged specimens.
counts, with the addition of large mononuclear cell categories Increased numbers of lymphocytes (lymphocytosis) are seen in
that are used for monocytes, macrophages, mesothelial cells, effusions from patients with tuberculosis (TB), malignancy, and
and malignant cells. At some institutions, monocytes and viral infections (see Table 30-6).
macrophages are counted together, with a separate category When present, plasma cells resemble those encountered
for mesothelial cells. These cell types are counted and in the bone marrow. Increased numbers can accompany the
reported as a percentage, with a comment describing the types increase in lymphocytes that is seen in patients with multiple
of cells present. This method of counting has the advantages myeloma or other disorders with associated lymphocytosis
of (1) cells that are difficult to identify morphologically (1.e., (Fig. 30-26). They are also seen in effusions from patients
monocytes and macrophages) may be grouped together and with TB, RA, malignancy, Hodgkin’s disease, and so forth. If
(2) no attempt is made to count nonhematologic malignant a malignancy is suspected, referral to pathology is mandatory
cells individually because they often occur in clumps and are and cytological examination must be performed.
very difficult to count. Malignant hematologic (leukemia/ Mononuclear phagocytes (monocytes, histiocytes, or
lymphoma) cells are counted and reported as in peripheral macrophages) are seen in variable numbers in both benign
blood differentials. All stained fluid slides with atypical, sus- and malignant effusions. Monocytes and mesothelial cells can
picious, or malignant cells must be reviewed by a pathologist transform into macrophages. The number of mononuclear
before reporting. cells increases as the inflammatory process becomes chronic.
Segmented neutrophils in pleural or pericardial fluid In vivo LE cell formation has been observed as well. If the
appear morphologically identical to those in the peripheral fluid remains at room temperature, in vitro LE cell formation
blood, although cytocentrifugation may introduce artifactual can occur. Tart cells are also frequently seen in serous fluids.
changes, as previously discussed. Immature neutrophils are These cells, which are morphologically similar to LE cells,
rarely seen except in chronic myelogenous leukemia or in are small macrophages that have phagocytosed a nonhomog-
leukoerythroblastic conditions. Neutrophils in chronic effu- enized nucleus of another cell.
sions can show signs of cellular degeneration, with vacuoliza- Pleural fluid eosinophilia (eosinophils seen in increased
tion and decreased numbers of granules in the cytoplasm. numbers greater than 10%) is a nonspecific finding, com-
They can also exhibit pyknosis or karyorrhexis and can be monly seen in idiopathic effusions. It may signify that air
mistaken for nucleated red blood cells (NRBCs) (Fig. 30-25). (pneumothorax) or blood (hemothorax) has been introduced
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 735

Figure 50-26 ® One plasma cell (A), one plasmacytoid lympho-

cyte, reactive and normal lymphocytes in a chronic effusion; note Figure 50-27 @ Reactive mesothelial cells: Pleural fluid; note the
the monocyte/macrophages (B). Wright stain, x 1000 magnifica- prominent nucleoli and multinucleation. Wright stain, x 1000 mag-
tion. (From Best, M: Body Fluids Examination. ASCP Workshop nification. (Courtesy of Judith Brody, M.D., North Shore-Long Island
9294, Boston, 1990, with permission.) Jewish Health System Laboratories, Lake Success, New York.)

into the pleural space. By itself, it is not diagnostically signif- having a more pronounced irregularity in size and shape of
icant; it may also be manifested in parasitic or fungal the nuclei and nucleoli, uneven chromatin distribution with
diseases, malignancy, connective tissue disorders, hypersensi- hyperchromasia, and irregular cellular arrangements (see
tivities, or pulmonary infarction. The presence of eosinophilia Table 30-7).
in an exudative effusion is an indication that the condition is Increased concentrations of mesothelial cells are seen in
probably not malignant or tuberculous. Mast cells and pneumonia, pulmonary infarction, and malignant disorders. A
basophils often accompany eosinophils, and 5% to 10% may marked increase, especially if the fluid is bloody or
be seen in pleural fluid eosinophilia’ (see Table 30-6). eosinophilic, suggests the presence of a pulmonary embolism.
Mesothelial cells in small numbers are normally However, they are usually markedly reduced in number in
sloughed into the serous cavities. During inflammatory cases of TB of the pleura (i.e., tuberculous pleurisy) or if there
processes, they proliferate and are shed in greater numbers. is massive infection of the pleural cavity with pyogenic (pus-
They often vary in appearance, manifesting reactive or atypi- producing) organisms; this may be related to the fibrinous
cal changes, which can create difficulty during the differential exudate that covers the lining of the cavity, trapping the cells?
examination (see Figs. 30-12 and 30-13); familiarity with (see Table 30-6).
these issues is necessary when examining the slides.
Mesothelial cells may appear as single cells, in clusters, or Peritoneal Effusions
sheets. Clustering can be an artifact caused by cytocentrifuga-
tion that may produce a resemblance to malignant cells. SPECIMEN COLLECTION AND PROCESSING
Clumps, or loose aggregates of benign mesothelial cells, can A peritoneal effusion is an accumulation of fluid in the peri-
be differentiated from malignant cells by comparing the toneal space, clinically termed ascites. The procedure to with-
appearance of those in the clump with other more easily dis- draw this fluid is known as paracentesis (see Table 30-9). The
tinguished mesothelial cells in the same smear. A uniform, collected fluid can be called peritoneal, ascitic, or paracente-
regular arrangement of cells that display fenestrations: open- sis fluid. Paracentesis is most commonly performed to rule
ings or windows between the cytoplasmic membranes of the out bacterial peritonitis and malignancy. Aspiration may be
cells, usually indicates that they are benign. Spheroidal or combined with /avage: a flushing of the peritoneal space with
multilayered cell formations are more likely to be malignant lactated Ringer’s solution. Evidence of blood in the lavage
(see Figs. 30-18 and 30-19). The cells are large, with scant to fluid is an indication for immediate surgical exploration
abundant light gray to deep blue cytoplasm, and may present (laparotomy) of the abdominal cavity for internal bleeding.
an area of perinuclear pallor; as such, they may resemble Because the abdominal cavity is large and distendable,
large plasma cells. There may also be variably sized cytoplas- more than approximately 500 mL of fluid must usually be
mic vacuoles. The nucleus occupies one third to one half the present before the effusion can be detected by radiologic or
cell’s diameter, and may be round to oval in shape with a physical examination. Diagnostic abdominal paracentesis,
smooth nuclear membrane. The chromatin distribution is uni- with the removal of 50 to 100 mL of fluid, is essential to
form, with a stippled, dark purple Wright stain effect. There determine a differential diagnosis. A portion of this is sent to
may also be one to three spherical nucleoli. Mesothelial cells the laboratory for analysis in the same manner as discussed
that could be classified as “reactive” have slightly irregular previously for pleural and pericardial fluids.
nuclei with prominent nucleoli (Fig. 30-27). Atypical The causes of peritoneal effusions are the same as those
mesothelial cells more closely resemble malignant cells, involved in pleural and pericardial effusions: increased capillary
736 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

pressure or permeability, abnormal colloid osmotic pressure, Chylous fluid is rare, but can be caused by the leakage of lym-
poor lymphatic drainage, or cardiac abnormalities. Additional phatic vessels due to trauma, hepatic cirrhosis, TB, lymphoma,
causes include abdominal conditions that do not directly involve or carcinoma.
the peritoneum, such as hepatic cirrhosis, intrahepatic and por-
tal venous obstruction, hypoalbuminemia, renal function, ovar- QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC
ian disease, pancreatic disease, or parasitic infection. Malignant ANALYSIS The total RBC and WBC counts are useful for
disease and alcoholic cirrhosis are the most common causes of diagnosis in peritoneal lavage, as they improve the accuracy
peritoneal effusion." and specificity of the diagnosis. A positive lavage fluid would
Patients in renal failure, who must undergo dialysis to be visibly bloody, and considered as such when the total RBC
cleanse the blood of waste products, can be treated with a pro- count is greater than 100,000/uL (or greater than 50,000/uL in
cedure known as continuous ambulatory peritoneal dialysis known penetrating trauma such as stab or gunshot wounds).
(CAPD). This procedure utilizes the natural properties of the The total WBC count would be greater than 500/uL. How-
peritoneal membrane and the infusion of a dialyzing fluid into ever, when indeterminate by eye, a lavage would be consid-
the cavity to remove the impurities in the bloodstream. This ered positive when the total RBC count is 50,000 to
procedure requires laboratory monitoring to measure its 100,000/uL (10,000 to 50,000/uL in cases of penetrating
effectiveness, and carries an inherent risk of recurrent peri- trauma).'! The total WBC count would be 100 to 500/uL. A
tonitis. The used dialysis fluid (dialysate) can be sent to the negative lavage fluid would show a total RBC count less than
laboratory for analysis. 50,000/uL (or less than 1000/uL in penetrating trauma). The
total WBC count should be less than 100/uL.
LABORATORY ANALYSIS AND CLINICAL Cell counts on nonlavage peritoneal fluids, specifically the
CORRELATIONS total WBC count, are of limited value in differential diagnoses,
QUALITATIVE ANALYSIS The hematology laboratory will but are useful in distinguishing peritoneal transudates from
receive’ the fluid in tubes containing EDTA, which prevents exudative effusions. A WBC count greater than 300/uL is con-
clotting and preserves the cell morphology for analysis. A sidered to be abnormal. It is extremely necessary to make a
clotted fluid cannot be analyzed. As with pleural and pericar- correct diagnosis as early as possible in suspected spontaneous
dial fluids, a partially clotted specimen would not provide an bacterial peritonitis (SBP) to reduce the morbidity and mortality
accurate cell count, but morphological examination for the associated with it. A WBC count greater than 500/uL is useful
presence of reactive or malignant cells should be performed, presumptive evidence in distinguishing between an exudate in
with the required physician notification and comment on the bacterial peritonitis and a transudate of cirrhosis. Serial WBC
report. counts and cultures are helpful in assessing the adequacy of
The criteria previously described to differentiate transu- treatment in patients with SBP and also in differentiating SBP
dates and exudates may not fully apply for ascitic fluid as they from nonperforation secondary bacterial peritonitis."
do for pleural and pericardial fluids. The total protein and LDH A Wright-stained cytocentrifuge preparation is used for
ratios may be better indicators in most cases (see Table 30-4). the differential examination. The same procedure, as described
A more reliable method is known as the serum-ascites albu- earlier in the discussion of pleural and pericardial effusions, is
min gradient (SAAG), calculated by subtracting the ascitic followed for the morphologic examination. An exudative peri-
fluid albumin concentration from the simultaneously col- toneal effusion characteristically contains a variable number of
lected serum albumin concentration.'*? The SAAG is signifi- neutrophils, lymphocytes, eosinophils, basophils, macrophages,
cantly greater than 1.1 g/dL in transudates (high-gradient and mesothelial cells.
ascites) than in exudates.'* It can also provide valuable infor- The presence of more than 25% segmented neutrophils is
mation to assist in the differential diagnosis of ascites: considered abnormal and suggestive of bacterial infection. The
e.g., patients with high-gradient ascites include those with absolute neutrophil count may also be helpful; counts greater
cirrhosis, alcoholic hepatitis, cardiac-related ascites, or mas- than 500 neutrophils/uL are a fairly sensitive indicator of spon-
sive liver metastasis. Low-gradient ascites (SAAG less than taneous or secondary bacterial peritonitis. In CAPD, peritonitis
1.1 g/dL) are associated with peritoneal malignancies and is defined by a WBC count of greater than 100/uL with more
nonmalignant diseases such as TB, pancreatitis, nephrotic than 50% neutrophils, or if microorganisms are seen on the
syndrome, biliary disease, or connective tissue diseases.'* cytocentrifuge preparation and/or Gram stain of the dialysate."!
Normal or transudative peritoneal fluid is pale yellow or straw Chronic, long-standing effusions may show neutrophils with
colored, and transparent. The visible appearance of the fluid decreased cytoplasmic granules, and nuclear pyknosis and kary-
may be helpful in determining the cause of the effusion. orrhexis as in pleural or pericardial effusions.
Abdominal blunt trauma, postoperative complications, rupture Lymphocytes transform in response to various stimuli
of the liver or spleen, intestinal infarction, pancreatitis, or malig- and exhibit a variety of morphologic features. Differentiation
nancies can cause the production of a grossly bloody fluid. of benign lymphocytes from malignant lymphoma in effusions
Effusions resulting from perforation of the gall bladder or may occasionally be difficult. A predominance of lymphocytes
intestines, duodenal ulcers, cholecystitis, or acute pancreatitis can be seen in transudates from patients with congestive heart
will appear green owing to the presence of bile. Exudative effu- failure, cirrhosis, or the nephrotic syndrome; they may also be
sions will be cloudy or turbid if they contain elevated protein seen in chylous effusions, tuberculous peritonitis, and other
concentrations, increased leukocytes, and/or microorganisms. malignant disorders.
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 137

Eosinophilia in peritoneal fluid is less commonly seen CSF collection is commonly performed by intervertebral
than in pleural fluid. Eosinophilic ascites is rare; when present, puncture in the lumbar region of the spinal column, between
it contains greater than 50% eosinophils. It is associated with vertebrae L3 and L4, known as a lumbar puncture (LP) or
congestive heart failure, vasculitis, and malignant lymphoma.'° “spinal tap” (see Table 30-9). The procedure must be per-
Patients with CAPD may exhibit peritoneal eosinophilia, usu- formed aseptically and without trauma. In patients with coagu-
ally greater than 10% or as high as 95%.'° The cause is lopathies or infection at the puncture site, LP is contraindicated.
unknown, but may be caused by hypersensitivity to the foreign If the patient has a normal intracranial pressure, determined at
material, or the possibility that air entered the cavity during the initiation of the LP, at least 20 mL of fluid can be removed
the connection of the catheter line to the infusion set!’ (see in an adult without complication. Significantly lower volumes
Table 30-6). Small numbers of basophils and mast cells may may be collected from infants, children, and neonates, or if the
also be seen in cases of peritoneal eosinophilia. intracranial pressure is elevated. Approximately 2-4 mL of fluid
It is possible to find choroid plexus cells from the CNS is collected into each of three to five sequentially numbered,
in the peritoneal fluid of a patient with a ventriculoperitoneal sterile, nonadditive tubes. The tubes must be filled in numerical
shunt: a drainage device inserted into the ventricles of the order. The first tube is used for chemical analysis, because it
brain to remove excess CSF in cases of hydrocephalus, neo- typically would be contaminated with peripheral blood and cel-
plastic conditions, or head injury. The catheter is placed under lular debris from the initiation of the puncture, and requires
the skin, from the skull to the abdomen, allowing the excess centrifugation before analysis. The second tube is used for
fluid to drain into the peritoneal cavity where it is reabsorbed. microbiological analysis. The third tube is sent to hematology
A one-way valve controls the flow of the fluid. for the cell count and differential analysis. Additional tubes
Mononuclear phagocytes are present in variable num- may be used for further chemistry analyses, serology, cytology,
bers. These cells, as well as mesothelial cells, may have a flow cytometry, immunocytochemistry, or molecular genetic
variable appearance. As discussed previously under pleural analysis. An LP may also be performed for therapeutic pur-
and pericardial fluid analysis, differentiation from malignant poses, such as to reduce intracranial pressure, or to administer
cells may be difficult. Atypical mesothelial cells, resembling anesthetics, radiographic contrast media, or antifungal or
malignant cells, are especially seen in chronic effusions and chemotherapeutic agents directly into the CNS. A whole blood
in ascitic fluid associated with cirrhosis.'* LE cells have also sample is also collected at the time of LP for a complete blood
been reported. Referral to pathology and cytological exami- count (CBC) and chemical analysis of the serum.
nation must be performed if a malignancy is suspected. On receipt in the laboratory, the CSF, also known as
“spinal fluid,’ must be processed immediately (STAT); analysis
must occur within | hour of collection owing to cellular degra-
Cerebrospinal Fluid dation and lysis, which will lead to falsely lower WBC and RBC
counts. The lower protein content of CSF leads to destabiliza-
The CSF is a selective ultrafiltrate of plasma, under the con- tion of the cell membranes. Any specimen remaining after
trol of the blood-brain and blood—CSF barriers, and differs analysis must be refrigerated in case additional tests are neces-
from serous fluids in that a greater volume is present in the sary. An exception to this procedure applies only to specimens
normal state. It is also actively secreted by cells in the ventri- for microbiological examination: these must not be refrigerated,
cles and subarachnoid space. Examination of CSF is an as some microorganisms (e.g., Niesseria meningiditis) are
extremely important diagnostic procedure, and its acquisition destroyed by low temperatures. It is preferable to place them in
is not without risk to the patient. All specimens must be han- a 37°C incubator until testing is performed, especially during
dled with universal precautions and never discarded until all off-hours when the bacteriology department may not be staffed.
testing has been completed.

Specimen Collection and Processing Laboratory Analysis and Clinical

Correlations
Proper specimen collection is of the highest importance in
order to provide the most clinically accurate information. The QUALITATIVE ANALYSIS
physician must perform a complete clinical history and physical The first step in CSF analysis is a gross inspection for the tube
examination prior to the collection procedure. Indications for number that was received, volume, clarity, color, and viscos-
the analysis of CSF are divided into four major categories: sus- ity of the fluid. A normal CSF is clear, colorless, sterile, and
picion of meningeal infection, subarachnoid hemorrhage, CNS watery in consistency. Any cloudiness or turbidity can be
malignancy, and demyelinating diseases.'” Imaging techniques caused by the presence of more than 200 WBC/uL, more than
such as computerized tomography (CT scan) and magnetic res- 400 RBC/uL, microorganisms, fat globules, or increased pro-
onance imaging (MRI) can provide clinical information without tein levels. The presence of more than the normal numbers of
the need for fluid analysis in cases of intracranial masses. How- cells in a CSF is referred to as pleocytosis. Radiographic con-
ever, laboratory analysis of CSF still remains critical to the trast media can give the fluid an oily appearance. Grossly
diagnosis of CNS infections and inflammatory conditions, espe- bloody specimens may result from a traumatic tap, which
cially in neonates or infants younger than 3 months of age with occurs in approximately 20% of LPs,’° or the presence of sub-
the presence of fever and lethargy. arachnoid or intracerebral hemorrhage. A common problem in
738 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

the analysis of CSF is distinguishing between a true CNS removal of large volumes of CSF during radiologic proce-
hemorrhage versus a traumatic LP. If the bloodiness decreases dures, hyperthyroidism, leukemias, and in normal children
with each successive tube, this would suggest a traumatic tap. 6 months to 2 years of age.
If the bloodiness is similar for all tubes, this could indicate a Albumin, a constituent of TP, is not synthesized in the
CNS hemorrhage. However, this method is not always CNS, but is transported across the blood—CSF barrier. A
reliable.*' method to evaluate the integrity (degree of permeability) of
A clotted CSF is an abnormal finding; it suggests an the blood—CSF barrier is to calculate a ratio of the serum
increased fibrinogen and/or protein concentration from periph- albumin to the CSF albumin, which should normally be less
eral blood contamination, an infectious condition within the than 9. If greater than normal, the albumin originated from the
CNS, or a markedly increased protein concentration in the fluid. blood due to a damaged blood—CSF barrier, intracerebral
The presence of any color should be noted. If the speci- bleeding, or traumatic LP. Electrophoretic analysis can
men is bloody, the color of the supernatant is interpreted after also be used to visualize and quantify the individual protein
centrifugation. This must be performed as soon as all the cell fractions.
counts are completed. Xanthochromia, a pink, orange, or yel- The IgG concentration is measured to determine the
low color of the supernatant, is caused by the breakdown of amount of production within the CNS, normally about 3 mg/
hemoglobin. It is usually thought to indicate a true CNS hem- day. It is the predominant immunoglobulin in the CSE. It is eval-
orrhage, and is present in more than 90% of patients within uated to assist in determining a diagnosis of MS or other
12 hours of subarachnoid hemorrhage onset.** Lysis of RBCs demyelinating diseases. The IgG may originate from the blood
present in the CSF begins approximately | to 2 hours after a if the integrity of the blood—CSF barrier is disrupted, or from
subarachnoid hemorrhage. Hemoglobin is converted to biliru- increased synthesis by lymphocytes and plasma cells within the
bin within 24 hours of RBC lysis, giving the supernatant a CNS. To correct for possible increased permeability, increased
yellow tint. If the fluid supernatant is clear and colorless, this serum IgG and albumin concentrations, and to isolate intrathe-
could suggest that either the LP was traumatic,, the CNS cal IgG production, the CSF IgG index is calculated.
hemorrhage occurred less than 2 hours before the LP, or min-
_ CSF IgG X serum albumin
imal RBC lysis has occurred. Xanthochromia will occur, acre eat
however, if a bloody fluid from a traumatic LP is not
processed within | hour of collection. Other causes of xan- All components must be from simultaneously collected
thochromia include jaundice (serum bilirubin levels of 10 to samples. An approximate normal index value would be less
15 mg/dL), extremely elevated CSF protein concentrations than or equal to 0.7; this is highly dependent on the methodol-
(greater than 150 mg/dL), and hyperbilirubinemia in prema- ogy and normal cut-offs for the four analytes. An elevated
ture infants who have an immature blood—CSF barrier. index greater than 0.7 would be seen with increased intrathecal
IgG synthesis, which could occur in MS, subacute sclerosing
BIOCHEMICAL ANALYSIS pan-encephalitis, bacterial or viral meningitis/meningoen-
Biochemical analysis of CSF routinely includes the measure- cephalitis, Guillain-Barré syndrome, neurosyphilis, SLE affect-
ment of the total protein (TP) and glucose levels, with com- ing the CNS, or other neurologic conditions. In addition, more
parison to the simultaneously collected serum levels. Other than 90% of patients with MS have two or more discrete bands
analyses may include albumin, IgG, and LDH; the clinical sit- in the gamma-globulin fraction on CSF electrophoresis which
uation would determine when these would be performed. is not present on serum protein electrophoresis. These are
There are many other more complex tests and electrophoretic known as oligoclonal bands; however, they are not specific for
procedures that can be performed, but these are beyond the MS. Diagnosis also requires neurologic history and physical
scope of this chapter. examination.
Protein enters the CSF by diffusion across the blood— Glucose in the CSF is entirely derived from the blood,
CSF barrier. Some CNS synthesis of protein also occurs, but is normally only about 60% to 70% of serum levels. It
mostly in the form of IgG. The normal CSF TP level in adults enters almost completely by facilitated diffusion in the
is approximately 15 to 45 mg/dL, which is less than 1% of the choroid plexus.'? Changes in the blood glucose concentration
serum level. Higher levels (up to 100 mg/dL) are possible in may require 2 to 4 hours to equilibrate with the CSF. Ideally,
neonates owing to immaturity of the blood—CSF barrier. CSF and blood specimens should be drawn in the fasting
These values are also method dependent and each laboratory state; however, this is not always clinically possible. The
must determine its own range. An increased TP is the most analysis must be performed immediately to avoid artifactual
frequent, although nonspecific, abnormality found in CSF decreases caused by the presence of leukocytes and/or bacte-
analysis. Abnormally increased CSF TP is seen in meningeal ria. Without knowing the actual serum glucose level, an
and CNS disease (e.g., increased permeability of the blood— acceptable range for CSF glucose should be 50 to 80 mg/dL,
CSF barrier, meningitis, traumatic LP, CNS hemorrhage, assuming a normal fasting serum glucose range of 65 to
multiple sclerosis [MS], obstruction to CSF circulation, 110 mg/dL.** A traumatic LP will cause a false elevation due
neoplasm, or cerebral infarction). As little as 0.1 mL of blood to the introduction of peripheral blood. Patients with hyper-
in | mL of CSF can increase the TP by 400 mg/dL. Low TP glycemia may have higher CSF glucose values depending on
levels are associated with CSF leakage due to skull fracture, the timing of the last carbohydrate load; however, the kinetics
increased intracranial pressure due to water intoxication, of glucose transport changes as the blood glucose level
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 739

increases.. Not knowing the serum glucose value at the time

of LP could cause a false interpretation of the CSF glucose eT Table 30 10 Normal Values
as normal when it may in fact be significantly decreased. for Cerebrospinal Fluid
By itself, an increased CSF glucose level has no diagnostic
significance. Appearance Clear, colorless, watery
Total Protein 15-45 mg/dL (adults)
Decreased glucose in the CSF, known as hypoglycor- (<1% of serum total protein)
rhachia, is a significant finding. It is considered to be present 15-100 mg/dL (neonates)
when the CSF glucose is less than 40 mg/dL in a patient with Glucose 50-80 mg/dL (approx. fasting)
normal fasting serum glucose. This could be caused by (~60-70% of serum glucose)
increased utilization by leukocytes, RBCs, microorganisms, RBC Count <J/uL
WBC Count 0-5/uL (adults)
or tissues within the CNS; it is more likely to be caused by
0-30/uL (infants up to | year)
impairment of glucose transport and increased anaerobic gly- WBC Differential 3% neutrophils
colysis by the brain and spinal cord. Low glucose levels are ~70:30% lymphocytes:monocytes,
often accompanied by increased lactate levels in the CSF, macrophages; rare ependymal
Hypoglycorrhachia is one of the most characteristic abnor- cells
malities in patients with bacterial, tuberculous, or fungal
meningitis, although it is not specific for infection. It may also
WBC counts greater than 1000/uL with turbid fluid sug-
be present in hypoglycemia, primary or metastatic tumors of
gest bacterial or fungal meningitis; very high counts (greater
the meninges, or subarachnoid hemorrhage with the release of
than 50,000 cells/uL) are unusual and suggest intraventricular
glycolytic enzymes from RBCs."° Viral meningitis, however,
rupture of a brain abscess. The total WBC count cannot be
is not associated with decreased CSF glucose.
interpreted without the total RBC count. In a traumatic LP, the
Lactate dehydrogenase (LDH) is one of many enzymes
WBC and RBC counts will reflect the same WBC/RBC ratio as
present in CSF; however, it appears to be the only enzyme
the peripheral blood of the patient. As a general rule, in a patient
that has clinical utility. LDH is present in CSF by diffusion
with a normal peripheral blood count, one can expect to find
across the blood—CSF barrier, and is also found in cells within
approximately one to two WBCs for every 1000 RBCs in the
the CNS such as leukocytes, bacteria, and tumor cells. Brain
CSF from a traumatic LP.*° For example, a CSF with a total
tissue is rich in LDH; therefore, damage to the brain or spinal
WBC count of 10 cells/uL and a RBC count of 10,000 RBCs/uL,
cord can result in increased levels of LDH in the CSF. The
the ratio is 1:1000. There is no significant increase in WBCs in
level of LDH in the CSF is approximately 10% of the simul-
this fluid; however, a traumatic LP is implicated, In a CSF
taneously collected serum value. Measurement of LDH has
with a total WBC count of 10 cells/uL and a RBC count of
been found to be useful in the differential diagnosis of bacte-
100 RBCs/uL, the ratio is 1:10, indicating a significant increase
rial versus viral meningitis.” In bacterial meningitis LDH
and a pathologic state.’ In a situation in which a patient has an
activity is found to be greater than three times the upper nor-
increased or decreased peripheral blood WBC or RBC count, a
mal limit, whereas with viral infection, the LDH level is less
correction to the count of the CSF must be performed to see if it
than two times the upper limit.
is significant, using the following formula'??’:
QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC Corrected CSF WBC = total WBC of fluid — WBC of
ANALYSIS blood X RBC of fluid
Next, the specimen is examined microscopically. The total RBC of blood
numbers of RBCs and WBCs in the undiluted fluid are man-
The types of WBCs present are the most important part
ually counted using a hemacytometer. Cloudy or bloody
in the interpretation of a CSF and their relationship to the
fluids may be diluted with saline or buffered cell diluent, cor-
clinical findings.
recting the final count by the dilution factor. As stated earlier
in the chapter, automated cell counters should not be used,
especially for CSF, because the allowable background limits Cellular Responses, Malignant Cells,
on the instrument diluent are higher than the normal range for and Microorganisms in CSF
CSF cell counts.
Normal CSF is practically acellular by the manual cham- The morphologic evaluation of the CSF must follow the cell
ber count (Table 30-10). No RBCs should be present (less count, regardless of whether the results are in the normal
than 1/uL); RBC counts are of limited value in a differential range. It is possible that stem cells or malignant cells may be
diagnosis. They can be present due to trauma during LP, and present, especially if the patient has been treated with
commonly seen in infants. WBC counts, however, are very chemotherapy for leukemia or other malignancies. The spec-
useful in developing a differential diagnosis because even imen is processed by cytocentrifugation and Wright staining,
low counts are suggestive of pathology. Normally, less than as previously discussed under pleural and pericardial fluid
5 mononuclear cells/uL can be seen in adults; in neonates and analysis. A normal CSF can yield 30 to 50 cells per 0.5 mL of
infants up to | year of age, up to 30 mononuclear cells/yL is cytocentrifuged fluid sample.'?
possible. These cells consist of lymphocytes, monocytes/ On differential analysis, a normal CSF contains mononu-
macrophages, or CNS tissue cells. clear cells (Fig. 30-28) (approximately 70% lymphocytes and
740 — Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

The fluid differential alone cannot differentiate between bac-

terial and nonbacterial meningitis.
Normal CSF contains lymphocytes, which are predomi-
nantly small, with intermediate and large forms present as
well. They appear similar to those in the peripheral blood, and
undergo transformation in response to antigens. Transformed
or reactive lymphocytes are variable in size, with moderately
abundant basophilic cytoplasm, moderately coarse chromatin,
and one or more nucleoli; some may appear plasmacytoid.
They may also occasionally contain azurophilic granules
(Fig. 30-29). When marked reactive changes occur it may be
difficult to identify them as lymphocytes. Lymphoblasts, or
immature-appearing lymphocytes, have scant cytoplasm, a
fine chromatin pattern, and may or may not have prominent
Figure 50-28 m Normal CSF; two monocytes and one lympho- nucleoli. Increased numbers of lymphocytes (lymphocytic
cyte (A). Wright stain, x1000 magnification. (From Best, M: Body pleocytosis; Fig. 30-30) are associated with viral, fungal,
Fluids Examination. ASCP Workshop 9294, Boston, 1990, with tuberculous, syphilitic, and parasitic infections.
permission.) Reactive lymphocytosis is seen in viral meningoen-
cephalitis, or after chemotherapy or radiation treatment. The
30% monocytes) and rare ependymal or choroid plexus cells reactive lymphocytes present in viral meningitis are morpho-
(see Table 30-10). Other cells that could be seen in a CSF logically very similar to those in the peripheral blood during
include granulocytes (mature and immature), lymphocytes infectious mononucleosis. Noninfectious and degenerative
(mature and/or reactive), mononuclear phagocytes (mono- causes of reactive lymphocytosis include MS, Guillain-Barré
cytes, histiocytes and macrophages), plasma cells, ependymal syndrome, drug therapy, polyneuritis, and sarcoidosis of the
and choroidal cells, leukemic blasts, and malignant cells. The meninges. In MS, the reactive lymphocytes are distinctly
cells can appear comparable to those in peripheral blood with plasmacytoid in appearance (Fig. 30-31). Their nuclei are
some variation in details. eccentric and lymphocytic in appearance, whereas their cyto-
Normally, very few segmented neutrophils should be plasm is plasma cell-like. Blast-like lymphocytes are
observed in CSF. They traditionally were considered patho- frequently seen in the CSF of newborn infants and can be
logical; the observation of more than an occasional neutrophil mistaken for leukemic cells. Reactive lymphocytes must be
classically suggested a bacterial infection. However, with the distinguished from lymphoma cells. The ability to differenti-
current method of cytocentrifugation that concentrates the ate benign and malignant lymphocytes is important. Benign
fluid and improves cell recovery, the appearance of a small cells appear as a mixture of variable sizes and a mixture of
number of neutrophils can be seen in normal CSF. These may normal and reactive cells. This is in contrast to malignant
be related to peripheral blood contamination from a traumatic cells which appear more homogeneous in size and shape.
LP; their concentration would be proportional to the extent of Cytocentrifugation produces artifactual changes to the
peripheral blood contamination and the neutrophil percentage nuclear and nucleolar shapes, therefore, these are not reliable
of the peripheral blood. There is no general agreement on indicators. There may also be difficulty in differentiating lym-
what constitutes a normal upper limit to the neutrophil phocytes from monocytes in some cytocentrifuge prepara-
count,'? but neutrophil counts greater than 3% should be tions; some monocytes may appear small and contracted,
regarded as potentially pathological and carefully investi-
gated. Their quantity must always be considered in the con-
text of the patient’s clinical condition and with results of other
laboratory tests. Neutrophils rapidly disintegrate; if the spec-
imen is not examined promptly, a falsely decreased count will
be reported. The cytoplasmic granules are often less promi-
nent than in the peripheral blood.**’? The nucleus may be
hyperlobulated with long and narrow filaments, an artifact of
cytocentrifugation (see Table 30-2). Older cells can exhibit
pyknosis or karyorrhexis, and could be mistaken for NRBCs.
Bacterial infections of the CNS cause neutrophilic pleo-
cytosis and increased total WBC counts. Early bacterial
meningitis may show only a slight increase in neutrophils.
Early viral meningitis (first 48 hours) may also present with
neutrophilia. Increased numbers of neutrophils can also be
seen in inflammatory or noninfectious conditions, such as tis-
Figure 30-29 m Reactive lymphocytosis in CSF. Wright stain,
sue infarction, foreign body or substance, metastatic tumor/ 1000 magnification. (From Best, M: Body Fluids Examination.
leukemic infiltration, or 3 to 4 days after a CNS hemorrhage. ASCP Workshop 9294, Boston, 1990, with permission.)
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 741

lymphocytes, and plasma cells. Monocytes can develop into

macrophages.
The mononuclear phagocytes consist of macrophages
and histiocytes. Histiocytes are fixed (immobile) tissue
macrophages. Macrophages are transformed monocytes.
These cells function as scavengers, consuming RBCs, leuko-
cytes, microorganisms, other macrophages, and lipids present
in the CSF. Ingested but undigested material is stored in vac-
uoles of the cytoplasm, many of which may fuse into one
large vacuole forming a signet ring cell. Foamy macrophages,
or lipophages, contain numerous small vacuoles that contain
lipid. These are associated with cerebral infarction, brain
abscess, and radiologic procedures They are also seen in
Tay-Sachs disease, Hunter’s, and Hurler’s syndromes. Many
Figure 30-30 @ Lymphocytic pleocytosis, CSF. Wright stain, x400 macrophages may fuse together into a large, multinucleated
magnification. (From Best, M: Body Fluids Examination. ASCP Work- syncytium known as a giant cell. These may be seen in gran-
shop 9294, Boston, 1990, with permission.) ulomatous disorders such as TB and sarcoidosis.'” Increased
numbers of macrophages are seen in patients with meningitis,
meningeal inflammation, infectious diseases, CNS leukemia,
while some lymphocytes can appear stretched, depending on and metastatic carcinomas. Other causes include CNS hemor-
their ___location on the slide. Cytochemical stains may be neces- rhage, ventricular shunts, irradiation of the brain, and intrathecal
sary in some situations. Any questionable morphology must chemotherapy (administered directly into the CNS.)
be referred to the pathologist for interpretation. A definitive sign of CNS hemorrhage is erythrophagocy-
Monocytes present in CSF appear similar to, and arise tosis; the phagocytosis of erythrocytes by histiocytes or
from, the peripheral blood. They normally constitute approxi- macrophages. It takes approximately 18 hours for histiocytes
mately 30% of the cells seen on differential examination; they or macrophages to mobilize and phagocytose RBCs after the
can be more numerous in infants and smail children. Differenti- hemorrhage, thus appearing as empty vacuoles within the
ation from lymphocytes can be problematic in some cases, macrophages. More than a single macrophage containing
especially in neonates. Cytocentrifugation may cause the mono- ingested RBCs should be present before considering a CNS
cytes to stick together and appear like choroidal, ependymal hemorrhage, as they may occur in vitro or in an LP repeated
cells, or tumor cells.'? They degenerate more rapidly in vitro, 8 to 12 hours after an initial traumatic LP. After approxi-
requiring prompt preparation of the slide. They are increased in mately 4 days, the degraded hemoglobin becomes hemo-
a variety of disorders, which include tuberculous meningitis, siderin, visible as dark brown or black granules (Fig. 30-32).
fungal meningitis, syphilitic meningoencephalitis, and viral If the hemorrhage is older, hematoidin crystals, also a product
meningoencephalitis. Other causes include subarachnoid hem- of hemoglobin catabolism, may be seen as bright yellow or
orrhage, cerebral infarction, multiple sclerosis, Guillain-Barré red crystals (Fig. 30-33). Iron-containing macrophages,
syndrome, foreign agents, and CNS malignancy. Pure monocy- known as siderophages, are visible with a stain for iron; the
tosis is rarely seen in the CSF. More commonly, they present as presence of numerous siderophages is a good indicator of a
part of a mixed cell reaction, which includes neutrophils, hemorrhagic event.

Figure 30-31 m Reactive (plasmacytoid) lymphocytes in CSF in Figure 50-32 ™@ Siderophages in CSF: hemosiderin pigment in
‘multiple sclerosis. Wright stain, x1000 magnification. (From Best, M: macrophages (A). Wright stain, 400 magnification. (From Best, M:
Body Fluids Examination. ASCP Workshop 9294, Boston, 1990, with Body Fluids Examination. ASCP Workshop 9294, Boston, 1990, with
permission.) permission.)
742 > Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

Figure 50-55 @ Siderophage with hematoidin (hematin) Figure 30-34 @ Eosinophils (A) and reactive lymphocytes (8) in
pigment. Wright stain, 1000 magnification. (From Best, M: Body CSF; ventricular shunt. Wright stain, 1000 magnification.
Fluids Examination. ASCP Workshop 9294, Boston, 1990, with (Courtesy of Judith Brody, M.D., North Shore-Long Island
permission.) Jewish Health System Laboratories, Lake Success, New York.)

Plasma cells are normally not present in CSF. In inflam- sheets or as single cells are helpful features in their identifica-
matory conditions associated with lymphocytic reactions, tion. Occasionally, the nucleus may be pyknotic and eccentri-
transitional stages of reactive lymphocytes, plasmacytoid cally placed. The cytoplasm is moderate to abundant, stains a
lymphocytes, and plasma cells can be seen. These would gray-blue with Wright stain, and may contain vacuoles;
include acute viral infections, and chronic inflammatory the cytoplasmic borders may be indefinite, and cilia may
states; TB, syphilis, sarcoidosis, Guillain-Barré syndrome, occasionally be present.'? When found in cytocentrifuged
and MS. preparations, they may resemble lymphocytes or monocytoid
Other granulocytes that may be seen include eosinophils cells. However, if the fluid contains an increased number of
and basophils. Eosinophils are rarely seen in a normal CSF. lymphocytes and/or monocytes, artifactual clusters of these
They may be increased in a variety of infectious and non- cells may resemble choroid plexus cells.'? Situations or con-
infectious disorders. Eosinophilic meningitis is defined ditions in which these cells may be seen include CSF obtained
when the total WBC count is greater than 10% eosinophils; by ventricular tap, following traumatic brain injury, brain
a parasitic infection should be suspected with this find- surgery, ischemic brain infarction, radiologic procedures, and
ing.° Idiopathic eosinophilic meningitis, without any in children with hydrocephalus and ventricular shunts.'* *? It
evidence of a pathogen, has also been described.*' Other is important to recognize these cells because they can be mis-
infectious causes include viral, fungal, or rickettsial infec- takenly identified as malignant cells; however, they have no
tions. Noninfectious causes include malignancy, intrathe- diagnostic significance.
cal therapy, radiographic contrast media, and systemic LE cells are rarely seen in CSR.**
drug reactions. Malignant cells can be seen in the CSF. Every Wright-
Eosinophilia is also associated with malfunctioning ven- stained CSF slide must be searched for the presence of
tricular shunts or foreign body reactions (Fig. 30-34).
Basophils are normally not seen in CSF. There are conditions
in which they can be found in small numbers, such as inflam-
matory diseases, foreign body reactions, parasitic infections,
convulsive disorders, and chronic myelogenous leukemia
(CML)."°
Ependymal or choroid plexus cells that line the cerebral
ventricles and choroid plexus, sometimes referred to as neu-
roectodermal cells, may be seen in CSF under certain circum-
stances. They are difficult to differentiate, but it is believed
that the majority of the cells seen in the fluid are choroid
plexus cells. They are rarely, if ever, seen in normal CSF
obtained by LP in adults; however, they may occasionally be
found in infants.'° These cells are medium in size, and may
appear in papillary clusters or sheets, or sometimes individu-
ally. The nuclei are the size of a small lymphocyte, round to
Figure 30-35 @ Ependymal or choroid plexus cells (neuroepithe-
oval in shape, with delicate, finely granular, and evenly dis-
lial tissue) in sheet formation. Wright stain, 400 magnification.
tributed chromatin. Nucleoli are not present (Figs. 30-35 and (From Best, M: Body Fluids Examination. ASCP Workshop 9294,
30-36). Their uniformity of nuclear size and appearance in Boston, 1990, with permission.)
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 743

with leukemic cells; if no RBCs are present, even 1% to

2% of blasts present may be indicative of CNS involvement.
Malignant lymphomas may involve the CNS, with the pres-
ence of lymphoma cells in the CSF (Figs. 30-38, 30-39, and
30-40). These may also be difficult to differentiate from
leukemic cells. The presence of lymphoma cells in the CSF
shows a direct correlation with bone marrow involvement and
leukemic conversion of the lymphoma. Any questionable cel-
lular morphology must be reviewed by the pathologist.
Other cells that could possibly be seen in normal CSF,
usually as a result of a traumatic tap, include:
1. Bone marrow cells: if the needle is advanced too far and
enters the vertebra; particularly seen in infants or patients
with spinal abnormalities; megakaryocytes and hematopoi-
Figure 50-36 @ Papillary cluster of neuroepithelial lining cells. etic precursors could be mistaken for signs of infection,
Wright stain, x1000 magnification. (Courtesy of Judith Brody, M.D., malignant cells, leukemia, or lymphoma.
North Shore-Long Island Jewish Health System Laboratories, Lake 2. Chondrocytes (cartilage cells): if the needle pierces an
Success, New York.)
intervertebral disc.**
3. Squamous epithelial cells (skin cells).
4. Starch particles from gloves: these may be confused as the
clumps of tumor cells. The presence of any large tissue cells
fungal organism Cryptococcus neoformans.
should be considered suspicious for malignancy. Carcinoma
5. Blast-like cells, or primitive cell clusters, may be seen in
cells tend to be less cohesive; they may simulate hematopoi-
the CSF of neonates, particularly premature infants with
etic malignancies. Free tumor cells can originate from pri-
subarachnoid or intraventricular hemorrhage and_ sec-
mary CNS neoplasms or metastases from tumors of the lung,
ondary hydrocephalus;"” these cells have scant to moderate,
breast, gastrointestinal tract, or melanoma. Cytology studies
lightly basophilic cytoplasm and nuclei with delicate chro-
are required for identification. Leukemic cells in the CSF are
matin and small single nucleoli; they frequently occur in
rarely an initial finding; their appearance usually indicates
clusters.'°
established disease. Their infiltration of the CNS presents a
treatment dilemma. The blood—CSF barrier is almost imper- Microorganisms may be present in CSF samples; they may be
meable to chemotherapeutic agents, allowing the disease to intra- or extracellular, and must be distinguished from stain
progress uninhibited. Intrathecal chemotherapy must be insti- precipitate. Gram stain, performed by the microbiology
tuted to kill the malignant cells and any latent cells within the department, is helpful in this situation. The most common
CNS from systemic disease. Acute lymphoblastic leukemia yeast organism to infect the CNS is Cryptococcus neofor-
involves the CNS more often than acute nonlymphocytic mans, an India ink preparation or serological analysis must be
leukemias (Fig. 30-37). The chronic myeloid and lymphoid performed to confirm the identity of the organism. Cultures
leukemias do not usually spread to the CNS. When RBCs are must be performed if any organisms are present. Viral cul-
present, this could suggest peripheral blood contamination tures can also be performed, although these can take up to
7 days to obtain a definitive result. Some viral studies can be
performed using the polymerase chain reaction (PCR), which

Figure 30-37 @ Acute lymphoblastic leukemia in CSF; note the

prominent nucleoli. Wright stain, x 1000 magnification. (Courtesy Figure 50-38 @ Burkitt's lymphoma in CSF. Wright stain,
of Judith Brody, M.D., North Shore-Long Island Jewish Health Sys- x 1000 magnification. (From Best, M: Body Fluids Examination.
tem Laboratories, Lake Success, New York.) ASCP Workshop 9294, Boston, 1990, with permission.)
744. Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

diseases. Normal values for routine synovial fluid analyses are

summarized in Table 30-11.
The analysis of synovial fluid provides important infor-
mation in the diagnosis of inflammatory and degenerative
joint diseases. These can be divided into five disease cate-
gories: Group I, non-inflammatory; Group II, inflammatory;
Group III, infectious; Group IV, crystal-associated; and
Group V, hemorrhagic.*> Overlap can occur, however, and
more than one diagnosis can be present simultaneously. Syn-
ovial fluid is most often analyzed when there is a suspicion of
infectious (septic) arthritis or a crystal-associated disease of
the joint (e.g., gout). A delay in diagnosing septic arthritis can
lead to serious complications which include joint destruction
and long term disability. Because there is no laboratory or
Figure 30-59 ™ Cleaved lymphoma cells in CSF. Wright stain, radiologic test that can rule out septic arthritis, this analysis is
1000 magnification. (From Best, M: Body Fluids Examination. critical to the proper management of the patient.*° This condi-
ASCP Workshop 9294, Boston, 1990, with permission.) tion is more common in children than adults, the highest inci-
dence occurring between 6 months and 2 years of age.*°
Trauma, which could introduce pathogenic organisms into the
joint space, is a possible cause; however, an upper respiratory
infection, otitis media, or other infected site can predispose
the formation of a septic arthritis, spread by the bloodstream.
Other diseases may not be able to be diagnosed by joint fluid
examination alone; clinical history, physical examination, and
radiologic studies may also be necessary. The various labora-
tory parameters for each disease category are compared in
Table 30-12.

Specimen Collection and Processing

The analysis of synovial fluid begins with the aspiration of
fluid in a procedure known as arthrocentesis; this must be
performed only by trained medical professionals by aseptic
Figure 50-40 = Lymphoma cells in CSF. Wright stain, x 1000 technique. Arthrocentesis may also be used therapeutically, to
magnification. (Courtesy of Judith Brody, M.D., North Shore-Long alleviate increased intra-articular pressure, thereby removing
Island Jewish Health System Laboratories, Lake Success, New York.) inflammatory products and limiting potential joint damage
(see Table 30-9).
can provide results within a day. PCR is used to detect viral For routine analysis, the aspirated fluid should ideally
DNA in a specimen, and is an extremely sensitive method. be divided into four samples. The first portion is placed into
Viruses frequently implicated in CNS infection are Herpes a nonadditive (plain) evacuated tube and observed for
simplex viruses (HSV), cytomegalovirus (CMV), human clot formation; a second portion placed into a sterile,
immunodeficiency virus (HIV), and enteroviruses.

Synovial Fluid
Normal synovial fluid is transparent, colorless, viscous, and
does not clot. The protein content is approximately one fourth Appearance Transparent, colorless to straw
that of plasma; glucose, electrolytes, and uric acid are similar to colored
Physical Qualities Viscous; does not clot
plasma in their concentrations. Age, as well as many inflamma-
WBC Count <200/uL
tory and pathologic joint disorders, can alter the volume and Differential Neutrophils: <25%; remainder as
composition of the synovial fluid affecting its function. An lymphocytes, monocytes,
increased volume of synovial fluid is considered to be an effu- synovial cells, macrophages
sion, and classified as inflammatory or noninflammatory, septic, RBC Count None
or hemorrhagic. Inflammatory responses caused by mechanical, Total Protein 1-3 g/dL
Glucose <10 mg/dL serum-fluid difference
chemical, immunological, or bacterial damage change its cellu- Crystals None
lar and chemical constitution. The impaired function of the fluid Uric Acid Equivalent to normal serum level
may play a role in the development of degenerative joint
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

Group I: Group II: Group III: Group [V: Group V:

Noninflammatory Inflammatory Infectious Crystal-induced Hemorrhagic

Color/Clarity Yellow/clear Yellow/translucent, Yellow/turbid Yellow/cloudy, Red-brown,

cloudy, turbid, or purulent turbid, or white- opaque,
or opaque opaque xanthochromic
(supernatant)
WBC Count <3000 3000-75,000 50,000- 500-200,000 50—10,000
(cells/uL) 200,000
Neutrophils <30 >50 >90 <90 <50
(%)
RBCs = = + = 4
Crystals = = = se =
Glucose ratio* N I I I I

(—) = negative, not present (+) = positive, present.

*Glucose ratio = serum-fluid glucose difference; N = normal (0-10 mg/dL); I = increased.
Modified from Kjeldsberg CR, Knight J. Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous
and Synovial Fluids, 3rd ed. Chicago: ASCP Press; 1993.

heparinized tube for microbiological culture and Gram stain; formation. Viscous fluids represent normal or noninflam-
a third portion placed into a sodium heparin-anticoagulated matory states; viscosity decreases with increasing inflam-
evacuated tube for chemical and/or crystal analysis; a mation, caused by lytic enzymes that depolymerize the
fourth portion into a liguid EDTA-anticoagulated tube for hyaluronic acid. This reduces the lubricating ability of the
cell counts and/or crystal examination (powdered EDTA fluid, promoting damage to the joint. If the physician
and lithium heparin can produce crystalline structures that requires, fluid viscosity would be assessed at the time of
can be mistaken for synovial crystals). A normal fluid does aspiration (not in the laboratory), by allowing the fluid to
not clot; this must be centrifuged as soon as possible to drip from the needle onto a microscope slide or by rapidly
remove all the cells. The supernatant fluid can be assayed lifting the needle from the drop of fluid on the slide and
for rheumatoid factor, antinuclear antibody, complement, observing the length of the “string” of fluid that forms. A
glucose, total protein, lactate, uric acid, and other compo- normal fluid forms a string of 4 to 6 cm in length.** As the
nents. Testing must be initiated within one hour of collec- protein—hyaluronic acid complexes are degraded by lytic
tion; cellular degradation and chemical changes occur with enzymes, the viscosity decreases and no string forms.*’
time. Refrigeration is required if any delay in testing is There is no clinically useful information derived by mea-
anticipated. For some analytes, the supernatant may need to suring the fluid’s viscosity.
be frozen if testing cannot occur immediately. If a sufficient The volume of the specimen bears little correlation
volume of fluid is submitted, a routine analysis should to the severity of the joint disease. Some effusions, though
include an examination of a wet preparation for crystals, apparently significant by the amount of swelling present,
a cell count and differential analysis, a Giam stain and may be difficult to aspirate if they contain considerable
culture, and chemical analysis for protein and glucose debris or fibrin content. If a limited volume of fluid is
concentrations. collected, only the most clinically relevant laboratory tests
are requested by the physician, such as a Gram stain and
Laboratory Analysis and Clinical culture, and microscopic examination for leukocytes and
crystals.°
Correlations
The color of the fluid changes with the disease
QUALITATIVE ANALYSIS process; it is dependent on the quantity of albumin, biliru-
The hematology laboratory will receive the fluid in tubes con- bin, cells, and other debris present. A normal fluid may be
taining EDTA, which should prevent clotting, although very colorless to straw colored. Noninflammatory, inflamma-
viscous fluids may form small clots if they are bloody and/or tory, and infectious fluids can have a yellow color, due to
not well mixed. If the specimen is partially clotted, the cell hemoglobin breakdown of RBCs that enter the synovial
count would be inaccurate, but an attempt should be made at cavity during inflammation, or from chromogenic products
performing the morphological examination for the leukocyte of bacteria within the joint cavity. A traumatic aspirate will
differential, with the required physician notification and com- show streaks of blood in the fluid. Hemarthrosis, hemor-
ment onthe report. If the fluid is completely clotted, no analy- rhage within a joint, produces a homogeneously bloody
sis can be performed. fluid. This can be caused by disease (e.g., hemophilia) or a
Once the specimen is received by the laboratory, it traumatic injury. Centrifugation is necessary to determine
is examined grossly for volume, color, clarity, and clot the origin of the blood. A xanthochromic supernatant
746 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

(similar to CSF) indicates the breakdown of RBCs has glucose in the fluid has no diagnostic significance, whereas
occurred; a bloody or dark red-brown supernatant is sug- the difference between the fluid and plasma concentration
gestive of hemarthrosis rather than a traumatic aspirate or can be clinically important. Normally, synovial fluid glu-
injury (see Table 30-12). cose is in equilibrium with the plasma level during a fast-
Fluid clarity also changes with the disease process; ing, or at least 6 hours postprandial state; the fluid glucose
an inflammatory fluid may be translucent or opaque. is usually equal to or not greater than 10 mg/dL less than
Turbidity increases with inflammation, which may be the plasma level. Generally, patients with noninflammatory
caused by leukocytosis, crystals, or cartilage debris. Septic joint disorders have fluid glucose levels that are within
arthritis will produce a purulent fluid, caused by the 25 mg/dL less than the plasma glucose level. Differences
presence of bacteria and pus. The presence of crystals can greater than 25 mg/dL lower than the plasma level are
create a cloudy, turbid, fatty, or milky appearance (see indicative of inflammatory or septic disorders, due to
Table 30-12). Large quantities of degenerated synovial consumption by microorganisms or increased cellular
lining cells can create the appearance of pus, commonly metabolism (see Table 30-12). Overlap of expected glucose
seen in patients with RA. differences exist between the different types of disorders
Spontaneous clotting of the fluid, the fibrin clot test, is and may not be present in all patients with a particular
observed in inflammatory conditions. Fibrinogen and other disorder.
clotting proteins are acute phase reactants, which can be pre- The concentration of protein in synovial fluid is
sent in synovial fluid. A grossly bloody fluid caused by a trau- dependent on several factors that include the synovial
matic aspiration or traumatic injury will produce spontaneous membrane permeability, the molecular weight of various
clotting due to plasma fibrinogen; a hemorrhagic effusion protein molecules, the plasma level of protein, local synthe-
would not clot. sis, and local consumption. Normal synovial fluid protein
A test that is now obsolete, but of historical interest, levels range from bk to 3 g/dL. Clinically, increased protein
was known as the mucin clot or Ropes test.’ This test levels (greater than 3 g/dL) simply indicate the presence of
was used when a very limited aspirate was obtained, to inflammation, which increases membrane permeability.
determine if the aspirated fluid was synovial, subcutaneous There may also be local synthesis of immunoglobulin
tissue fluid, or local anesthetic. It also was used to discern that increases the amount of total protein present. Elevated
if the effusion was inflammatory or noninflammatory by protein is commonly associated with RA, gout, septic
evaluating the integrity of the molecular interactions arthritis, SLE, or other systemic disorders that can affect
between the hyaluronic acid and protein in the fluid. A the joints.
few drops of the fluid were added to a solution of 2% to Uric acid levels in normal serum and synovial fluid are
5% acetic acid. After a few minutes, a clot formed (mucin essentially in equilibrium. The levels are increased in both
clot) if the fluid is synovial, due to the polymerization of the serum and synovial fluid of patients with gout: a
the hyaluronic acid. The density and friability of the metabolic disorder in which excess uric acid precipitates in
clot after shaking evaluated the molecular interactions. A the synovial cavity causing an arthritic condition. The
dense clot that remained intact when shaken indicated a measurement of uric acid in synovial fluid is not the recom-
normal or noninflammatory (e.g., osteoarthritic) fluid. mended method for the diagnosis of gout; definitive diag-
A clot that fragmented or shredded easily on shaking indi- nosis is achieved by the detection of monosodium urate
cated that enzymatic degradation of the hyaluronic acid (MSU) crystals in the fluid by polarizing microscopic
occurred; this is associated with inflammatory effusions. examination (see discussion on crystals). In some cases,
Use of this test has been discontinued because it is nonspe- however, synovial fluid uric acid levels may be diagnosti-
cific, there are no standard criteria for comparison, and cally significant even if no crystals are observed; values
end-point interpretation is subjective. significantly greater than the upper reference range for
serum uric acid may be diagnostic.** *° It may also verify
the presence of MSU crystals in laboratories without the
Quantitative Analysis: Biochemical Analysis proper polarizing microscopic equipment or if the analyst is
and Microscopic Examination inexperienced.°

BIOCHEMICAL ANALYSIS MICROSCOPIC EXAMINATION

Synovial fluid composition, as an ultrafiltrate of plasma, is The microscopic examination of synovial fluid is crucial to
greatly dependent on the permeability of the synovial mem- determining the presence of crystals and leukocytes. Septic,
brane. A whole blood sample must be collected at the time of inflammatory, and noninflammatory effusions are classified
arthrocentesis for comparative purposes. Analytes routinely by the quantity and types of leukocytes present; the crystal-
measured in the evaluation ofjoint disorders include glucose, induced joint diseases are characterized by the presence of
protein, and in certain cases, uric acid. crystals. A fresh synovial fluid must be screened by per-
Synovial fluid glucose levels are usually compared to forming a wet preparation: a drop of well-mixed, anticoag-
the plasma glucose level at the time of arthrocentesis. Ide- ulated fluid is placed on an exceptionally clean microscope
ally, the blood should be collected in a tube containing slide with a clean coverslip, and the edges sealed with clear
sodium fluoride to inhibit glycolysis. The concentration of nail polish or petroleum jelly to prevent drying. It is then
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 747

examined by routine microscopy for cells, crystals, and and histiocytes. In RA, synovial fluid may contain many
other particulates. plasma cells and moderate numbers of lymphocytes, histio-
A total WBC and RBC count must be performed man- cytes, synovial cells, cartilage cells, and rarely, multinucleated
ually in a hemocytometer; debris within the specimen may giant cells.
clog the flow cells of automated analyzers, and crystals or Certain diagnoses can be suspected when comparing
fat globules may be erroneously counted as cellular mater- the total WBC count with the percentage of segmented neu-
ial. Viscous fluids are difficult to pipette. They must be pre- trophils present. If the differential count reveals 90% or
treated with either 0.05% hyaluronidase in phosphate buffer more segmented neutrophils, then an infectious agent is
(i.e., one drop per milliliter of fluid), or with a pinch of most likely present (see Table 30-12). A Gram stain and
lyophilized hyaluronidase, to liquefy the sample. Fluids culture must be obtained; microorganisms can be seen in
should be counted undiluted and allowed to settle in the synovial fluid if present in sufficient numbers, and should
counting chamber for at least 30 minutes. If the appearance be seen as intracellular organisms. However, if bacteria
of the fluid suggests that dilution is necessary (i.e., bloody are absent on Gram stain this would not exclude the possi-
and/or turbid), only isotonic (0.85% or 0.9%) or phosphate bility that there is sepsis in the synovial cavity. Bacterial
buffered saline must be used as the diluent. Other diluents organisms are more common; pathogenic yeasts are seen
may contain acetic acid that will precipitate the hyaluronic only rarely. If fluids have a total leukocyte counts in the
acid, trapping cells and falsely lowering the WBC count. If range of 2000 to 200,000/uL, with greater than 50% neu-
the fluid is bloody, it may be necessary to lyse the RBCs trophils, a possible diagnosis of RA, SLE, or Reiter’s
with 0.3% saline or 0.1 N hydrochloric acid in order to syndrome should be considered. Reiter’s syndrome is a
count the WBCs more accurately.” The reason is that a suf- reactive arthritis caused by intestinal bacteria that also
ficient dilution of the RBCs may overdilute the WBCs; the affect the skin, eyes, and muscles. The presence of LE cells
greater the dilution factor, the more error that may be intro- is suggestive of SLE but not diagnostic, as they can also be
duced. A separate count must then be performed for the seen in RA.
RBCs. A normal synovial fluid does not contain red blood Eosinophilia of the synovial fluid, defined as greater than
cells; however, an RBC count less than 2000/uL is consid- 2% of the total WBC count, can be associated with RA,
ered normal; the concentration of RBCs is rarely of clinical rheumatic fever, metastatic carcinoma, parasitic infestation of
significance unless there is a need to determine if a trau- the joint, Lyme disease, radiologic procedures, and radiation
matic tap has occurred. therapy.°
The WBC count is necessary to classify the type of Other types of cells that can be observed in inflammatory
effusion present, but by itself is not diagnostic. A normal synovial fluids are known as RA cells and Reiter’s cells. RA
fluid will have less than 200 WBC/uL. Noninflammatory cells are associated with RA but are not specific for a diagnosis
effusions may contain 200 to 3,000 WBC/uL. Inflammatory of RA. These cells may also be called “ragocytes” or “inclusion
effusions may contain 3000 to 75,000 WBC/uL. Infectious body cells”. These are neutrophils that may contain from | to
disorders may contain 50,000 to 200,000 WBC/uL and the 20 granules in the cytoplasm, and are known to contain
crystal-induced disorders are associated with WBC counts immune complexes such as IgG, IgM, complement, and
of 500 to 200,000/uL.° There is obviously a considerable rheumatoid factor. Reiter’s cells, also nonspecific and not
amount of overlap to the leukocyte counts among disorders. diagnostic for Reiter’s syndrome, are vacuolated macrophages
The counts must be considered in the context of the clinical with intracytoplasmic inclusions or debris of ingested neu-
presentation; and most importantly, the differential cell trophils, which may appear as unrecognizable blue material by
count and morphology, which is the most important factor Wright stain.
in the differential diagnosis in synovial fluid analysis (see Synovial lining cells are mononuclear cells with mor-
Table 30-12). phology resembling that of the mesothelial cells in serous
Morphologic examination is performed using Wright- fluids. Their nuclei may have small, regular nucleoli. They
stained cytocentrifuge preparations. If a cytocentrifuge is not may become proliferative in a reactive setting similar to
available, or if fluids have extremely high cell counts, standard mesothelial cells. Reactive synovial cells may be multinu-
push smear or slide-on-slide methods may be used. The fluid cleated and may occur in clusters. Synovial lining cells may
should be liquified and appropriately diluted, as previously also be difficult to differentiate from monocytes and histio-
mentioned, within | hour of collection owing to cellular degra- cytes. Their presence does not have any specific diagnostic
dation and the risks of misdiagnosis. The cells that are normally significance.
present include lymphocytes, monocytes, macrophages, and Malignant cells can be seen in synovial fluid, although
synovial cells. Neutrophils are also present, but do not exceed this is extremely rare; they are usually derived from metasta-
25% of the total cells. Abnormal synovial fluids can contain tic disease.
neutrophils, lymphocytes (normal and reactive), plasma cells,
monocytes, eosinophils, histiocytes, macrophages, synovial Crystal Examination
cells, and LE cells. In acute inflammatory conditions, synovial
fluid can contain many neutrophils and a moderate number of Every synovial fluid sent to the laboratory for cell counts
synovial cells; bacteria may also be present. In chronic inflam- must be examined for crystals, especially if infection is
mation, synovial fluid contains lymphocytes, plasma cells, not a consideration. This aspect of fluid analysis has the
748 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

greatest impact on diagnosis and therapy; if incorrect or present in the crystals. A drop of the stain is added to a drop of
improperly performed it can result in permanent disability the synovial fluid, placed on a slide, and cover slipped. Crys-
for the patient. As previously mentioned, the specimen tals containing calcium appear orange with light microscopy
must have been collected into liguid EDTA or sodium or bright red under polarized light.
heparin, to avoid confusion with crystalline structures Laboratories that perform crystal analysis and identifica-
caused by the powdered forms of these anticoagulants. tion must use appropriate quality control materials, to align
Evaluation should be performed on a wet preparation; how- the polarized microscopes properly and for comparative pur-
ever, examination of the fluid by standard centrifugation or poses in identification.
cytocentrifuge preparation is also necessary. The cytocen-
trifuge concentrates the fluid; a sample that is negative by a TYPES OF CRYSTALS, ANALYSIS, AND CLINICAL
wet preparation may actually show crystals on the cytocen- CORRELATION
trifuge slide. If sufficient numbers of crystals are present, The causes of crystal deposition are not well understood,
they can be seen with plain light microscopy on the Wright- but certain predisposing factors may exist such as increas-
stained slide. It is important to note, however, that some ing age, the presence of joint damage, and familial inheri-
Wright staining techniques may result in the dissolution of tance. Others include metabolic disorders, osteoarthritis
some crystals. Extra cytocentrifuge slides should be pre- (OA), and SLE (see Table 30-12). The most common types
pared and left unstained to determine if crystals can be seen of crystals that may be present in synovial fluid include
before staining. If few crystals are present, phase-contrast monosodium urate (MSU), calcium pyrophosphate dihy-
or polarized light microscopy may be necessary to see them drate (CPPD), basic calcium phosphates (BCP), oxalates,
initially by wet preparation or cytocentrifugation. Synovial cholesterol and lipids, and steroids. Less common crystals
fluids should be thoroughly examined for crystals, for and artifacts also occur, which may be confused with clini-
at least 15 minutes, before they are reported as negative. cally significant synovial fluid crystals or interfere in their
Artifaéts such as cell clumps or fibrin strands may obscure detection.
or trap the few crystals that may be present. Excellent images of synovial fluid cells and crystals are
Polarized light must be used for the detection and con- readily available on the Internet. The reader is urged to visit
firmation of birefringence, the ability of a particular mater- the following Web site(s) for useful educational material:
ial to refract light. This can be determined with a polarized
www.archrheumatol.net/ASF/index.html
light microscope that contains a rotating filter, known as a
“polarizer” situated below the slide stage in the light path. www.palmslab.com.au/.../ Infolink/issue9.shtml
A fixed filter, a second polarizer known as the “analyzer,” is http://www-medlib.med.utah.edu/WebPath/
located above and between the objectives and the oculars. BONEHTML/BONE063.html
Both filters will allow only light of one direction or polarity
to exit. When the polarizer is rotated 90 degrees with MONOSODIUM URATE CRYSTALS The presence of MSU
respect to the analyzer, it creates a block, or maximum crystals is pathognomonic for gout, causing a condition
extinction, of the polarized light, yielding a dark field. If known as gouty arthritis. These crystals are seen in the ma-
the specimen contains birefringent material, the direction of jority of patients during acute attacks. Between attacks,
the light is refracted, allowing this light to pass through the they may be seen in approximately 75% of patients.> They
analyzer and is seen as a bright particle or crystal against may also be present, occasionally, in cases of inflammation.
the dark field. Not all materials possess birefringent charac- MSU crystals are typically long, thin, and needle-like with
teristics, but this attribute assists in identification of the pointed ends. They may be seen singly or in aggregates;
particles present in the synovial fluid. Another filter, known rarely, they may appear as spherules that are composed of
as a “red compensator,” changes the velocity of the trans- clusters of many individual needle-shaped crystals.2 Some
mitted light, allowing birefringent crystals to display differ- Wright staining methods can cause MSU crystals to dis-
ent colors depending on their position in relation to the axis solve, which is why the wet preparation must always be
of the compensator. This is used to further identify and examined first. These crystals may be found within neu-
confirm the existence of certain types of crystals. The com- trophils or macrophages during attacks of acute gouty
pensator is placed at a 45-degree angle to the axis of the arthritis.” It is important to report their appearance as intra-
polarizing lenses, which causes the background to become and/or extracellular; phagocytosis of crystals suggests that
a reddish color when properly aligned. The slide (or the they are responsible for the acute arthritis.2* MSU crystals
stage, depending on the microscope’s design) is rotated to are strongly birefringent and appear white in polarized
bring a crystal into a parallel orientation with the compen- light, however, they lose their birefringence with compen-
sator. The color is noted; then the crystal is oriented perpen- sated polarization. These characteristics are sufficient to
dicular to the axis of the compensator and the color change identify them as MSU crystals. If these crystals are parallel
is noted. If many crystals are present, only one crystal to the axis of the compensator they will appear yellow (neg-
needs to be found that fits the identification criteria for that ative birefringence); if perpendicular they will appear blue.
particular crystal. As previously mentioned, an increased concentration of
In addition, if unknown crystals are seen, alizarin red S uric acid in the serum and/or synovial fluid, even without
can be used, which selectively stains any calcium that may be the presence of MSU crystals, is diagnostic of gout.
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 749

CALCIUM PYROPHOSPHATE DIHYDRATE CRYSTALS CHOLESTEROL AND LIPID CRYSTALS Cholesterol crys-
CPPD crystals are characteristically present in a group of tals can be present in chronically inflamed joints, and are
disorders known as CPPD deposition disease (CPDD).*' It not diagnostic of any particular disorder. They may also be
is also known as pseudogout or chondrocalcinosis. These found in chylous effusions which contain a high concentra-
crystals are a common cause of arthritis, most frequently seen tion of lipids. Although rarely observed, they are most
in the elderly, and in patients with degenerative arthritis. often seen in cases of patients with long histories of RA,”
Patients with hereditary forms of CPDD, as well as meta- OA, and ankylosing spondylitis.** Crystal formation is
bolic disorders, may also possess CPPD crystals. The thought to involve the cholesterol from the cell membranes
symptoms of this condition may imitate gout or RA. Occa- of degenerating cells associated with impaired drainage of
sionally, a synovial fluid may contain both MSU and CPPD the joint.° They have characteristically large, rectangular
crystals; the presence of one does not exclude the other. notched-plate shapes that are easier to see by regular light
This may be an indication of septic arthritis, especially if microscopy, although strongly birefringent with polariza-
the WBC count is extremely elevated. These crystals may tion. However, they may also resemble the needle or rhom-
also be seen intra- and/or extracellularly. They can be boid shapes of MSU or CPPD crystals; this can lead to
more difficult to identify, typically appearing as short misidentification. Compensated polarization is not reliable
rectangular shapes, but they may also assume multiple as a method of differentiation because cholesterol crystals
three-dimensional forms such as rods and rhomboids. are variable in their appearance. The presence of lipophages
Needle-shaped forms are also possible, causing misidentifi- (lipid-laden macrophages) during the microscopic exami-
cation as MSU crystals. They are more easily seen with nation may provide additional evidence of the type of
light microscopy; they are only weakly birefringent, crystal present.
and therefore may be difficult to see with polarized light. Lipids can exist in crystalline and noncrystalline forms
With compensated polarization, these crystals have the and are typically found in both chylous and chronic effu-
opposite appearance of MSU. They appear blue if parallel sions.** Noncrystalline lipids occur as fat globules that
to the axis (positive birefringence), and yellow if perpen- are round and nonbirefringent. Lipid crystals are round
dicular, thereby differentiating them from MSU crystals. when seen by light microscopy. With polarized light,
The use of alizarin red S staining will indicate the presence the crystalline forms appear as strongly birefringent
of calcium. ‘ Maltese crosses. Under compensated polarization, they
appear as red and blue crystals. Lipid crystals can be mis-
BASIC CALCIUM PHOSPHATE CRYSTALS The BCP crys- taken for MSU spherules by an inexperienced analyst.
tals are a group of compounds consisting mainly of calcium However, observation under high-power magnification
hydroxyl phosphate, also known as hydroxyapatite (HA); (< 100) would reveal they are not composed of numerous
others include octacalcium phosphate, and less frequently, individual crystals.
tricalcium phosphate. These crystals are very small in
size and form aggregates, which are occasionally visible CORTICOSTEROID CRYSTALS Patients with known non-
by light microscopy on a wet preparation as shiny infectious joint disorders, such as OA and RA, may occasion-
irregular cytoplasmic inclusions. These crystals may also ally receive treatment with corticosteroid injections into the
appear extracellularly. They will appear as purple inclu- joint cavity or surrounding connective tissue, to reduce the
sions on a Wright-stained smear. They are too small and inflammatory response. Knowledge of a history of intra-
too weakly birefringent to be identified by polarized articular steroidal injection is important in the evaluation of
light microscopy. Positive staining does occur with alizarin synovial fluid. The synthetic corticosteroids form artifactual
red S, but this is nonspecific. HA crystals are not crystals of varying sizes, shapes, and birefringent qualities.
pathognomonic for any specific disorder, but they are They may persist in the synovial fluid for several weeks or
associated with arthritic syndromes such as RA and OA; months after injection. Steroids do not have characteristic
these involve calcification in and/or around the joint, with crystalline shapes. They may be amorphous with irregular
periarticular inflammation, calcific deposits, and the pres- outlines and ragged edges, or they can resemble MSU and
ence of HA in the synovial fluid. Most typical clinical CPPD crystals. They can be strongly birefringent or not at all.
laboratories will not be able to definitively identify these They can also be phagocytosed by leukocytes and/or synovial
crystals. However, diagnosis, prognosis, and treatment are cells, appearing intracellularly. These multiple factors
not dependent on it. should alert the analyst to the probability that they are
steroidal crystals, one of the more common artifacts that can
OXALATE CRYSTALS Crystals of calcium oxalate may be be found in synovial fluid.
found in the synovial fluid of patients with primary oxalosis,
arare inborn error of metabolism, but is more commonly seen HEMATIN OR HEMATOIDIN CRYSTALS Red blood cells
in patients with chronic renal failure who undergo hemodial- that are present in the synovial cavity are phagocytosed by
ysis. The deposition of these crystals causes arthropathy. The macrophages. Enzymatic degradation of the hemoglobin
crystals appear as small, bipyramidal or pleomorphic shapes, results in the formation of hemosiderin, which may subse-
with a wide variation in birefringence and stain positively quently form hematin or hematoidin crystals. Their presence
with alizarin red S. suggests that a significant hemarthrosis has occurred. These
750 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

crystals are rhombohedral shaped, but occasionally may between the layers of the pericardium, pleura, and peri-
be irregularly shaped. They appear gold in color with regu- toneum, respectively.
lar light microscopy and are not birefringent. It is possible The central nervous system (CNS) consists of the brain
for these to be mistaken for CPPD crystals by an inexperi- and spinal cord, which are lined by the meningeal mem-
enced technologist; however, their appearances differ with branes, Or meninges, that consist of three layers: the dura
polarization. mater, arachnoid mater, and pia mater. Between the arachnoid
mater and pia mater is the subarachnoid space, containing the
ARTIFACTS Numerous particulates can appear in synovial cerebrospinal fluid (CSF).
fluid during microscopic analysis that are clinically Joints (articulations) are junctions between two or more
insignificant and/or not originally of synovial origin. These bones. They are enclosed by the joint capsule and lined by the
are known as artifacts and must be differentiated from clin- synovial membrane, or synovium, composed of a single layer
ically significant synovial particulates. Most can be of mononuclear synovial cells. Synovial fluid is contained
avoided with the use of clean microscope slides and cover- within the synovium and joint capsule.
slips, such as dust, glass fragments, lint, paper fibers, and Manual cell counts of WBCs and RBCs are the methods
so forth. Cartilage fragments, collagen fibrils, or fat glob- used for body fluids. Slides for morphologic analysis are pre-
ules may be present due to a disease process or as a result pared via cytocentrifugation. Advantages and disadvantages
of the arthrocentesis; metallic fragments may arise from of this method are summarized in Tables 30-1 and 30-2.
articular prostheses. As previously mentioned, therapeutic The most common types of cells encountered in body
intra-articular steroid injections can cause the formation of fluids are neutrophils, lymphocytes, monocytes, and tissue
crystalline structures. Wright or Wright-Giemsa-—stained cells. Other cells less frequently seen include eosinophils,
slides may contain stain precipitates. Artifactual crystals basophils, mast cells, plasma cells, and malignant cells.
typically have variable appearances by regular light and An abnormal* collection of fluid is known as an
polarized light microscopy. They do not have definite crys- effusion. Effusions that accumulate due to systemic dis-
tal morphology. Clinically significant crystals have regular eases are known as transudates; those that accumulate
outlines with smooth, parallel edges. Polarized light due to pathology within a compartment are known as
microscopy can create confusion in differentiating artifact exudates. The differences between the two are outlined in
from pathologic crystals, most commonly when starch Table 30-4. Effusions can occur due to a wide variety of
granules enter the specimen from surgical gloves. They local or systemic disease processes. They may be further
appear as variably to strongly birefringent Maltese crosses, described as normal cellular, benign/reactive, or malignant,
similar to lipid crystals. In addition, they can be mistaken based on the cell types present. The types of effusions, their
for MSU spherules. However, starch granules have irregu- locations, and the methods of collection are described in
lar outlines and a central depression visible by light Table 30-9.
microscopy. The CSF is a selective ultrafiltrate of plasma. Indica-
tions for its analysis include suspicion of meningeal infec-
Summary tion, subarachnoid hemorrhage, malignancy within the
CNS, or demyelinating diseases. CSF collection is per-
The analysis of fluids from normally sterile body compart- formed by lumbar puncture (LP) or “spinal tap.” A normal
ments is an important element in the diagnosis and treatment CSF is clear, colorless, sterile, and watery in consistency. A
of disease. Body fluids are a diverse group of non-blood, non- common problem in the analysis of CSF is distinguishing
urine fluids that are found in closed body cavities. The types between a true CNS hemorrhage and a traumatic LP.
of cells, their concentrations, and the presence and types of Grossly bloody specimens may result from a traumatic tap,
crystals are useful in disease diagnosis. It is important for the or the presence of a subarachnoid or an intracerebral hem-
laboratory professional to be able to distinguish benign from orrhage. Xanthochromia, a pink, orange, or yellow color of
reactive or malignant cells and appropriately refer these find- the CSF supernatant, is caused by the breakdown of hemo-
ings to the pathologist. All stained fluid slides with atypical, globin. Other causes of xanthochromia include jaundice,
suspicious, or malignant cells must be reviewed by a pathol- extremely elevated CSF protein, and hyperbilirubinemia in
ogist prior to reporting. premature infants.
The aspirated fluid is placed into evacuated, anticoagu- Biochemical analysis of CSF routinely includes the mea-
lated, and/or sterile tubes to be sent to the laboratory. The surement of the total protein (TP) and glucose levels. An
specimens must be processed immediately. The cells are usu- increased TP is seen in meningeal and CNS disease. Glucose
ally less numerous than in peripheral blood and they begin to in the CSF is normally about 60% to 70% of serum levels.
deteriorate once they are removed from the body. Decreased CSF glucose is characteristic of bacterial, tubercu-
Fluids from the thoracic and abdominal cavities are lous, or fungal meningitis. Viral meningitis, however, is not
referred to as serous fluids. The heart and lungs within the associated with a decreased CSF glucose. Normal findings in
thoracic cavity, and various organs in the abdominal cavity, CSF are summarized in Table 30-10.
are lined by a membrane composed of mesothelial cells. CSF WBC counts are clinically significant; even low
Pericardial, pleural, and peritoneal fluids are contained counts are suggestive of pathology. The types of WBCs
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis = 751

present are most important in the interpretation of a CSF, and leukocytes. The cells normally present include lymphocytes,
their relationship to the clinical findings. The morphologic monocytes, macrophages, synovial cells, and neutrophils,
evaluation of the CSF must be performed regardless of a which should not exceed 25% of the total nucleated cells
normal cell count. present. Every synovial fluid must have an examination for
Normal synovial fluid, aspirated by arthrocentesis, is crystals. Polarized light must be used for the detection and
transparent, colorless, viscous, and does not clot. Impaired confirmation of birefringence. The most common types of
function of the fluid may play a role in the development of crystals that may be present in synovial fluid include
degenerative joint diseases. Synovial fluid is most often ana- monosodium urate (MSU), calcium pyrophosphate dihydrate
lyzed when there is a suspicion of infective (septic) arthritis (CPPD), basic calcium phosphates (BCP), oxalates, choles-
or crystal-associated diseases. Analytes routinely measured terol and lipids, and steroids.
in the evaluation of joint disorders include glucose, protein, The procedural name, collection requirements, routinely
and uric acid. The microscopic examination of synovial ordered tests, and storage conditions for each body fluid type
fluid is crucial to determining the presence of crystals and are summarized in Table 30-13.

Table 30-15 Body Fluid Col

Pie

Tube Types
Fluid Type Procedure(s) Collected* Tests Storage

Serous:
Pleural Thoracentesis 1. EDTA—lavender top 1. Cell counts, morphology, 4°C
differential count
Pericardial Pericardiocentesis 2. Heparin—green top 2. Color, clarity, glucose, total 4°C
Peritoneal Paracentesis protein, LDH, cholesterol,
(Ascites) bilirubin, specific gravity
3. Sterile, heparinized 3. Culture, Gram stain, AG
acid-fast stain
4. Nonadditive tube; plain 4. Clot formation; gross 4°C
red top examination
Cerebrospinal Lumbar puncture Sterile, nonadditive, Tube 1: Chemical/Serological 4°C
sequentially numbered Studies
tubes Color, clarity, glucose, Total
Protein, IgG, Albumin
Tube 2: Microbiological SHC
Studies
Culture, Gram Stain, India Ink
Preparation
Tube 3 or 4: Cytology or 4°C
Hematology
Cell counts, morphology,
differential count
Synovial Arthrocentesis 1. Liquid EDTA— 1. Cell counts, morphology, AXE
lavender top differential
count, crystal
analysis
2. Sodium heparin— 2. Color, clarity, glucose, AL@
green top total protein, uric acid,
lactate, rheumatoid factor,
complement, ANA, crystal
analysis
3. Sterile (heparinized) 3. Culture, Gram stain AG
4. Plain, nonadditive 4. Clot formation; gross 4°C
examination

*A whole blood specimen for serum preparation must be drawn at the time of fluid collection for comparative studies.
**Microbiological specimens must be kept at 37° C until cultures are prepared; thereafter, remaining specimen must be stored
at 2-8°C until all testing is completed.
ANA = antinuclear antibody.
oe Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

WeThe CSF RBC count is elevated at 15/L. Normally, RBCs

Case Study I should not be present, but can be elevated in infants. The
CSF WBC count is elevated at 1300/uL. Normally, the
A |-month-old female infant was brought to the pediatrician WBCs should be less than 30/uL in infants younger than
with a 2-day history of fever of up to 100°F (37.7°C). No other 1 year of age. The CSF glucose level is decreased at
symptoms were evident. The physical examination was nor- 35 mg/dL. Normally, the CSF glucose is 60% to 70% of
mal. The infant was sent home with a diagnosis of a mild viral the fasting serum glucose (110 mg/dL). A level less than
illness. Two days later, the fever spiked to 103°F (39.4°C). The 40 mg/dL indicates hypoglycorrhachia, which is signifi-
infant was irritable, lethargic, and rejected feedings. There cant. This may be due to increased utilization by WBCs,
were no other signs or symptoms of a rash, cough, vomiting, RBCs, microorganisms, or CNS tissues.
or diarrhea. She was brought to the local emergency room. A . The results suggest a viral, or aseptic, etiology. The WBC
lumbar puncture was promptly performed, and a peripheral differential does not show an increase in neutrophils,
blood sample was simultaneously collected. A strong suspi- which would be increased in a bacterial infection. The
cion of meningitis existed and the patient was admitted to the Gram stain and bacterial cultures were also negative.
pediatric intensive care unit. An aggressive course of antibiotic . The CSF cultures were negative for bacterial or viral
and antiviral therapy was started. pathogens. However, the stool culture was positive for
The laboratory values obtained on the spinal fluid and Enterovirus, which is a known etiological agent for asep-
peripheral blood are indicated: tic meningitis. Aseptic meningitis is diagnosed when there
is no growth of bacteria by standard culture methods.
Whole blood (WBC): 14,000/uL
Differential: Neutrophils = 32%
Lymphocytes = 61%
Monocytes = 2%
Serum glucose: 110 mg/dL
Ger aac : Case Study 2
Appearance: Cloudy, colorless ; =
RBC: 15/uL A 50-year-old man with no prior medical history sought med-
WBC: ; 1300/uL : ical attention for flu-like symptoms. The patient has a current
Differential: Neutrophils = 1% history significant for a persistent cough and fever of 2 weeks’
OO
Monocytes
ae
= 67%
duration. Antibiotic therapy
Rane Eas
wa olypee
ibed by the physici
no oe
Cincose: 35 mg/dL and the patient was sent home with the directive to increase
Total protein: 79 mg/dL fluid intake and bedrest. After 2 days, severe left-sided chest
Gram stain: Negative pain and shortness of breath developed. The patient was
brought to the emergency room. A radiologic exam revealed a
collection of fluid in the left chest cavity. The fluid was with-
Viral and bacterial cultures were performed on the CSF drawn for analysis and a simultaneous peripheral blood speci-
and a stool specimen. The results of these analyses were: men was collected.
The laboratory values obtained on the chest fluid and
CSF bacterial culture: Negative for 72 hours peripheral blood are indicated:
CSF viral culture: Negative for 7 days
CSF viral PCR analysis: Negative
Stool viral culture: Positive for enterovirus ae ee oe e ae
Fluid appearance: Thick, turbid, yellow
The patient made an uneventful recovery and was dis- es Se ae ee
charged after 10 days of hospitalization. Fluid we , 40 U/L
LDH ratio: 0.8
QUESTIONS Fluid WBC: 20,000/uL
Differential: Neutrophils = 90%
1. Why was a lumbar puncture performed? Macrophages = 10%
2. What abnormal results are present, if any, and what do Many degenerating cells present
they represent?
3. Do the results suggest a bacterial or aseptic (viral)
ee QUESTIONS
4. What is the significance of the culture results? 1. What is the name of the fluid collection in the chest cavity?
2. What is the name of the procedure to withdraw this fluid?
ANSWERS 3. Is the fluid specimen considered a transudate or an
exudate?
1. Whenever there is a suspicion of meningeal infection, sub-
. Is the fluid specimen chylous?
arachnoid hemorrhage, CNS malignancy, or demyelinating
disease. In newborns or infants less than three months of . What further tests should be performed on the fluid
specimen?
age, the presence of fever is significant. Fever, irritability,
. Are these findings representative of an acute or chronic
and lethargy are highly suggestive of meningeal infection.
reactive process?
continued
continued
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 753

ANSWERS QUESTIONS
1. A fluid collection in the chest cavity is known as a . What type of procedure was performed to obtain the
pleural effusion. joint fluid and what terminology applies to the speci-
2. The procedure to withdraw a pleural effusion is known men?
as a thoracentesis. 2. Into what types of collection tubes should the aspirated
3. The pleural effusion collected is considered to be an specimen be placed?
exudate, because the WBC count is greater than eS). What abnormal results are present, if any, and what
1000/uL, the total protein is greater than 3 g/dL, the could they indicate?
total protein ratio is greater than 0.5, and the LDH ratio 4. What additional tests should be performed to aid in
is greater than 0.6. diagnosis?
4. This specimen is not chylous, as the predominant cell type
in the differential examination are neutrophils, whereas
ANSWERS
chylous effusions predominantly contain lymphocytes. It
could be considered pseudochylous by its appearance, but 1. The procedure to obtain fluid from a joint space is
the laboratory studies for triglycerides are not available. known as arthrocentesis; the fluid collected is known as
5. Further tests to be performed should include a Gram synovial fluid.
stain and culture. 2. The specimen should be placed into sterile nonadditive,
6. These findings are representative of an acute reactive sodium heparin, and liquid EDTA tubes.
process, which present with greater than 50% neu- 3. The child’s peripheral blood WBC count and erythrocyte
trophils on differential examination. sedimentation rate are elevated but consistent with the
presence of an acute upper respiratory infection or other
inflammatory process. However, the fluid analysis
reveals abnormalities in the clarity, cell counts and dif-
ferential, and the glucose level. These parameters indi-
cate the presence of an active inflammatory process: a
normal synovial fluid would not contain RBCs, there
would be less than 200 WBC/uL, and neutrophils would
Case Study 3 not exceed 25% of the WBCs. The fluid WBC count
falls within the Group II/Inflammatory disease category,
A 2-year-old male child was brought to the emergency room however, other parameters fit into the Group III/
to determine why he suddenly was refusing to walk and Infectious disease category. The presence of neutrophilia
complaining of pain in his right leg. There was no history of (greater than 80%) would be highly suggestive of septic
trauma. He had been seen by his pediatrician earlier in the arthritis, regardless of the WBC count. Results can over-
day due to the presence of fever and an upper respiratory lap disease categories, and it is possible for more than
infection; he was diagnosed with otitis media, prescribed an one type of disease process to be present simultaneously.
antibiotic, and sent home. Clinical presentation and the microscopic findings, most
Physical exam in the emergency room revealed pain and importantly the differential cell count and morphology,
tenderness of the right ankle. There was also some edema will determine what process is involved and what course
and erythema. X-ray exams of the lower extremities did not of action should be taken. The glucose level is not in
reveal any fractures or displacements. Laboratory tests were equilibrium with the plasma; differences greater than
ordered on his peripheral blood, and a fluid aspirate was 25 mg/dL lower than the plasma level are indicative of
taken from the ankle. The results were as follows: inflammatory or septic disorders, due to consumption by
microorganisms or increased cellular metabolism.
Peripheral blood: 4. Additional testing must include examination of a Gram
_ WBC: 26,000/uL stain and culture of the fluid to identify the presence of
Differential: Neutrophils = 75% bacterial organisms. If positive, this would point to a
Bands = 2% diagnosis of septic arthritis.
Lymphocytes = 19%
Monocytes = 4%
_ RBC parameters: Normal
Erythrocyte sedimentation rate: Elevated
Plasma glucose:
Fluid analysis:
96 mg/dL
Case Study 4
Color: Yellow
Clarity: Purulent A 50-year-old man with a history of alcohol abuse was seen
WBC: 22,000/uL in the emergency room for symptoms of nausea, vomiting,
Differential: Neutrophils = 95%
and abdominal pain. On physical exam, the abdomen
Macrophages = 5%
appeared distended. A computerized tomographic (CT) scan
RBC: 375,000/uL
Fluid glucose: 39 mg/dL of the abdomen revealed a collection of fluid. About
1200 mL of fluid was drained and portions were sent to the
continued continued
754 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

ANSWERS
Case Study 4. conta
1. Paracentesis is the procedure to drain and collect fluid
from the peritoneal cavity. This collection of fluid is
laboratory for analysis. Peripheral blood was collected
known as a peritoneal effusion, or ascites. The collected
simultaneously. The laboratory findings were as follows:
fluid may be referred to as peritoneal fluid, paracentesis
fluid, or ascitic fluid.
Serum values:
. This fluid is a transudate.
Glucose 99 g/dL
_ Transudative ascitic fluids can be caused by increased
Why
Total protein 7 g/dL
Albumin 4.0 g/dL capillary pressure or permeability, abnormal colloid
LDH 60 U/L osmotic pressure, poor lymphatic drainage or cardiac
Fluid analysis: Clear, pale yellow abnormalities. Additiona! causes include abdominal con-
Glucose 100 mg/dL ditions that do not directly involve the peritoneum, such
Total protein 1.9 g/dL as hepatic cirrhosis, intrahepatic and portal venous
TP ratio 0.3 obstruction, hypoalbuminemia, renal function, ovarian
LDH 25 U/L disease, pancreatic disease, or parasitic infection. Alco-
LDH ratio 0.4 holic cirrhosis is one cause of peritoneal effusion, which
Albumin 2.4 g/dL appears to be the case in this patient. Malignant disease
SAAG 1.6
can also produce an effusion.
RBC 500/uL
WBC 60/uL 4. The SAAG, or serum-ascites albumin gradient, is a
Differential: Neutrophils = 10% value calculated by subtracting the ascitic fluid albumin
Lymphocytes = 80% concentration from the simultaneously collected serum
Monocytes/Macrophages = 10% albumin concentration. This value is useful in differenti-
‘ Atypical mesothelial cells seen ating whether the fluid is transudative or exudative in
nature; transudative ascitic fluids have an SAAG signifi-
cantly greater than 1.1 g/dL, whereas exudative fluids
QUESTIONS are found to be less than 1.1 g/dL. In addition, patients
1. What type of procedure was performed to drain and col- with high-gradient ascites include those with cirrhosis,
lect the fluid, what body cavity was involved, and what alcoholic hepatitis, cardiac-related ascites, or massive
terminology applies to the specimen? liver metastasis. Low-gradient ascites are associated
2. Is the fluid a transudate or an exudate? with peritoneal malignancies and non-malignant diseases
3. What process(es) could cause the production of this type such as TB, pancreatitis, nephrotic syndrome, biliary
of fluid? disease, or connective tissue diseases.
4, What is the SAAG and what is its significance? 5. The presence of atypical mesothelial cells are especially
5. What does the presence of atypical mesothelial cells seen in chronic effusions and in ascitic fluid associated
indicate? with cirrhosis.
continued

Acknowledgments
The author gratefully acknowledges the contributions of
Judith P. Brody, M.D., and Michele L. Best, MT (ASCP).

Direct iome
1. A specimen is sent to the laboratory labeled “synovial 3. All of the following are serous fluids except:
fluid.” What procedure was used to obtain the specimen? a. Synovial
a. Thoracentesis b. Peritoneal
b. Paracentesis c. Pleural
c. Arthrocentesis d. Pericardial
d. Lumbar puncture
4. The anticoagulant used for hematological body fluid
i) . The presence of MSU crystals is pathognomonic for analysis Is:
which disorder? a. Heparin
a. Pseudogout b. Oxalate
b. Osteoarthritis c. EDTA
c. Rheumatoid arthritis d. Citrate
d. Gout
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis 755

5. Another term for “ascites” is: c. Peripheralization, distortion, and segmentation of

a. Pleural fluid nuclei
b. Thoracentesis fluid d. Normal, reactive, and malignant cells can be identified
c. Peritoneal fluid
10. Normal CSF contains:
d. Pericardial fluid
a. Lymphocytes and ependymal cells
6. The cell type that forms the lining of the pleural, pericar- b. Ependymal and choroidal cells
dial, and peritoneal cavities is: c. Mesothelial and ependymal cells
a. Epithelial d. Erythrocytes and leukocytes
b. Endothelial
11. Match the following gross examination findings of CSF
c. Mesothelial
with the appropriate diagnosis:
d. Ependymal
1. Cloudy and turbid
7. Which of the following best characterizes a transudate? 2. Grossly bloody specimen
a. Total protein greater than 3.0 g/dL; total WBC less 3. Xanthochromia
than 1000/uL 4. Gel formation
b. Total protein less than 3.0 g/dL; total WBC less than a. Increased fibrinogen
1000/uL b. Subarachnoid hemorrhage
c. Total protein less than 3.0 g/dL; total WBC greater c. Subarachnoid hemorrhage (more than 12 hours after
than 1000/uL the bleed)
d. Total protein greater than 3.0 g/dL; total WBC greater d. Pleocytosis
than 1000/uL
12. When should a pathologist review a fluid smear?
8. A turbid peritoneal fluid is collected from a patient with
13. Differentiate between a traumatic LP and a CNS hemor-
suspected peritonitis and a cell count is performed. A
rhage. What is xanthochromia?
1:100 dilution is prepared and 6 cells are counted in each
of the 4 WBC corner squares of the hemacytometer 14. What is birefringence? Why is a polarizing microscope
chamber. The final WBC count would be: necessary when examining synovial fluid for crystals?
a. 37,500/uL 15. What types of cells are normally found in synovial fluids?
b. 1500/uL
c. 150/uL See answers at the back ofthis book.
d. 6000/uL
9. Which of the following is a disadvantage of cytocen-
trifugation for the preparation of body fluid slides?
a. Cell differentiation is determined by Wright stain
b. Cell differentiation is done on a concentrated
preparation

ARY cH
d ms SUMM @ Synovial fluid is secreted by the synovial cells, also pro-
ducing hyaluronic acid.

mw Fluids from the thoracic and abdominal cavities are re- m The large joints may normally contain approximately
ferred to as serous fluids; they are ultrafiltrates of plasma. 1 mL of synovial fluid.

gw The pleural, pericardial, and peritoneal cavities are lined by gwAll body fluids are potentially infectious and they
a membrane composed of a single layer of mesothelial cells. frequently are nonretrievable.

m Normal pleural, pericardial, and peritoneal cavities do not maImportant terms: Thoracentesis = removal of pleural
fluid; Pericardiocentesis = removal of pericardial fluid;
contain appreciable amounts of fluid.
Paracentesis = removal of peritoneal fluid; Lumbar
= The central nervous system (brain and spinal cord) is lined puncture = collection of CSF; Arthrocentesis =
by the meningeal membranes/meninges. removal of synovial fluid; Effusion = an abnormal col-
mThe subarachnoid space contains cerebrospinal fluid lection of fluid in a body cavity; Transudates = effusions
(CSF), which is a selective ultrafiltrate of plasma. caused by a systemic disease state; Exudates = effusions
caused by a primary pathologic state within the compart-
gw The normal total volume of CSF in adults is 90 to 150 mL,
ment; Chylous effusions = exudates resulting from leak-
and 10 to 60 mL in neonates.
ing or blocked lymphatic vessels.
w Joints (articulations) are enclosed by a joint capsule; the syn-
ovium or synovial membrane is composed of synovial cells.
continued
756 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis

m To distinguish a true CNS hemorrhage versus a traumatic

LP: If the bloodiness decreases with each successive tube,
this would suggest a traumatic LP.
mw Transudates result from increased capillary hydrostatic
pressure or decreased plasma oncotic pressure. = Xanthochromia = a pink, orange, or yellow color of the
CSF supernatant, caused by the breakdown of hemoglo-
mwExudates result from increased capillary permeability bin. It is usually thought to indicate a true CNS hemor-
and/or decreased lymphatic reabsorption. rhage. Xanthochromia will occur, however, if a bloody
mw Malignancy is a major cause of serous effusions. fluid from a traumatic LP is not processed within | hour
g All body fluid specimens must be processed immediately of collection.
upon receipt in the laboratory and all specimens are eval- mwNormal CSF is practically acellular by the manual
uated for the appropriate collection container and visually hemacytometer count. No RBCs should be present (less
inspected for volume, color, clarity, and/or the presence of than 1/uL).
fibrin clot. Serous fluid is collected into EDTA for hema- w On differential analysis, normal CSF contains predomi-
tological analysis. nantly mononuclear cells; very few to no segmented neu-
w Laboratory tests performed by the hematology laboratory trophils should be observed.
include: Total cell count, morphological and differential @ Cells that could potentially be seen in a CSF include gran-
analysis. Synovial fluids are additionally evaluated for ulocytes (mature and immature), lymphocytes (mature
crystals. Total cell count is performed manually in a and/or reactive), mononuclear phagocytes (monocytes,
hemacytometer. histiocytes, and macrophages), plasma cells, ependymal
@ Body fluid slides for morphological analysis are prepared by and choroidal cells, leukemic blasts, and malignant cells.
cytocentrifugation. This method markedly improves the The cells can appear comparable to those in peripheral
quality of the cell morphology obtained but may create arti- blood with some variation in details.
facts. These slides are used for the WBC differential count gw A definitive sign of CNS hemorrhage is erythrophagocy-
and for detection of any reactive or malignant cells. tosis (the phagocytosis of erythrocytes by histiocytes or
mwCells that can be found in body fluids include WBCs, macrophages).
RBCs, tissue cells, and in some cases tumor cells. The # Normal synovial fluid is transparent, colorless, viscous,
most common types of cells encountered are neutrophils, and does not clot.
lymphocytes, monocytes, and tissue cells. Other cells that
are less frequently seen are eosinophils, basophils, and w The analysis of synovial fluid provides important informa-
mast cells. tion in the diagnosis of inflammatory and degenerative
joint diseases.
m The general features of malignant cells are: multilayered
formations; three-dimensional spheroidal arrangements; m For routine analysis, the aspirated fluid should ideally
large cell size (often greater than 50 um diameter); an be divided into four samples: nonadditive (plain)
irregular nuclear membrane; multinucleation; nuclear evacuated tube; sterile, heparinized tube; sodium
molding; Indian file arrangements nuclear hyperchroma- heparin-anticoagulated evacuated tube; liquid EDTA-
sia, unevenly distributed chromatin; prominent nucleoli anticoagulated tube.
with irregular size and shape; high/variable N:C ratio; m The microscopic examination of synovial fluid is crucial
increased/abnormal mitotic activity; bizarre vacuolation; to determining the presence of crystals and leukocytes.
abnormal inclusions; cannibalism; uneven staining of Every synovial fluid sent to the laboratory for cell counts
cytoplasm. must be examined for crystals. Evaluation should be per-
m CSF is collected into each of three to five sequentially formed on a wet preparation as well as a cytocentrifuge
numbered, sterile, nonadditive tubes. The tubes must be preparation. If sufficient numbers of crystals are present,
filled in numerical order. they can be seen with plain light microscopy on the
Wright-stained slide.
m@CSF analysis must occur within | hour of collection;
remaining fluid is refrigerated immediately; CSF requir- = The most common types of crystals that may be present in
ing microbiological analysis must be incubated at 37°C. synovial fluid include monosodium urate (MSU), calcium
pyrophosphate dihydrate (CPPD), basic calcium phos-
gwA normal CSF is clear, colorless, sterile, and watery in
phates (BCP), oxalates, cholesterol and lipids, and steroids.
consistency.
@ The presence of MSU crystals is pathognomonic for gout.
mGrossly bloody specimens may result from a traumatic
MSU crystals are typically long, thin, and needle-like
lumbar puncture (LP) or the presence of subarachnoid or
with pointed ends. MSU crystals are strongly birefringent.
intracerebral hemorrhage.
continued
 Chapter 30 Body Fluid Examination: Qualitative, Quantitative, and Morphologic Analysis TEST

m CPPD crystals are characteristically present in a group of Cholesterol crystals have characteristically large, rectan-
disorders known as CPPD deposition disease (CPDD), gular notched-plate shapes and are strongly birefringent.
also known as pseudogout or chondrocalcinosis. CPPD
g Lipid crystals are round when seen by light microscopy.
crystals typically appear as short rectangular shapes, but
With polarized light, the crystalline forms appear as
they may also assume multiple three-dimensional forms
strongly birefringent Maltese crosses.
such as rods and rhomboids. CPPD crystals are more eas-
ily seen with light microscopy; they are only weakly bire- m Patients with OA and RA may occasionally receive treat-
fringent. ment with corticosteroid injections into the joint cavity or
surrounding connective tissue.
= The BCP crystals are a group of compounds consisting
mainly of hydroxyapatite (HA). @ Steroids do not have characteristic crystalline shapes.

g HA crystals are not pathognomonic for any specific disor- @ Septic, inflammatory, and noninflammatory effusions are
der, but they are associated with arthritic syndromes classified by the quantity and types of leukocytes present;
such as RA and OA. HA crystals are too small and too the crystal-induced joint diseases are characterized by the
weakly birefringent to be identified by polarized light presence of crystals.
microscopy. @ A normal fluid will have less than 200 WBC/uL and does
= Calcium oxalate crystals may be found in the synovial not contain red blood cells; the concentration of RBCs is
fluid of patients with primary oxalosis, a rare inborn error rarely of clinical significance unless there is a need to
of metabolism. determine if a traumatic tap has occurred.

g Cholesterol crystals can be present in chronically inflamed

joints, and are not diagnostic of any particular disorder.

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 Chapter

Hematology Methods
Virginia C. Hughes, MS, MT(ASCP)SBB, CLS(NCA)I

Introduction OBJECTIVES
Specimen Collection At the end of this chapter, the learner should be able to:
Patient Identification
Safety if Calculate a manual white blood cell count, red blood cell count, and platelet count.
Verification of Laboratory 2 . Calculate a percentage of reticulocytes and an absolute/corrected reticulocyte count.
Requisitions
Labeling the Blood 3 . Calculate a reticulocyte production index (RPI).
Specimen . List errors that could occur in the performance of a centrifugal microhematocrit
Specimen Accessioning determination.
Method 1. Venipuncture
. Caiculate the mean corpuscular volume, mean corpuscular hemoglobin, and a mean
Method 2. Capillary Blood
corpuscular hemoglobin concentration.
Collection
Unopette Technology— . Identify factors that affect the erythrocyte sedimentation rate.
Manual Counts . Identify hemoglobins separated at alkaline and acid pH in hemoglobin electrophoresis.
Method 3. Red Blood Cell
. Name a method for quantifying hemoglobin A, and hemoglobin F.
Counts
Method 4. White Blood Cell . Describe the differential solubility test for hemoglobin S.
Counts
. Describe a procedure for detecting Heinz bodies.
Method 5. Platelet Counts
Evaluation of Peripheral . Identify conditions that show increased and decreased osmotic fragility.
Blood Smear . Describe the basic principle for the Ham’s test and the sugar water test for
Method 6. Slide Preparation paroxysmal nocturnal hemoglobinuria.
and Wright Stain
Method 7. The White Blood . Explain the basic principle of the cyanmethemoglobin method for hemoglobin
Cell Differential determination.

Methods Used in Detection . Calculate a correct white blood cell count for a peripheral blood smear containing
and Monitoring of more than 10 nucleated red blood cells per 100 white blood cells.
Anemia . Differentiate between a venipuncture and capillary puncture.
Method 8. Hemoglobin
Determination . Identify the appropriate site for capillary puncture on an infant.
Method 9. Microhematocrit
. Compare and contrast hereditary spherocytosis and pyruvate kinase deficiency in the
Determination autohemolysis test.
Method 10. Red Blood Cell
Indices
Method 11. Reticulocyte
Counts
Method 11A. Reticulocyte
Counts Using the Miller
Disc
Standard Methods for
Specific Anemias
Method 12. Sickle Screen
Method 13. Helena SPIFE®
Alkaline Hemoglobin
Electrophoresis
159
760. Chapter 31 Hematology Methods

Method 14. Helen SPIFE®

Acid Hemoglobin
Electrophoresis
Method 15. Hemoglobin A,
Determination
Method 16. Isoelectric
Focusing
Method 17. Hemoglobin F
Acid Stain
Method 18. Screening Test
for Glucose-6-Phosphate
Dehydrogenase
Deficiency
Method 19. Staining for
Heinz Bodies
Method 20: Screening
Method for Detection
of Red Cell Pyruvate
Kinase
Methods to Detect Red Cell
Membrane Disorders
Method 21. Sugar Water
Test
Method 22. Ham’s Test
Method 23. Osmotic
Fragility
Method 24. Autohemolysis
Test
Nonspecific Tests of
Inflammation
Method 25. The Westergren
Erythrocyte Sedimentation
Rate
Method 26. The Automated
Streck ESR-100
Case Study

Introduction used most often in routine hematology methods, excluding

coagulation analysis, is ethylene diaminetetraacetic acid
Standard methods in hematology are a heterogeneous collec- (EDTA). There are many preanalytical steps to follow in the
tion of tests that are utilized in the hematology laboratory. process of blood collection, such as patient identification, requi-
Some of these methods may be performed on a routine basis, sition verification of laboratory orders, and biohazard safety, to
while others may serve as a backup method when automation name a few. These steps, as well as venipuncture and capillary
is not available or as a specialized test to confirm a particular puncture methodology, are discussed here.
type of anemia. The results of hematology tests aid the clini-
cian in diagnosing a plethora of hematologic disorders.
Patient Identification
HOSPITALIZED PATIENTS
Specimen Collection Hospitalized patients wear a wristband with the following
information:
Blood collection for hematologic studies can be performed via
venipuncture (blood collected from a vein) of by capillary punc- 1. Patient’s first and last name, middle initial
ture (blood collected from the heel or finger). The anticoagulant 2. Hospital number
 Chapter 31 Hematology Methods 76]

3. Date of birth or age patient’s results can be detrimental and sometimes fatal.
4. Gender Many hospitals and laboratories have implemented preprinted
5. Name of physician patient labels that contain the information in Table 31-1. An
6. Location or room number example of a patient label is depicted in Figure 31—1. There
All of this information should be cross-checked with the lab- are many varieties of barcode printers. One example is the
oratory requisition form or laboratory orders for each patient. A-50 by Argox (Fig. 31-2). The A-50 is a direct thermal
printer compatible with 128 and Codabar.* The tubes in which
If the patient is not wearing a wristband, a nurse should be
blood is collected should be labeled after the blood is col-
notified to verify patient identification.
lected and not before; this alleviates labeling another tube if
OUTPATIENTS
blood cannot be collected successfully on the first venipunc-
ture. If preprinted labels are not available, the patient’s first
For outpatients who have blood collected, the phlebotomist
and last name and social security number should be written
should ask the patient to recite his or her full name, address,
indelibly on the tube of blood.
social security number, and date of birth. The phlebotomist
must check this information with the laboratory requisition
form or physician’s record of laboratory orders. Specimen Accessioning
Safety Once the specimen has been collected, it is accessioned and
delivered to the hematology or appropriate department of the
Universal precautions should be in practice at all times when
collecting blood specimens.' This means that all specimens
should be handled as if they were infected with an infectious AGE, GENDER DEPT y PATIENT ID#
agent (e.g., human immunodeficiency virus). Such safety pre- PATIENT —DOEJOHN M51YM ER 123450 02/2299
—— DATE OF DRAW
cautions include wearing a new pair of latex gloves for each
patient, washing hands between patients, and disposing of
needles, and contaminated supplies in a puncture-resistant
UNIQUE BAR CODE
biohazard container (sharps).

Verification of Laboratory Requisitions ah STAT aN CBCW/DIFF SHI) R

With each request for blood collection and laboratory analysis

there must be a laboratory requisition that is either written or \. PHYSICIAN
contained in the laboratory information system (LIS). Infor- LAB ID PRIORITY BLOOD TUBE TEST ORDERED
mation contained on the requisition is outlined in Table 31-1.
The phlebotomist needs to verify that the patient information
Figure 51-1 ™ Example of a specimen label with bar code.
on the requisition form or specimen label is correct before
blood is collected. He or she should be aware of the appropri-
ate anticoagulants for the tests being performed as well as the
volume of blood required.

Labeling the Blood Specimen

A properly labeled specimen is essential to patient care. The
consequence of a patient being treated based on another

Patient’s full name

Age of patient
Social security number
Hospital number I
PN i
if
Date/time of collection
Physician’s name
Accession or specimen number
Location where order was placed
Ordered tests Figure 351-2 @ Argox A-50 Barcode printer. (Courtesy of Argox
USA, Ventura, CA.)
762 Chapter 31 Hematology Methods

laboratory to be analyzed. In specimen accessioning, the _ Assemble necessary venipuncture equipment (Fig. 31-4).
information on the printed label is transmitted to the LIS, _Apply tourniquet 3 to 4 inches above phlebotomy site.
Oo
Ww

which can be done manually or with the aid of abarcode scan- Proper application of tourniquet is illustrated in Figure 31—5.
ner (Fig. 31—3).* The patient identifiers as well as the time the . Locate the vein using the index and middle fingers and
specimen was collected and received in the laboratory are gently palpate the vein The most common vein entered
permanently entered into the LIS as well as the initials of is the median cubital vein (Fig. 31-6). Remove the
the phlebotomist. tourniquet.
5. Cleanse the venipuncture site using 70% isopropyl alcohol.
Method 1: Venipuncture Cleansing is done using a circular motion, starting at the
inside of the venipuncture site to the outside area.
The most common anticoagulant used in the hematology lab- 6. Once the alcohol has dried the tourniquet is reapplied to
oratory is EDTA and the most common technique for obtain- the patients arm as illustrated in Figure 31—5. The plastic
ing the specimen is venipuncture using evacuated tubes, which cap of the needle is removed and fastened to the vacu-
allow for collection of multiple tubes. tainer holder. The phlebotomist’s nondominant hand
holds the skin taut while the dominant hand holds the nee-
Principle dle apparatus using the thumb, index, and middle fingers.
After aseptic cleansing of the antecubital fossa, a vacutainer The needle is inserted bevel up using an angle of 15 to
needle is inserted into the patient’s vein. An evacuated tube is 30 degrees.* Once the vein is entered, the evacuated tube
placed into the vacutainer holder breaching the rubber sheath is pushed completely into the tube holder. Allow tubes to
needle which facilitates flow of blood. fill completely because “short draws” may lead to erro-
neous results. The tourniquet is removed before the tube
Specimen or all tubes are filled. Remove the last tube from the
EDTA-anticoagulated blood needle holder and invert all tubes several times for proper
mixing. Place a sterile gauze pad over the site and remove
Equipment the needle in a quick motion. Place the clean gauze pad on
Vacutainer tubes the venipuncture site and ask the patient to hold it on his
Tourniquet or her arm.

Vacutainer holder
Alcohol pad
Vacutainer needle
A sterile
Adhesive bandage multiple-use needle

Procedure
1. Properly identify patient by cross-checking unique identi-
fiers (e.g., social security number) found on phlebotomy req-
uisition form with unique identifiers on patient wristband.
A holder

An evacuated
glass tube

Figure 31-4 @ Evacuated tube system consisting of a double-

pointed needle, a plastic holder, and a vacuum tube with a rubber
Figure 351-3 ™ Argox AS-8250 CCD Barcode Scanner. (Courtesy of stopper. (From Wedding, ME, and Toenjes, SA: Medical Laboratory
Argox USA, Ventura, CA.) Procedures. FA Davis, Philadelphia, 1998, p 95, with permission.)
 Chapter 31 Hematology Methods 763

Method 2: Capillary Blood Collection

Principle
Using a standard skin puncture device, an aseptic area is
punctured approximately 2.0 mm deep and proximal area of
skin is massaged to facilitate adequate blood flow.

Specimen
EDTA anticoagulated blood or capillary blood

Equipment
Microtainer tubes
Alcohol pad
Automated puncture device
Adhesive bandage

Procedure
|. Properly identify patients by cross-checking unique identi-
fiers on requisition form with those found on patient wrist-
3 4 band or ankle band (infants).
2. Cleanse the selected site with 70% isopropyl alcohol.
Figure 351-5 @ Application of the rubber tourniquet. (From Perform this task using a circulation motion and allow to
Wedding,
L ME,t and Toenjes, SA: Medical; Laboratory Procedures, air dry. For infants, the most common site used is the
FA Davis, Philadelphia, 1998, p 102, with permission).
plantar surface of the heel (Fig. 31—7) and for pediatrics,
adolescents, and adults the third and fourth fingers on
7. Label the blood tube or tubes. This inust be done prior to the palmar side fleshy surface of the distal phalanx
leaving the patient’s side. Most blood specimens are (Fig. 31-8).
labeled with printed bar code labels (see Fig. 31-1) con-
taining patient demographic and test information.
8. Apply an adhesive bandage to the patient’s arm and deliver HEEL PUNCTURE SITES
specimens to the appropriate laboratory department.

Cephalic vein
Basilic vein

Median
cubital vein
Accessory
cephalic vein
Basilic vein

CALCANEOUS
Cephalic vein (HEEL BONE)
Median
antebrachial vein

(9) PUNCTURE ZONE

Figure 51-7 @ Acceptable heel puncture sites for a dermal punc-
Figure 31-6 @ The path of veins from the arm. (From Wedding, ture. (From Strasinger, SK, and DiLorenzo, MS: The Phlebotomy
ME, and Toenjes, SA: Medical Laboratory Procedures. FA Davis, Workbook, ed 2. FA Davis, Philadelphia, 2003, p 192, Fig. 16-5,
Philadelphia, 1998, p 100, with permission.) with permission.)
764 Chapter 31 Hematology Methods

Unopette Technology—Manual Counts

The Unopette system of manual cell counting has replaced the
antiquated Thoma pipet method. Manual cell counts are per-
formed on a hemacytometer counting chamber (Fig. 31-10),
which is constructed so that the distance between the bottom
NO
(White Area) of the coverslip and the surface of the counting chamber is
0.1 mm (Fig. 31-11). The surface of the chamber contains
two square-ruled areas separated by an H-shaped moat. These
two squares are identical, allowing the technologist to dupli-
cate cell counts. Each has a total of 9 mm? (3 mm on each
side).5 These squares are divided into nine primary squares,
each with an area of | mm? (1 mm on each side). The four cor-
ner primary squares are used when counting leukocytes. These
four corner primary squares are further divided into 16 smaller
secondary squares, each with an area of 0.04 mm’. The four
corner and center secondary squares of the center primary
square are used to count erythrocytes. All 25 secondary
Figure 31-8 m Acceptable finger puncture sites and correct
puncture angle. (From Strasinger, SK, and DiLorenzo, MS: The squares of the center primary square are used to count platelets,
Phlebotomy Workbook, ed 2. FA Davis, Philadelphia, 2003, p 269, and each of the 25 squares is further divided into 16 smaller
Fig. 16-6, with permission.) tertiary squares (see Fig. 31-10). Traditionally, glass hemacy-
tometers have been used for manual blood counts. Recently,
3. For the heel stick, using the nondominant hand, grasp the InCyto (Seoul, Korea) has developed the C-Chip disposable
heel between the thumb and index finger. For the finger hemacytometer made of thermoplastics for manual cell
stick, grasp between the thumb and index finger. Gentle counting (Fig. 31-12). The C-Chip consists of two surface
massaging may be performed on both types of sticks to patterned enclosed chambers with two ports for sample injec-
increase blood flow to the chosen puncture site. Place the tion. The grids consist of nine large squares, with each square
automated device firmly on the area to be punctured and measuring | X | mm for a total 3 X 3 mm counting area. The
arm the trigger device. After the puncture has been made
place the used blade in an appropriate biohazard sharps
container. Wipe the first drop of blood with a sterile gauze 3: Omm

pad. Position the Microtainer ™ tube (Fig. 31-9) directly

esa iiiiim ee
beneath puncture site and proceed to fill the tube by touch-
ing the tip of the Microtainer™ to the drop of blood. When
eT
|
1.0mm
tube is filled, remove the tip and secure the cap on the

= ea
Microtainer™ tube. Invert tube six to seven times to allow
proper mixing with anticoagulant.
4. Label Microtainer ™ tube appropriately and deliver the
specimen to respective laboratory department.

innHIMHIN
yun IAA

see! ae
MMs
Figure 31-10 @ Spencer Bright-Line double counting system with
improved Neubauer ruling. This represents an enlarged view of one
of the two ruled squares of the hemacytometer. The four corner pri-
mary squares are used for counting white blood cells. The arrows in
the upper left corner square represent the suggested counting
pathway of cells. Five secondary squares (labeled RBC) of the center
primary square are used for counting red blood cells. In platelet
enumeration, all 25 squares of the center primary square are
counted. (From Wedding, ME, and Toenjes, SA: Medical Laboratory
Figure 31-9 @ Microtainer tube containing EDTA.
Procedures. FA Davis, Philadelphia, 1998, p 277, with permission.)
 Chapter 31. Hematology Methods 765

HEMACYTOMETER

Side View

Figure 31-11 m@ Hemacytometer, side view.

depth of each chamber is 0.10 mm. An automated reader for

the C-Chip, the C-reader, is also available from InCyto for
optical measurements.
The boundary lines of the central primary square are
either double or triple. When the boundary line is double, all SSA

the cells within the square and those touching the innermost
line are counted. If the boundary line is triple, all of the cells Figure 31-13 @ Unopette system, consisting of a reservoir
containing a diluent, a pipet used to deliver the specimen, a
within the squares and those touching the middle line inward
reservoir, and a pipet shield used to puncture the plastic seal on
are counted. reservoir, and serve as a cap for the pipet to prevent evaporation.
Hemacytometers and coverslips should meet the specifi-
cations of the National Bureau of Standards and are so Specimen
marked by the manufacturer. A standardized coverslip must EDTA-anticoagulated blood
be used that has been ground to fit the specifications of the
hemacytometer, ensuring a uniform depth and, therefore, a Equipment
constant volume. An ordinary coverslip must not be used. Unopette reservoir No. 5851
Manual cell counts are most often performed in cases of
Sodium chloride 8.5 ¢g
thrombocytopenia.
The Unopette system consists of calibrated pipets, Sodium azide O.l g
reagent-filled reservoirs to ensure precise dilutions, and a Distilled water 1000 mL
pipet shield to puncture the Unopette reservoirs (Fig. 31-13). Capillary pipet (10-uL)
Hemacytometer and coverslip
Method 3: Red Blood Cell Counts Microscope

Principle Hand counter

EDTA-anticoagulated blood or capillary blood is pipetted into Petri dish lined with moist filter paper
a reservoir diluting vial containing sodium chloride, sodium
azide, and distilled water. The diluted sample is loaded onto a Procedure
hemacytometer for red blood cell enumeration. 1. Draw venous or capillary blood into 10-uL capillary pipet.
2. Using a pipet shield, puncture the reservoir. Load the
sample into the reservoir vial by squeezing the vial with
one hand and plugging the top of the capillary tube with
the index finger of other hand. As the capillary tube is
inserted and tightened onto the reservoir, release the grip
on reservoir to facilitate blood being drawn into reservoir
(Fig. 31-14A—D).
3. Place an index finger again on top of the capillary tube and
squeeze the reservoir gently to mix the sample with
reagents. Incubate for 10 minutes at room temperature.
4. Charge the hemacytometer by converting the capillary tube
to a dropper assembly (Fig. 31-15). Discard the first three
to four drops of the sample (Fig. 31-16). Carefully charge
the hemacytometer by gently squeezing the reservoir vial.
5. Place the hemacytometer in a moist Petri dish and allow
cells to settle for 10 minutes.
6. Place the hemacytometer on the microscope stage. Switch
to the 10X objective and lower the condenser. Once cells
Figure 31-12 @ InCyto C-Chip disposable hemacytometer. are in focus switch to the 40X objective.
766 Chapter 31 Hematology Methods

Figure 351-16 ® Charging of hemacytometer by Unopette inver-

sion of reservoir dropper assembly. The first three or four drops are
discarded before hemacytometer is loaded. (From Wedding, ME,
and Toenjes, SA: Medical Laboratory Procedures. FA Davis,
Philadelphia, 1998, p 236, with permission.)

7. Count RBCs in the four corner squares and center

square of the primary center square of hemacytometer
(Fig. 31-17). Count all cells that touch any of the upper
and left lines; to avoid duplicate counting of cells do not
count any cell that touches a lower or right line. Repeat
the count using the other side of hemacytometer and
Figure 51-14 ™ Schematic for using Unopette system. A. Using average results.
shield of capillary pipet, puncture diaphragm of reservoir. B. Fill capil-
lary with sample from fingerstick or venous blood specimen. C. Trans-
8. Calculate the RBC count using the following formula:
fer sample to reservoir by squeezing reservoir slightly to force out No. of RBCs X Depth factor X Diluting factor
some air. Do not expel any liquid. Cover opening of over flow cham- Fe = = RBCs/mm°*
ber with index finger. Maintain pressure until pipet is secured in Area
reservoir neck. Squeeze reservoir several times to rinse capillary bore
The depth factor is 10 and the dilution is 200. The area
without expelling any liquid. D. Place index finger over upper open-
ing and gently invert several times to thoroughly mix sample with counted is 0.2. This can be formulated to a factor of 10,000;
diluent. (From Wedding, ME, and Toenjes, SA: Medical Laboratory therefore if the average count is 600. The RBCs per mm*
Procedures. FA Davis, Philadelphia, 1998, p 235, with permission.) 6.0 X 10°/mm? or 6.0 X 10!7/L.

Limitations
The hemacytometer must be properly filled to avoid erro-
neous results.

Figure 51-15 @ Unopette conversion to dropper assembly by Figure 31-17 @Red blood cells are illustrated at <400 using
withdrawing pipet from reservoir and securing it in reverse position Unopette methodology.
 Chapter 31 Hematology Methods 767

Reporting Results Limitations

RBCs may be expressed as RBCs/mm}? or RBCs X 10!2/L. To The hemacytometer must be properly filled to avoid erroneous
convert mm? to 10'*/L multiply by a factor of 10°. results caused by underfilling or overfilling. A markedly high
leukocyte count may make accurate counting difficult, and a
Normal Values secondary dilution should be made.
Newborn AllS) 8 S€ {OVPAL,
Infant/child Zesousy IO} AL, Reporting Results
Adult men: ALTE6 dl SK AOL, White blood cell count is reported in thousands per uL.

Adult women: AlSal SS OYA

Normal Values
Newborn 9000-30,000/uL 9.0-30.0 X 10°/L
Method 4: White Blood Cell Counts 1 week 5000-2 1,000/uL 5.0-21.0 10°/L
Principle 1 month 5000—19,500/uL 5.0-19.5 10°/L
Free-flowing capillary or well mixed anticoagulated venous 6—12 months 6000—17,500/uL 6.0-17.5 X 10°/L
blood is added in a diluent at a specific volume in the 2 years of age 6200—17,000/uL 6.2-17.0 * 10°/L
Unopette reservoir. The diluent lyses the erythrocytes but
Child/adult 4800—10,800/uL 4.8-10.8 * 10°/L
preserves leukocytes. The diluted blood is added to the hema-
cytometer chamber. Cells are allowed to settle for 10 minutes
before leukocytes are counted.
Method 5: Platelet Counts
Specimen Principle
EDTA-anticoagulated blood Free-flowing capillary or well-mixed anticoagulated venous
blood is added to a diluent at a specific volume in the
Equipment Unopette reservoir. The diluent lyses the RBCs but preserves
Unopette reservoir no. 5854/5855 (1.98 mL of diluent) the platelets. The diluted blood is added to the hemacytome-
Ammonium oxalate 11.45 ¢ ter chamber. Cells are allowed to settle for 10 minutes before
platelets are counted.
Sorensen’s phosphate buffer 1.0 g
Thimerosal 0.1 g
Specimen
Purified water qs to 1000 mL EDTA-anticoagulated blood
Unopette capillary pipet, 20-uL
Hemacytometer and coverslip Equipment
Microscope Unopette reservoir no. 5854/5855 (1.98 mL of diluent)

Lint-free wipes Ammonium oxalate 11.45 g

Alcohol pads Sorensen’s phosphate buffer 10¢

Hand counter Thimerosal O.l g
Petri dish lined with moist filter paper Purified water qs to 1000 mL
Unopette capillary pipet, 20-uL capacity
Procedure Hemacytometer and coverslip
1. Puncture the diaphragm of the Unopette reservoir and add
Microscope
sample using a 20-uL capillary pipet (see Fig. 31-14).
2. Let diluted blood mixture stand 10 minutes to allow RBCs Lint-free wipe
to lyse. Alcohol pads
3. Clean the hemacytometer using alcohol pad and wipe with Hand counter
lint-free paper. Charge the hemacytometer.
Petri dish lined with moist filter paper
4. Place hemacytometer in a Petri dish lined with moist filter
paper and let stand for 10 minutes to allow cells to settle.
5. Under 10X magnification, count WBCs in four large Procedure
squares of hemacytometer (see Fig. 31-10). Repeat count 1. Puncture diaphragm of Unopette reservoir and add sample
on opposite side of chamber and average results. using a 20-uL capillary pipet (see Fig. 31-13).
6. Total WBC/mm? = (No. of WBCs X 10 X 100)/4, where 2. Let the diluent—blood mixture stand 10 minutes to allow
10 is the depth factor and 100 is the dilution factor. For RBCs to lyse.
example, if the average number of WBCs is 50, the num- 3. Clean the hemacytometer with an alcohol pad and
ber of WBCs per mm’ is 12.5 X 10°; the number of WBCs wipe dry with a lint-free wipe. Charge both sides of the
per liter is 12.5 X 10”. hemacytometer.
768 Chapter 31 Hematology Methods

4. Place the hemacytometer in a Petri dish lined with moist fil- stain, is the most common dye. A well prepared blood smear
ter paper and let sit for 10 minutes to allow platelets to settle. provides valuable information concerning the patient’s hemato-
5. Under 40X magnification using bright-light or phase logic status and is a vital component of the CBC (complete
microscopy, platelets are counted in all 25 small squares blood count) in the event a slide preparation is warranted.
within the center primary square (see Fig. 31-10). Count both
sides of the hemacytometer and average the results. Platelets Principle
appear as dense, dark bodies and can be round, oval, or rod- Blood from an EDTA-anticoagulated specimen or capillary
like, sometimes showing dendritic processes (Fig. 31-18). blood is applied to a standard glass slide using the slide-
6. Multiply the average number of platelets counted on both to-slide method. The blood smear is then stained using Wright
sides of hemacytometer by 1000 to get total platelet stain. Blood smears are fixed using methanolic fixative solu-
count/mm*. This step simplifies multiplication of dilution tion to stabilize cellular components. Eosin Y is an anionic
factor (100) and depth factor (10), which is equal to 1000. dye that stains cytoplasm pink to yellowish-red and methyl-
For example, if an average of 200 platelets are counted, the ene blue, which stains the nucleus and RNA blue.°
number per mm? is 200,000, while the number per liter is
200 X 10°.
Specimen
EDTA-anticoagulated blood
Limitations
A delay in mixing blood sample (anticoagulated sample) may
result in platelet clumping and subsequent erroneous results. Reagents/Equipment
The hemacytometer must be properly filled; underfilling or Clean glass slides
overfilling may lead to erroneous results. In the event of Diff-Quik® Stain Set®
platelet satellitosis (neutrophils ringed with adhesive platelets), Fixative Solution: 1.8 mg/L of triaylmethane dye in
obtain correct platelet counts by collecting a fresh specimen methyl! alcohol.
with sodium citrate as the anticoagulant.
Solution I: 1 g/L of xanthene dye, buffer and sodium
Reporting Results azide 0.01%
Results are expressed as number of platelets per uL or mm* Solution II: 1.25 g/L of thiazine dye mixture
(0.625 g/L of azure A and 0.625 g/L of
Normal Values methylene blue) and buffer
150,000—450,000/uL 150-450 x 10°/L Coplin jars
Immersion oil
Evaluation of the Peripheral Blood Smear
Procedure
Method 6. Slide Preparation 1. Place a small drop of EDTA-anticoagulated blood or
and Wright Stain capillary blood on the end of a clean glass slide
(Fig. 31-19A).
The wedge blood smear is the most common smear preparation
in the hematology laboratory and Wright stain, a Romanowsky
2. Place a second slide (spreader) in front of the drop of blood
at a 30 to 45 degree angle (Fig. 31-198).
3. Draw the spreader slide into the drop of blood and push the
spreader slide forward, spreading blood across the length of
the first slide. The smear at the opposite end of where the
drop of blood was placed should have a feathered edge
appearance (Fig. 31-19D). An example of an improperly
made blood smear is illustrated in Figure 31—19E. An unac-
ceptable unstained blood smear is shown in Figure 31-19C.
Acceptable and unacceptable stained smears are shown in
Figure 31-19F.
4. Discard the spreader slide into an appropriate biohazard
sharps container.
5. Gather 3 Coplin jars. Label as follows: fixative, solution I,
solution II.
6. Dip slide in Diff-Quik® fixative solution five times for
about | second each. Allow excess to drain.
7. Dip slide in Diff-Quik® solution I five times for about
Figure 351-18 ™ Photograph of platelets loaded onto a hemacy- | second each. Allow excess to drain.
tometer using a x40 objective (phase contrast). The platelets
appear as dense, refractile, dark bodies that can be round, oval, or Dip the slide in Diff-Quik® solution II five times for about
rod shaped with a diameter of approximately 2 to 4um. 1 second each. Allow excess to drain.
 Well-made peripheral blood smear

4
vad

Unacceptable peripheral blood smea

Figure 31-1 A. A drop of blood is placed on glass slide. B. A spreader slide is situated at a 30- to 45-degree angle. C. Draw back the
slide against the drop of blood and spread the blood smoothly. D. Adequate blood smear with feathered edge. £. Unacceptable blood smear.
F. Acceptable and unacceptable stained smears.
770. Chapter 31 Hematology Methods

Limitations
Only precleaned slides should be used for staining blood
smears. Characteristic staining patterns are described in
Table 31-2.
A popular automated slide stainer used in the hematol-
ogy laboratory is the Hema-Tek Slide Stainer (Fig. 31—20).
This automated slide stainer has positions for 25 slides on a
metal platen. Slides are stained at a rate of one slide per
minute. This instrument has excellent reproducibility and is
nearly maintenance free. The Wright’s stain is contained in a
“stain-pak” from a variety of manufacturers and sits within
the instrument connected by stylets and tubing. The Wright
stain is triggered to pump and deliver a precise amount of
solution to the slide via capillary space on the instrument.
Once stained, the slide is rinsed and dried on the platen and
eventually drops into a separate compartment.

Method 7. The White Blood Cell

Differential oo
‘ secon cnt hese ANSE SN NES RECONelt

Principle
One hundred white blood cells are counted and classified

on a Wright-stained peripheral blood smear using 1000x Pigure 351-20 m The Hema-Tek Slide Stainer and Stain-Pak.
magnification. (Courtesy of the Bayer Corporation, Tarrytown, NY.)

Reagents/Equipment
Microscope
Differential counter a. Scan the edges using low power (10X objective) and
Hand counter center of the slide to be sure there are no clumps of
RBCs, WBCs, or platelets.
Procedure b. Scan the edges for abnormal cells.
1. Determine the overall staining quality of the blood smear. 3. Find an optimal area for the detailed examination and enu-
2. Determine if there is a good distribution of the cells on the meration of cells.
smear. a. The RBCs should not quite touch each other.
b. There should not be areas containing large amounts of
broken cells or precipitated stain.
c. The RBCs should have a graduated central pallor.
4. Switch to high power (40X objective). Determine the
WBC estimate.
: ott a. Count the number of white blood cells in ten fields and
GalalacCanctitient divide by 10. Multiply this number by 2000. This factor is
peg SIRE ea based on the fact that each WBC seen in a high power field
ee Sones (hpf) is equivalent to approximately 2000 cells per uL.
Picts roePee Correlate the WBC estimate with the WBC counts per
MEAG oi ese oT ; mm’. Other methods correlate the average number of
Metamyelocyte cytoplasm i WBCs/hpf with a range of WBC counts from a previously
Neutrophil cytoplasm i established table found in the procedure manual used in
Lymphocyte cytoplasm the laboratory.
ee pte 5. Perform a 100 WBC differential count.
Bone Pink Evaluate RBC anisocytosis, poikilocytosis, hypochroma-
Basophilic granules Purple-black sia, polychromasia, and RBC inclusions (see Chap. 5).
Neutrophilic granules Pink-purple 6. Perform a platelet estimate and evaluate platelet
Platelet granules Pink-purple morphology.
fo cece pee purple a Count the number of platelets in 10 oil immersion fields.
Howell—Jolly bodies Purple b. Divide by 10.
Cabot rings Pink-purple c. Multiply by 20,000/mm? if a wedge smear is
used; multiply by 15,000 if it is an automated smear.
 Chapter 31. Hematology Methods 771

Report platelet estimate as adequate, increased, or Drabkin’s reagent: Dissolve | g of sodium bicarbonate,
decreased. 0.2 g of potassium ferricyanide, 0.05 g of sodium
7. Correct any total WBC count per mm} that has greater than cyanide in distilled water, qns to | liter.
10 nucleated red blood cells (NRBCs) per 100 WBCs. Hemoglobin standard
a. When performing the WBC differential, do not include
NRBCs in the count; NRBCs are reported as the number Procedure
of NRBCs per 100 WBCs. 1. Acquire reference material for hemoglobin at an approxi-
b. Use the following formula to correct a WBC count: mate value of 20 g/dL. Make four dilutions of reference
Corrected WBCs/mm* = WBC/mm? X 100 material using Drabkin’s reagent so that hemoglobin values
100 + No. of NRBCs/100 WBCs are four to six increments apart (e.g. 6, 10, 16, and 20 g/dL
of hemoglobin). Mix the contents of tubes and analyze at
8. The absolute WBC count may be determined by multiply- 540 nm using a spectrophotometer. Plot hemoglobin values
ing the differential (relative) count (%) by the total WBC versus absorbance on standard graph paper.
count.’ For example, to determine the absolute count of 2. For patient testing, add 5 mL of Drabkin’s reagent to a
segmented neutrophils when 60% of segmented neu- clean test tube.
trophils are counted in the differential and the total WBC 1SS). Add 20 uL of EDTA-anticoagulated whole blood to the tube.
count is 10,000/uL: 4. Mix contents of tube and allow to stand at room tempera-
Absolute segmented neutrophils = 10,000/uL < 60%/100 = ture for 5 minutes. Read absorbance at 540 nm on a spec-
6000/uL trophotometer.
5. Determine patient hemoglobin using standard curve pre-
Normal Values pared in step 1.
Relative counts
Lymphocytes 20-44% Limitations
Drabkin’s reagent should be kept away from direct sunlight.
Monocytes 2-9%
Adding Drabkin’s reagent to samples containing hemoglobin
Neutrophils 50-70% C or S will yield a turbid suspension and will fail to lyse the
Bands 2-6% red cells. To circumvent this, add 5 mL of water to mixture
Eosinophils 04% and incubate at room temperature for 5 minutes. Read off of
the spectrophotometer and double the result.
Basophils 0-2%
Absolute counts Reporting Results
Lymphocytes 1.2-3.4 XX 10°/uL 1.2-3.4 XX 10°/L Results for hemoglobin are reported in units of g/dL.
Monocytes 0.11-0.59 XK 10°/uL 0.11-0.59 x 10°/L
Normal Values
Neutrophils 1.4-6.5 x 10°%/uL s1.4-6.5 10°/L Newborn: 17-23 g/dL
Bands 0-0.7 X 10?/uL 0-0.7 X 10°/L Infant child: 9-14 g/dL
Eosinophils 0-0.5 X 10°/uL 0-0.5 X 10°/L Adult men: 14-18 g/dL
Basophils 0-0.2 X 10°/uL 0-0:2 x 107/L Adult women: 12-16 g/dL

Methods Used in Detection Method 9: Microhematocrit Determination

and Monitoring of Anemia
Principle
Method 8: Hemoglobin Determination EDTA-anticoagulated whole blood or capillary blood is cen-
trifuged and the total packed red cell volume is expressed as
Principle a percentage of the whole blood volume.
Hemoglobin is oxidized to methemoglobin by the reagent
potassium ferricyanide or Drabkin’s reagent. Methemoglobin
Specimen
is converted to cyanmethemoglobin in the presence of potas-
EDTA-anticoagulated blood or capillary blood
sium cyanide. The absorbance of cyanmethemoglobin is mea-
sured at 540 nm.*
Reagents/Equipment
75 mm X 1.5 mm glass capillary tubes (nonheparinized
Specimen for EDTA anticoagulated blood; heparinized for
EDTA-anticoagulated blood
capillary blood).
Microhematocrit centrifuge (Fig. 31-21)
Reagents/Equipment
Spectrophotometer Hematocrit reader
Pipet Critseal
772. Chapter 31 Hematology Methods

Figure 31-22 m Securing the capillary tube with sealing clay.

(From Wedding, ME, and Toenjes, SA: Medical Laboratory Proce-
TIMER dures. FA Davis, Philadelphia, 1998, p 220, with permission.)
‘3 8 ; BRAKE
NNT
\\\ : Iy2
12> m
Ra 3 BEEASE SE WHEN
Wen HEAD
near StORe
ee
i— eed
- moet
0% ~ Normal Values
q :) fo A \\S ;
Newborn: 53-65%
B QDWONecOvsicn
RCMB CENTRIFUGE
OCHET
Infant child: 30-43%
Adult men: 42-52%
Adult women: 37-47%

Figure 31-21 ® Microhematocrit centrifuge. (Photo courtesy of

International Equipment Co.) (From Wedding, ME, and Toenjes, SA: Method 10. Red Blood Cell Indices
Medical Laboratory Procedures. FA Davis, Philadelphia, 1998,
p 220, with permission.) The RBC indices are calculations used to classify anemia.
The indices include the mean corpuscular volume (MCV),
Procedure mean corpuscular hemoglobin (MCH), and mean corpuscular
1. Fill the capillary tubes three-fourths of the way with hemoglobin concentration (MCHC). The MCV is a measure
EDTA-anticoagulated blood or capillary tube using appro- of the average volume of RBCs per liter and expressed in
priate tubes as described above. Samples should be run in femtoliters (10!>):
duplicate.
Isler <I
i). Cap the tubes with Critseal by plugging the dry end of the MCV = ——— = fL
RBC count/mm°*
tube with Critseal (Fig. 31—22).
3. Wipe the exterior of the tube free of blood and place in So that with a Hct of 44% and an RBC count of
microhematocrit rotor (Fig. 31-23), making sure the capillary 4.2 million/mm*, the MCV = 105 fL
tubes are balanced. Centrifuge for 5 minutes at 14,500 rpm.
This separates RBCs from plasma and leaves a band of
buffy coat consisting of WBCs and platelets at the interface
(Fig. 31-24).
4. Read the packed red cell volume of tubes using a microhe-
matocrit reader (Fig. 31-25). Duplicate samples should be
within 1%.

Limitations
If capillary blood is used, the first drop should be wiped away
in capillary blood collection to avoid contamination with
interstitial fluid, which will dilute out the sample.
The buffy coat should not be included in the packed red
cell volume reading.
If the patient is on chloramphenicol or penicillin, a
decreased hematocrit level may be found.’

Reporting Results
The hematocrit is reported as a percentage of red cells to Figure 31-235 ™ Microhematocrit rotor. (Photo courtesy of Inter-
total blood volume. national Equipment Co.)
 Chapter 31 Hematology Methods 773

The MCHC is a measure of the average hemoglobin

concentration per unit volume of RBCs and expressed as a
percentage:
— Capillary tube
Hgb x 100 _
MCHC =—=— %

So that with a Hgb of 14 g/dL and an Het of 42% the

— Plasma MCHC = 33%

Normal Values
MCV 80-94 fL
MCH 28-31 pg
— Buffy coat MCHC 32-36%

Interpretation
Determination of the MCV, MCH, and MCHC gives valuable
information that helps to characterize RBCs. According to the
— Red blood cells
MCV, erythrocytes may be classified as normocytic, micro-
cytic, or macrocytic. Based on the MCHC, erythrocytes may
be classified as normochromic or hypochromic (Table 31-3).
The MCH expresses only the mean weight of hemoglobin per
erythrocyte.

— Sealing clay RBC DISTRIBUTION WIDTH

The RBC distribution width (RDW) is the coefficient of vari-
ation of the red cell volume distribution. It is provided by
Figure 31-24 ™ Diagram of a centrifuged microhematocrit tube
automated hematology instruments and is used as an indica-
of whole blood. (From Wedding, ME, and Toenjes, SA: Medical
Laboratory Procedures. FA Davis, Philadelphia, 1998, p 220, with tion of anisocytosis. An elevated RDW may be seen on blood
permission.) smears with varying degrees of anisocytosis. This parameter
is useful in distinguishing iron-deficiency anemia (increased)
from the hemoglobinopathies (normal).
The MCH is a measure of the average hemoglobin con-
tent of the RBC and expressed in picograms (10'”): RDW = Standard deviation of RBC volume Xx 100
Mean MCV
Heb X 10 Normal values for RDW = 11.5% to 14.5%
oe RBC count/mm?

So that with a Hgb of 14 g/dL and an RBC count of 4.2 MEAN PLATELET VOLUME
million/mm*, the MCH = 33 pg. The mean platelet volume (MPV) represents the average size
of platelets. It is determined from the arithmetic mean of the
platelet histogram provided by the hematology analyzer (See
chapter 32). The normal values may range from 7.4 to 10.4 fL.
The MPV may be increased in idiopathic thrombocytopenic
purpura (ITP) and in hypoxic states of chronic obstructive pul-
monary disease (COPD).'°

MCV (fL) MCHC (%) Classification

80-100 32-36 Normocytic,

normochromic
<80 <3) Microcytic,
hypochromic
> 100 32-36 Macrocytic,
Figure 31-25 @ Microhematocrit reader. (Photo courtesy of Inter- normochromic
national Equipment Co.)
774 Chapter 31 Hematology Methods

Method 11. Reticulocyte Counts Reporting Results

The reticulocyte count is reported as a percentage. A corrected
Reticulocytes are immature RBCs that contain remnant cyto- reticulocyte count compares the patient hematocrit value with
plasmic ribonucleic acid (RNA) and organelles such as mito- a normal hematocrit value:
chondria and ribosomes. Reticulocytes are visualized by
Reticulocyte (%) X Patient Hct (%)
staining with vital dyes (such as new methylene blue) that
45(%)
precipitate the RNA and organelles, forming a filamentous
network of reticulum (Fig. 31-26). On Wright stain, the retic- For example, if a patient presenting with a reticulocyte
ulocyte appears polychromatophilic or as a macrocytic blue count of 10% with a hematocrit of 22%, the correct reticulo-
red cell. The reticulocyte is a means of assessing the erythro- cyte would be:
poietic activity of the bone marrow. 10% X 22%
= 4.9%
Principle 45%
Ribonucleoprotein present in young red cells (reticulocytes) The reticulocyte count can also be expressed as an
react with dyes such as new methylene blue or brilliant cresyl absolute reticulocyte count by multiplying the patient reticu-
blue, forming a blue precipitate. locyte count times the patient red blood cell count:
Specimen Reticulocyte count (%) * RBC count (10!7/L)
EDTA-anticoagulated blood 100
Reagents/Equipment For example, a patient with a reticulocyte count of 7%
1% New methylene blue or Brilliant Cresyl Blue and a RBC count of 2.5 x 10!’/L the absolute reticulocyte
Test tubes and pipets count would be:

Procedure To X25 702

100 =
[7S 1078
9

1. Add equal volumes of basic dye and EDTA-anticoagulated

whole blood to a test tube. Mix and incubate at room tem- The reticulocyte count can also be expressed as the RPI
perature for 5 minutes. (reticulocyte production index) which accounts for the presence
2. Prepare a blood smear and let air dry. of “stress or shift” reticulocytes in the peripheral blood. These
3. Using the oil immersion objective, count 1000 red cells. premature reticulocytes contain more filamentous reticulum and
Note the number of red cells that are mature (non-reticulo- result in a longer maturation time in circulation. The RPI calcu-
cytes) and the number that are reticulocytes. lation takes into account the corrected reticulocyte count as well
4, Determine percent reticulocyte count by the following as the maturation time of the reticulocyte based upon patient
formula: hematocrit (Table 31-4). The RPI is calculated as follows:
Number of reticulocytes < 100 Reticulocyte count (%) Patient Hct (%)/45(%)
1000 Red cells 2 (Stress reticulocyte maturation time at 25% Hct)

Limitations For example, a patient with a reticulocyte count of 12%

Samples should be analyzed with 72 hours of collection to and a hematocrit of 25% would yield an RPI of:
avoid falsely decreased counts." 79% X (25%/45%)
5 = 3.3

Normal Values
Reticulocyte Count
Newborn: 2.5-6.0%
Adult: 0.5-2.0%
Absolute reticulocyte count: 24-80 X 10°/L
RPI 3 or greater

Maturation Time

1 day
1.5 days
2 days
Figure 31-26 @ Photograph of a reticulocyte using new methyl- 3 days
ene blue stain.
 Chapter 31 Hematology Methods 775

Method 11A. Reticulocyte Counts Using Specimen

the Miller Disc EDTA-anticoagulated blood

Principle Reagents/Equipment
Using the Miller ocular disc, reticulocytes and mature Reaction vials prefilled with sodium hydrosulfite
red cells are separated and counted in separate squares of
Phosphate buffer
the disc. Using a factor of 9, reticulocytes can be easily
calculated. SickleScreen control set
Deionized water
Reagents and Equipment Tube reading rack
New methylene blue (NMB)
Miller ocular disc Procedure
Microscope slides 1. Bring all reagents to room temperature.
2. Label one reagent tube for each patient and control.
10 X 75 mm test tubes
3. Place tubes in reading rack and add phosphate buffer to
6-inch capillary tubes each tube until it reaches the “Fill to this level” mark.
Hand counter 4. Add 50 ub of patient sample and controls to respective
tubes.
Procedure 5. Incubate at room temperature for 10 to 20 minutes.
1. Dissolve 10 g of NMB and 8.9 of NaCl (0.152 mol/L) in
1000 mL of distilled water. Filter through Whatman No. | Interpretation
filter paper before use. Store at room temperature. Positive: If hemoglobin S or another sickling hemoglobin is
2. Add an equal number of drops of thoroughly mixed EDTA present the solution will be turbid and the dark lines of the
anticoagulated blood and NMB mixture to a glass 10 X reading rack will not be visible when viewing through the
75 mm tube. tubes.
3. Make a conventional wedge smear and let air dry. Do not Negative: If no sickling hemoglobin is present, the solu-
counterstain with Wright’s stain. tion will be clear to slightly cloudy and the lines of the tube
4. Use a 100X objective and a 10 ocular secured with reading rack will be visible through the tubes. Both positive
Miller disc or similar apparatus and count the reticulocytes and negative reactions are illustrated in Figure 31—27.
and RBCs on the smear. As in differential enumeration of
WBCs, choose a section of the smear where cells are close Limitations
to each other but not touching or overlapping. Count a If the hemoglobin level is 8 g/dL or less, a false-negative
total of 20 fields. result may occur.
5. In each field, count RBCs in the smaller square and reticu- Patients with multiple myeloma or other plasma cell
locytes in the larger square. Determine percent reticulo- dyscrasias may yield false-positive results
cytes by the following formula: Elevated hemoglobin F levels may yield false-negative
results and therefore this test should not be performed on
% Reticulocytes = Total reticulocytes in larger square _
infants 6 months of age or younger.'*
Total RBC in smaller square X 9 oe
For example, if 10 reticulocytes are counted in the larger
square and 900 mature RBCs in the smaller square, the %
reticulocytes would be:
10 X 100 = 0.12%
900 x9

Standard Methods for Specific

Anemias
Method 12. Sickle Screen

Principle
Red blood cells are lysed by a surfactant and the released
hemoglobin is reduced by sodium hydrosulfite. While reduced
hemoglobin A is soluble in this medium, hemoglobin S is insol-
uble and forms a turbid suspension in phosphate solution. Both
sickle cell disease and sickle cell trait can be detected with the Figure 351-27 @® Positive (left) and negative (right) results of sickle
SickleScreen® test. solubility test.
776 Chapter 31 Hematology Methods

A patient that has been recently transfused may yield 5. Remove one disposable applicator blade assembly from
false-negative results. the packaging. Remove the protective guard from the
Patients with hemoglobin variants such as hemoglobin blade by gently bending the protective piece back and
Charlem OF Coeorgetown May yield positive reactions (Table 31-5). forth until it breaks free.
6. Place the applicator blade into the vertical slot numbered
Reporting Results 2 in the applicator assembly. Note that the blade assembly
Results are reported as positive or negative. will fit into the slots in the applicator assembly only one
way; do not try to force the applicator blade into the slots.
7. Slide the disposable sample cup strips into the top chan-
Method 13. Helena SPIFE® Alkaline nel of the cup tray (numbered | to 20).
Hemoglobin Electrophoresis 8. Pipet 17 uL of patient or control hemolysate into the
disposable cups. Cover until ready for use.
Principle
Small samples of hemolysates prepared from packed cells are
Gel Application
automatically applied to the SPIFE alkaline hemoglobin gel."* 9. Remove the gel from the protective packaging and dis-
The hemoglobins in the sample are separated by electrophore-
card the overlay.
sis using an alkaline buffer and are stained with acid blue
10. Using a REP blotter C, gently blot the entire gel with each
stain. The patterns are scanned on a densitometer and the rel-
blotter using slight fingertip pressure on the blotter.
ative percentage of each band is determined.
Remove the blotter.
11. Dispense approximately 2 mL of REP prep onto left side
Specimen
of SPIFE chamber.
EDTA-anticoagulated blood
12. Place the left edge of the gel over REP prep, aligning the
round hole on the left pin. Gently lay the gel down on
Reagents/Equipment
the REP prep, starting from the left side and ending on the
SPIFE Alkaline hemoglobin gel
right side, fitting the obround hole over the right pin. Use
Acid blue stain paper towel or absorbent paper to wipe around the edges
REP/SPIFE hemolysate reagent of the gel backing, especially next to electrode posts to
Citric acid destain remove excess REP prep. Make sure that the gel lays flat
in the chamber and that no bubbles remain under the gel.
AFSC Hemo Control
13. Clean the electrodes with deionized water and wipe with
AA, Hemo Control lint-free tissue before and after use.
SPIFE instrument 14. Place a carbon electrode on the outside ledge of each gel
block outside the magnetic posts.
Procedure 15. Press the Test Select/Continue buttons located on the elec-
1. Centrifuge anticoagulated blood for 10 minutes to separate trophoresis and stainer sides of the instrument until the
cells from plasma. alkaline hemoglobin option appears on the displays.
2. Remove plasma.
3. Wash packed cells three times by resuspending in 5 to Electrophoresis/Staining
10 volumes of normal saline solution (0.85% NaCl), cen- 16. With alkaline hemoglobin on the display, press the start/stop
trifuging and aspirating supernatant as before. button. An option to either begin the test or skip the opera-
4. After washing the samples, prepare the samples by mixing tion will be presented. Press start/stop again to begin.
10 uL of sample with 100 uL of hemolysate reagent. 17. The auto applicator speed should be set at 4. Place the
Vortex-mix or shake vigorously for 15 seconds. REP auto applicator assembly onto the tray alignment
pins. Lower the applicator tips down into the sample wells
by flipping the toggle switch to the down position. Press
test select/continue to time sample loading.
18. After 30 seconds, lift the applicator tips out of the wells
by flipping the toggle switch to the up position.
19. Carefully lift the entire applicator assembly away from the
Positive Negative cup tray and immediately place it onto the alignment pins
HAbCyartem HbA
located on the SPIFE electrophoresis chamber. Lower the
HbS HbF applicator blades down onto the gel by flipping the toggle
ADS Gravis HbC switch to the down position. Lower the chamber lid.
HbCziguinchor HbD
20. Press the test select/continue button. Sample application
HbG
HbE will be timed for 30 seconds.
HG resee
21. After sample application is complete, raise the lid and
HbA, carefully lift the applicator blades by flipping the toggle
switch to the up position.
 Chapter 31. Hematology Methods 777

22. Remove the applicator assembly from the electrophoresis CELLULOSE ACETATE pH 8.4
chamber. Discard the applicator blade and sample cups as
biohazardous.
oaNormal
23. Close the chamber lid, and press the test select/continue
Sickle trait
button to start electrophoresis. SPIFE will beep when
electrophoresis is complete. Hemoglobin D trait
24. After electrophoresis is complete, use the Gel Block SC disease
Remover to remove both gel blocks from the gel. Lift the
SE disease
gel holder from the stainer chamber. Attach the gel to
the holder by placing the round hole in the gel mylar over Normal cord blood
the left pin on the holder and the obround hole over the C Harlem trait
right pin on the holder. Control
25. Place the gel holder with the attached gel facing back-
wards into the stainer chamber. OoCA;A2 S FA
26. With alkaline hemoglobin on the display, press the C D
E G
start/stop button. An option to either begin the test or skip
O
the operation will be presented. Press start/stop again to
begin. The instrument will stain, destain, and dry the gel. CITRATE AGAR pH 6.0-6.2
27. When the process is complete, the instrument will beep. QaES

Normal
Remove the gel holder from the stainer. Take the gel off
of the holder and replace the holder. Sickle trait

Hemoglobin D trait
Interpretation
The hemoglobin gels may be inspected visually for the presence SC disease
of hemoglobin bands. The Helena Hemo controls provide a SE disease
marker for the band identification. The controls AA, and AFSC
Normal cord blood
Hemo Control should be used as markers for the ___location of
hemoglobin bands. Hemoglobin electrophoresis on cellulose C Harlem trait
acetate and citrate agar is depicted in Figure 31—28. In addition,
Control
the relative percentage of each hemoglobin band by scanning
the dried gels in the densitometer using a 595-nm filter.
o = Designates origin
Limitations
Citrate agar electrophoresis may be necessary to confirm
abnormal hemoglobins
Anion-exchange column chromatography is a more
Figure 31-28 ™ Comparative hemoglobin electrophoresis. Hemo-
accurate method for quantitating HgbA).
globin electrophoresis on cellulose acetate and citrate agar, indicat-
ing patterns of mobility. The width of the band is not indicative of
Normal Values hemoglobin concentration.
Birth 100% Hgb F
Adult 98% Hgb A
3.5% Hgb A; Reagents/Equipment
<2% Hgb F SPIFE Acid Hemoglobin gel
Acid blue stain
REP/SPIFE Hemolysate Reagent
Method 14. Helena SPIFE® Acid
Citric acid destain
Hemoglobin Electrophoresis
AFSC Hemo Control
Principle SPIFE instrument
Very small samples of hemolysates prepared from packed
cells are automatically applied to the SPIFE Acid Hemoglo- Procedure
bin gel.'* The hemoglobins in the sample are separated by 1. Centrifuge anticoagulated blood for 10 minutes to separate
electrophoresis using a citrate buffer and are stained with acid cells from plasma.
blue stain. 2. Remove plasma.
3. Wash packed cells three times by resuspending in 5 to
Specimen 10 volumes of normal saline solution (0.85% NaCl),
EDTA-anticoagulated blood centrifuging and aspirating supernatant.
7718 Chapter 31 Hematology Methods

4. After washing samples, prepare the samples by mixing a Lower the Applicator blade down onto the gel by flipping
10-uL sample with 100 uL of hemolysate reagent. Vortex- the toggle switch to the down position.
mix or shake vigorously for 15 seconds. 22. Press the test select/continue button. Sample application
Nn . Prepare control (AFSC) mixing | part contro! to | part will be timed for 30 seconds.
hemolysate reagent. _ After sample application is complete, carefully lift the
. Remove one disposable applicator blade assembly from Applicator blade by flipping the toggle switch to the up
the packaging. Remove the protective guard from the position.
blade by gently ending the protective piece back and forth 24. Remove the Applicator assembly from the electrophoresis
until it breaks free. chamber. Discard the applicator blades and sample cups
. Place the applicator blade into the numbered vertical as biohazard waste.
slots in the applicator assembly. Note that the blade _ Close the chamber lid, and press the test select/continue
assembly will fit into the slots in the applicator assem- button to start electrophoresis. SPIFE will beep when
bly only one way; do not try to force the applicator electrophoresis is complete.
blade into the slots. . After electrophoresis is complete, use the gel block
. Raise the applicator blades by flipping the toggle switch remover to remove the gel blocks. Place one electrode
to the up position. Using the adjustment knob, set the across each end of the gel to prevent curling during
Auto Applicator speed to 4. drying.
. Slide the disposable sample cups strip into the center . Close the chamber lid and press the test select/continue
channel of the cup tray (numbered 21 to 40). button to dry the gel.
. Pipet 17 uL of patient or control hemolysates into each of . After the gel has been dried, carefully remove the gel
the disposable cups. Cover until read for use. from the electrophoresis chamber.
Oe Remove the gel from the protective packaging and discard . Remove the gel holder from the stainer chamber. Attach
the overlay. the gel to the holder by placing the round hole in the gel
I, Using a REP Blotter C, gently blot the entire gel with each mylar over the left pin on the holder and the obround hole
blotter using slight fingertip pressure on the blotter. over the right pin on the holder.
Remove the blotter. . Place the gel holder with the attached gel facing back-
. Dispense approximately 2 mL of REP Prep onto left side wards into the stainer chamber.
of SPIFE chamber. Si Press the test select/continue button until acid hemoglobin
. Place the left edge of the gel over REP Prep, aligning the appears on the display.
round hole on the left pin. Gently lay the gel down on 52) With acid hemoglobin on the display, press the start/stop
the REP Prep, starting from the left side and ending on button. An option to either begin the test or skip the oper-
the right side, fitting the obround hole over the right pin. ation will be presented. Press start/stop to begin. The
Use paper towel or absorbent paper to wipe around the instrument will stain, destain, and dry the gel.
edges of the gel backing, especially next to electrode . When the gel has completed the process, the instrument
posts, to remove excess REP Prep. Make sure that the will beep. Remove the gel holder from the stainer. Take
gel lays flat in the chamber and that no bubbles remain the gel off the holder and replace the holder.
under the gel.
. Clean the electrodes with deionized water and wipe with Interpretation
lint-free tissue before and after use. The hemoglobin gels should be inspected visually for the
. Place a carbon electrode on the outside ledge of each gel presence of abnormal hemoglobin bands (see Fig. 31-28).
block outside the magnetic posts. Glycated hemoglobin migrates with HbF. The Helena AFSC
. Press the test select/continue buttons located on the elec- Hemo Control provides a marker for band identification. This
trophoresis and stainer sides of the instrument until the control should be run on each SPIFE Acid Hemoglobin gel.
acid hemoglobin option appears on the display. The control verifies all phases of the procedure and acts as a
. With acid hemoglobin on the display, press the start/stop marker to aid in the identification of the hemoglobins in the
button. An option to either begin the test or skip the oper- unknown samples.
ation will be presented. Press start/stop to begin.
. The Auto Applicator speed should be set at 4. Place Limitations
the REPAuto Applicator Assembly onto the tray align- Anion-exchange column chromatography is the most accurate
ment pins. Lower the applicator tips down into the method for quantitating HgbA,. Low levels of HgbF may be
sample cups by flipping the toggle with to the down accurately quantitated using radial immunodiffusion.
position. Press Test Select/Continue to time sample
loading. Normal Values
. After 30 seconds, lift the applicator tips by flipping the Birth 100% Hgb F
toggle switch to the up position.
. Carefully lift the entire Applicator Assembly away from
Adult 98% Hgb A
the Sample Tray and immediately place it onto the align- 3.5% Hgb A,
ment pins located on the SPIFE electrophoresis chamber. <2% Hgb F
 Chapter 31 Hematology Methods 779

Method 15. Hemoglobin A, Determination aspirate the undrained supernatant (making sure not to
disturb the resin) and discard.
Principle 6. Slowly and carefully apply 100 uL of the patient
The anion-exchange resin is a preparation of cellulose cova- hemolysate to the column. During application, do not
lently coupled to small positively charged molecules. The pos- allow the sample to form bubbles or run down the side of
itively charged cellulose attracts negatively charged molecules. the tube. Excessive force used during application will
Proteins, such as the hemoglobins, contain many positive and disturb the resin and may cause erroneous results.
negative charges due to the ionizing properties of the compo- 7. Immediately after sample application to the column, add
nent amino acids. In the anion-exchange chromatography of 100 uL of the same patient hemolysate to a Total Fraction
HbA,, buffer and pH levels are controlled to cause different collection tube. QS the tube to the scribed line using
hemoglobins to possess different net negative charges. These deionized water. Total volume = 15 mL.
negatively charged proteins are attracted to the positively 8. Allow the sample to completely absorb into the resin. The
charged cellulose and bind accordingly. After binding, the pro- hemolysate will have a glossy appearance when viewed
teins are removed selectively from the resin by altering the pH from above until the sample is completely absorbed by the
or ionic strength of the elution buffer. Because of the pH of the resin. On complete absorption, the top of the resin will
resin and the ionic strength of the HbA, developer, HbA, does have a dull, mat-like appearance.
not bind to the positively charged cellulose and is eluted as the 9. Following the absorption of the sample into the resin bed,
developer moves through the column. The other normal and slowly apply 2.5 mL of HbA, developer to the column.
most abnormal hemoglobins are retained by the resin. The Excessive force when applying the developer may disturb
HbA, fraction is compared to a total hemoglobin fraction by the resin and cause invalid results.
determining the absorbance of each using a spectrophotometer 10. Allow all of the developer to pass through the column into
and then calculating the percentage of HbA,.'° the HbA, collection tube (approximately 30 minutes to
2 hours). The eluate contains the HbA,.
Specimen 11. QS the small collection tube to the scribed line with
EDTA-anticoagulated blood deionized water. Total volume = 3 mL.
12. Invert both collection tubes several times to mix contents
Reagents/Equipment thoroughly.
Beta-Thal HbA, Quik Columns 13. Determine the HbA, percentage in each sample using a
HbA, Developer standard spectrophotometer.
A. Adjust the wavelength to 415 nm
Hemolysate Reagent C
B. Zero the instrument with deionized water.
Spectrophotometer C. Read and record the optical density (O.D.) of both the
HbA, and TF collection tubes.
Procedure D. Determine the HbA, % as follows:
1. For each patient quantitation, obtain a Quik Column, HbA,
Absorbance of HbA, fraction X 100
Collection tube, and total fraction tube. = HbA, %
5 (Absorbance of TF solution)
.
i) Allow the appropriate number of columns and reagents to
come to room temperature. where HbA2% = percentage of HbA2 in sample
3. Prepare patient samples by placing 50 uL of whole blood Absorbance of HbA2 fraction = absorbance of the con-
into a small laboratory test tube. Add 250 ul of hemolysate tents of the small collection tube at 415 nm (HbA2 fraction).
reagent C to test tube. Absorbance of the TF solution = absorbance of the con-
4. Shake the tube to achieve complete hemolysis of sample. tents of the large collection tube at 415 nm (other hemoglobin
Complete lysis of the sample is essential for accurate fractions).
results. Allow the sample to stand 5 minutes before use. 5 = dilution factor (15 mL of TF tube/3 mL of HbA2
5. Preparation of columns. tube 5 5)
A. Upend each column twice to remove any resin from the 100 = percentage conversion factor
top cap closure. Remove the top cap closure and gently
resuspend the entire contents of the column using a Pas- Interpretation
teur pipet with a small rubber bulb. Make sure all the Results of HbA, should be interpreted in conjunction with
resin is resuspended above the filter. patient history, total hemoglobin values, and other clinical
B. Immediately after resuspension of each column, hold and lab findings. Any value between 3.5% and 8% is consid-
the column over a sink or absorbant paper and remove ered indicative of beta (8)-thalassemia trait. Values above
the bottom tip closure, allowing the buffer to drain. 8% indicate the presence of additional hemoglobin variants
If the column is allowed to stand with the bottom tip such as HbC, E, O, D, G, S, or S—G hybrid.
closure in place, resuspension must be repeated.
C. As the resin repacks, you will see an interface slowly Limitations
move up the tube. As soon as the slurry settles to form 1. Failure to completely resuspend the contents of the column
an interface of resin and remaining supernatant above, may cause slow flow and erroneous results. Time must be
780. Chapter 31 Hematology Methods

allowed after resuspension for the formation of a distinct Hemolysate reagent: 0.07% potassium cyanide and
interface between the resin and supernatant. Any trapped 0.005 M EDTA
bubbles may be removed with a Pasteur pipet. AFSC Control (cat. no. 5331)
2. The bottom tip closure must be removed immediately after
AA, Control (cat. no. 5328)
resuspension. Resuspension must be repeated if the column
is allowed to site with the bottom closure in place after A rapid electrophoresis (REP) unit used to elec-
resuspension. Failure to do so may cause slow flow and trophorese, dry, and scan gels
erroneous values.
3. As soon as the resin repacks, the remaining supernatant Procedure
must be aspirated and discarded. 1. Prepare a 1:4 dilution with IEF hemolysate reagent. Mix
4. To avoid back pressure in the column, do not remove the 1 part (25 uL) of whole blood with three parts (75 wL)
bottom tip closure before removing the top cap closure. hemolysate reagent.
Any bubbles trapped in the column resin may slow or stop 2. Make a 1:8 dilution by mixing one part (10 uL) packed
the flow rate, leading to erroneous results.
cells to 7 parts (70 uL) hemolysate reagent.
5. If the developer ceases to flow through the column the col- 3. Place three 1/8-inch diameter discs into a test tube.
umn must be discarded and the quantitation repeated with Reconstitute with 25 uL of hemolysate reagent. 2 uL must
a fresh column. be applied by hand to the gel.
6. Any disturbance of the resin during the procedure may 4. Vortex-mix briefly and allow the sample to stand at least
cause erroneous results. No more than 5 minutes should 5 minutes before use. The cells should be completely
elapse from the time the column stops flowing until the lysed before gel application.
developer is added so that the resin does not dry out. 5. Place 30 sample cups into the sample tray. Place 75 uL of
7. Some of the abnormal hemoglobins (HbS, C, E, O, E, G, sample or control lysates into appropriate sample cups.
S-G hybrid) are eluted with HbA, in this method. The pres- 6. Place REP blotter A on the sample tray in area adjacent to
ence of the abnormal hemoglobins should be confirmed by the sample cups. Place approximately 4 mL of Sureprep®
electrophoretic techniques. HbF does not interfere with this into outside wash well of sample tray. Place approxi-
method. mately 4 mL of water into the inside wash well of the
sample tray.
Normal Values 7. Remove the gel from the protective packaging and dis-
Normal HbA, range = 2.2% to 3.3% card overlay. Use the REP prepper to remove excess
moisture from the sample wells and trough.
8. Place left edge of gel over the chamber, aligning the round
Method 16. Isoelectric Focusing hole on the left pin. Gently lay the gel down on the cham-
ber starting from the left side and ending on the right side,
Principle fitting the obround hole over the right pin.
The isoelectric point (pI) is the pH at which a molecule carries 9. Insert the IEF electrode adapter marked (Front) between
no net electrical charge. In order to have a sharp isoelectric the two magnetic posts located at the front of the chamber
point, a molecule must be amphoteric, meaning it must have floor. Insert the IEF electrode adapter marked (Back)
both acidic and basic functional groups. Proteins and amino between the two magnetic posts located at the back of the
acids are common molecules that meet that requirement. Pro- chamber floor.
teins can be separated according to their isoelectric point in a 10. Clean and wipe the IEF electrodes with a lint-free tissue.
process known as isoelectric focusing (IEF). At a pH below the 11. Place an electrode into the slots created by the adapter—
pl, proteins carry a net positive charge. Above the pI they carry one on each outside edge and one in the middle. Be sure
a net negative charge. The pH of an electrophoretic gel is deter- all three electrodes are seated firmly against the electrode
mined by the buffer used for that gel. If the pH of the buffer is gel blocks.
above the pI of the protein being run, the protein will migrate 12. The REP unit will automatically apply samples and elec-
to the positive pole. If the pH of the buffer is below the pI of trophorese and dry the gel.
the protein being run, the protein will migrate to the negative 13. Remove the gel from the REP unit for evaluation. If the
pole of the gel. If the protein is run with a buffer pH that is gel is to be used for future evaluation, it will be necessary
equal to the pl, it will not migrate at all. By using ampholyte to dry down the electrode gel blocks by placing the gel
buffers in the pH range of 6 to 8, the separation and identifica- into an I.0.D. at 60°C for 10 to 15 minutes.
tion of abnormal hemoglobins are possible based on migration 14. Gels may be visually examined or scanned using a
of proteins based on their isoelectric points." 415-nm interference filter.
Specimen
Interpretation
EDTA-anticoagulated blood or heparinized blood
HbE and Hb,-Arab migrate slightly anodal to HbA,. HbG-
Reagents/Equipment Philadelphia and HbD-Los Angeles are separated from HbS
REP Hemoglobin IEF gel: each gel contains 1% wt/vol as 18 Hby epore-HbA, HbF are clearly separated from each other,
agarose, 5.3% vol/vol carrier ampholytes, and allowing identification between sickle cell trait, sickle cell
0.01% thymol as a preservative. anemia, and HbS/f-thalassemia (Fig. 31-29). The AFSC
 Chapter 31 Hematology Methods 781

Specimen
EDTA-anticoagulated specimen.

Reagents/Equipment
Fetal cell fixing solution (80% reagent alcohol)
Fetal cell buffer solution (citrate buffer, 0.081 M)
Fetal cell stain (erythrosin-B, fast green)
Microscope
Glass slides
0.85% saline
Ey ve G Philadelphia Glass test tubes
Coplin jars
~S D Los Angeles
Procedure
HbA»
O-Arab, E 1. Mix the blood sample well by gentle inversion.
pe > —— 2. Place three drops of 0.85% saline and two drops of mater-
Constant
Spring
pring — A> nal blood into a glass test tube. Mix by gentle agitation.
3. Place one drop of diluted blood on a glass slide near one
pH 8.0 Cathode end. Prepare thin film by drawing the edge of another
slide through the drop of blood, and across the slide.
a - denatured Hb
b - ferrous/ferric hybrid 4. Air dry the slide at room temperature.
c - ferrous/ferric hybrid 5. Place the smears in a clean vessel containing sufficient
d - methemoglobin fetal cell fixing solution to cover the smears. Let stand for
5 minutes. Note: the slides should be placed in the groove
Pigure 51-29 ™ Schematic of isoelectric focusing pattern. (Helena of the Coplin jars singly, so that the smear is adequately
Laboratories, Beaumont, TX.) exposed to solution.
6. Remove the smears and rinse thoroughly in distilled
water. Allow the slides to drain dry.
Control and AA, control should be run with each gel. Dilute 7. Place the smears in a clean vessel containing sufficient fetal
the AFSC Control 1:2 with hemolysate reagent before use. cell buffer solution to cover the smears. Allow to remain in
solution at room temperature for 8 to 10 minutes.
Limitations 8. Remove the slides from the solution and immediately
1. HbGgaiveston ANd HbGy, 44, Cannot be distinguished from HbS. place in a clean vessel containing fetal cell stain. Stain for
2. HDpammersmithy Hbgethesdas Hbgrigham Carmot be distinguished 3 minutes.
from HbA. 9. Remove the slides from the fetal cell staining solution and
. HbE, HbCyaiem, HbO,,., and Hbx,;, cannot be separated.
rinse thoroughly in distilled water. Dry slides at room
temperature.
WwW HbNopattimore and HbL;,.,., cannot be separated.
5. HbCC cannot be distinguished from HbC/B-thalassemia and 10. Examine slides under the oil-immersion lens. Fetal cells
HbEE cannot be distinguished from HbE/B-thalassemia. will stain a dark reddish-pink while adult cells contain-
6. If the gels exhibit drying under low ambient humidity, ing hemoglobin A will appear white to light pink with a
darker center (Fig. 31—30). Slides should be read within
bands will be skewed and exhibit uneven migration. A band
24 hours. A positive control may be prepared from a
will migrate in an arched manner if the concentration of
mixture of | part cord blood to 9 parts normal adult
hemoglobin is too high. A sample application of 1 uL
blood.
should rectify the problem.
7. Bands from fresh whole blood may electrophorese at a Interpretation
slightly slower rate than the control. Older samples may The common means of reporting fetal cells is as a percentage
electrophorese at a slightly faster rate than the control. If of normal adult cells. This ratio is achieved by randomly
controls do not perform as expected, test results should be observing 8 to 10 fields of cells. Count the number of adult
considered suspect or invalid. cells in each field and total them. Count the number of fetal
cells in each of the same fields and total the fetal cells. Deter-
mine the percentage of fetal cells to adult cells by dividing the
Method 17. Hemoglobin F Acid Stain
total number of fetal cells counted by the number of adult
Principle cells counted."
Red cells containing fetal hemoglobin are resistant to acid For example, 25 fetal red cells and 4000 adult cells are
elution whereas red cells containing hemoglobin A will counted. The percentage of fetal cells to adults cells is
appear as ghost cells on exposure to acid. 25/4000 X 100 = 0.6%.
782. Chapter 31 Hematology Methods

3. Prepare saturated digitonin solution by adding | g of digi-

tonin to 100 mL of distilled water. Shake the mixture and
filter.
4. Prepare potassium phosphate buffer (0.25 M, pH 7.4) by
adding 8.25 mL of | M stock dipotassium hydrogen phos-
phate and 1.75 mL of potassium dihydrogen phosphate and
QNS up to 100 mL with distilled water.
5. Mix together the following which constitutes the reac-
tion mixture: 0.10 mL of glucose-6-phosphate (0.01 M),
0.10 mL of NADP (0.0075 M), 0.20 mL of saturated digi-
tonin, 0.30 mL of potassium phosphate buffer, and 0.30 mL
distilled water.
6. Add | volume of whole blood to 10 volumes of the reaction
Figure 31-50 @ Kleihauer—Betke stain of blood from a newborn. mixture and incubate 5 to 10 minutes at 37C.
Red-staining cells contain hemoglobin F; clear-staining cells contain 7. Place a drop of blood reaction mixture on a Whatman No. 1
hemoglobin A. (From Diggs, LW: Hematology. In Listen, Look, and
Learn. Health and Education Resources, Inc., Bethesda, MD, with
filter paper and view under a UV lamp.
permission.)
Interpretation
If normal G6PD activity is present, the spot will fluoresce
Limitations
brightly. If the blood is G6PD deficient, no fluorescence is
1. Hematological disorders (e.g., thalassemia) may produced
seen. .
increased levels of fetal cells.
2. The degree of elution of adult hemoglobin may vary from
patient to patient. Limitations
3. Lymphocytes may take up stain in varying degrees, albeit Blood with hematocrits below 25% are contraindicated. If
less than fetal cells. anemic bloods are tested, adjust the hematocrit to within 40%
to 45%.

Method 18. Screening Test

for Glucose-6-Phosphate Method 19. Staining for Heinz Bodies
Dehydrogenase Deficiency
Principle
Principle Heinz bodies are denatured hemoglobin precipitated in the
Glucose-6-phosphate is oxidized to 6-phosphogluconate, and RBC and attached to the RBC membrane. They are not visi-
NADP is reduced to NADPH in the presence of glucose- ble with Wright’s stain but show up with supravital staining
6-phosphate dehydrogenase (G6PD) from the red cell (crystal violet) and phase microscopy (Fig. 31-31).
hemolysate. The red cells also contain 6-phosphogluconate
which reduces more NADP. The NADPH, when activated by
Specimen
UV light, produces a vivid fluorescence.'*
EDTA-anticoagulated blood
Specimen
Heparinized blood

Reagents/Equipment
Glucose-6-phosphate (0.01 M)
Triphosphopyridine nucleotide (0.0075 M)
Digitonin solution
Potassium phosphate buffer
UV light
Whatman No. | filter paper

Procedure
|. Prepare 0.01 M glucose-6-phosphate by dissolving 42 mg
of glucose-6-phosphate in 10 mL of distilled water.
2. Prepare 0.0075 M triphosphopyridine nucleotide by dissolv-
ing 18.75 mg of triphosphopyridine nucleotide in 1.0 mL of
1% sodium bicarbonate solution. Figure 31-31 @ Heinz bodies.
 Chapter 31 Hematology Methods 783

Reagents/Equipment 2. Prepare ADP (0.03M) by dissolving 0.1508 g of ADP in

Crystal violet solution: 1.0 g of crystal violet dissolved 8.5 mL of distilled water. Neutralize with 0.25 N NaOH
in 50 mL of 0.85% saline solution, which is (pH 7.8) and dilute to 10 mL with distilled water.
shaken for 5 minutes and filtered before storage 3. Prepare 0.015 M NADH by dissolving 0.1063 g of NADH
Methyl! violet solution: 0.5 g of methyl violet dissolved in 8.5 mL ofdistilled water. This reagent should be freshly
in 100 mL of 0.85% saline solution, which is prepared.
shaken for 5 minutes and filtered before storage 4. Prepare magnesium sulfate by dissolving 1.9720 g of
this salt in 100 mL of distilled water. Aliquot in 5-mL
Glass slides and coverslip
volumes.
5. Prepare 0.25 M potassium phosphate buffer (pH 7.4) by
Procedure
dissolving 3.403 g of potassium phosphate in 85 mL of
1. Mix equal volumes of EDTA blood and stain in a small test
distilled water. Adjust to pH 7.4 and dilute to 100 mL.
tube. Either methyl violet or crystal violet stain may be used.
6. Prepare the reaction mixture by adding 0.03 mL of PEP,
2. Incubate for 20 minutes at room temperature.
0.10 mL of ADP, 0.10 mL of NADH, 0.10 mL of magne-
3. Remix the blood stain solution and transfer one drop to a
sium sulfate, 0.05 mL of potassium phosphate buffer, and
slide.
0.62 mL of distilled water.
4. Place a coverslip on the slide and examine for Heinz bod-
7. Centrifuge the anticoagulated blood at 2000 rpm for
ies under oil immersion.
5 minutes and remove the buffy coat.
8. Remove | volume of the red cell mass using a Pasteur
Results
pipet and add to 4 volumes of saline.
Heinz bodies appear as irregular, refractile, purple inclusions,
9. Add approximately 0.02 mL of this cell suspension to
1 to 3 um in diameter, located on the periphery of the cell.'°
0.20 mL of the reaction mixture. Add a drop of mixture to
They may even seem to be outside the cell. Reticulocytes are
filter paper by spotting, and incubate remainder of mix-
not stained by this technique.
ture at 37C for 30 minutes.
10. After incubation, a second spot is made on filter paper.
After drying the paper, examine the two spots using a UV
Method 20. Screening Method
light source.
for Detection of Red Cell Pyruvate Kinase
Principle Interpretation
A phosphate group is transferred to ADP, forming pyruvate The first spot from normal blood sample will fluoresce
and ATP in the presence of red cell pyruvate kinase. Lactate brightly, but the second spot will not fluoresce. Red cells,
dehydrogenase in the red cell hemolysate catalyzes the reduc- deficient in pyruvate kinase, fluoresce in both spots.?°
tion of pyruvate to lactate with the resulting oxidation of
diphosphopyridine nucleotide (DPNH) to diphosphopyridine Limitations
(DPN). DPNH has the property of fluorescence, while DPN All frozen reagents are stable for | month.
does not fluoresce under UV light. Reaction mixture is stable at room temperature for
7 hours.
Specimen EDTA blood samples are stable for 5 days at room
EDTA-anticoagulated blood temperature.

Reagents/Equipment
Phosphoenolpyruvate (trisodium salt) Methods to Detect Red Cell Membrane
0.03 M Adenosine diphosphate (ADP) Disorders
0.015M Nicotinamide-adenosine dinucleotide phos-
Method 21. Sugar Water Test
phate (NADH)
Magnesium sulfate Principle
PNH (paroxysmal nocturnal hemoglobinuria) red cells
0.25M Potassium phosphate buffer
hemolyze when incubated with compatible normal serum in a
UV light source sucrose solution of low ionic strength. The principle of this
Whatman No. | filter paper test is that sucrose provides a medium of low ionic strength
0.85% Sodium chloride that promotes binding of complement to red cells.*' PNH cells
undergo lysis in these conditions by virtue of their increased
Procedure sensitivity to complement.
1. Prepare phosphoenolpyruvate (PEP) by dissolving 0.6984 g
of PEP in 8.5 mL of distilled water. Neutralize with 0.25N Specimen
NaOH (pH 7.8) and dilute to 10 mL with distilled water. Clotted blood from patient
784 Chapter 31 Hematology Methods

Reagents/Equipment
Clotted blood from both patient and ABO compatible
donor
0.005 M Phosphate buffer (pH 6.1): Sodium dihydrogen
phosphate (NaH,PO,H,O): Dissolve 690 mg of
monobasic sodium phosphate in | L of distilled
water.

0.005 M Disodium hydrogen phosphate

(Na,HPO,7H.O): Dissolve 135 mg of dibasic
sodium phosphate in | L of distilled water. Mix
910 mL of 0.005 M sodium dihydrogen phosphate
with 90 mL of disodium hydrogen phosphate. The
buffer can be stored indefinitely at 4°C.
Isotonic sucrose solution: Dissolve 924 mg of sucrose
(0.27 M) in 8 mL of double phosphate buffer.
Adjust the pH to 6.1 with 0.75 N sodium hydrox-
ide or with 0.75 N hydrochloric acid and make up
to 10 mL with buffer.
0.75 N Sodium hydroxide: Dissolve 3.0 g sodium
hydroxide in 100 mL of distilled water.
0.75 N Hydrochloric acid: Add 6.25 mL of 12 N (con-
centrated) hydrochloric acid to 100 mL of distilled Figure 31-52 @A positive (right) and control (/eft) sugar water test.
water.
Centrifuge.

Procedure Specimen
|. Separate the serum from the clotted blood samples of both Defibrin
patient and control.
2. Prepare a 50% saline suspension of cells for each sample Reagents/Equipment
from the residual clots. 0.2 N Hydrochloric acid
3. Add to each of these tubes 0.85 mL of sucrose solution and 0.85% Sodium chloride
0.05 mL of unacidified autologous serum.
0.1-mL and |!.0-mL graduated pipets
4. Add 0.1 mL of the 50% suspension of patient’s red cells to
one tube. 37°C Water bath
5. Add 0.1 mL of the control cells to the second tube. 20 mL of patient’s defibrinated blood. Place two bent
oO. Mix both the tubes and incubate at 37°C for 30 minutes. paper clips in the bottom of a 50-mL Erlenmeyer
7. Centrifuge both tubes at 2000 rpm for | to 2 minutes and flask and swirl the flask continuously for 10 to
examine the supernatant for hemolysis. 15 minutes.

Interpretation 10 mL of normal defibrinated blood of the same blood

A positive test for PNH is indicated when the patient cells are group (ABO) as the patient’s.
hemolyzed. The control cells should show no more than a Centrifuge
trace of lysis (Fig. 31-32). 12 X 75 mm test tubes
Limitations
Blood collected in EDTA or heparin is contraindicated Procedure
in this test. 1. Centrifuge the defibrinated blood samples at 2000 rpm for
5 minutes. Save and label the serum from each tube.
No hemolysis is found in other types of hemolytic anemia.
2. Wash the cells three times with an equal volume of saline.
Normal Values Centrifuge the last washing at 3000 rpm for 10 minutes to
Less than 5% red cell hemolysis achieve adequate cell packing.
3. Prepare a 5% suspension of both patient and normal red
cells by adding 0.5 mL of packed cells to 9.5 mL of saline.
Method 22. Ham’s Test 4. Set up seven tubes as follows:
! Z 3) 4 5 6 7
Principle Patient 0.05 0:05910,05.90:05 90.05
The patient’s red cells are exposed at 37°C to the action of RBC
normal serum or to the patient’s own serum, acidified to a pH Control 0.05 0.05
of 6.5 to 7.0. RBC
 Chapter 31 Hematology Methods 785

0.2 N OO Sa0505 0.05 0.05 Disodium hydrogen phosphate dihydrate 27.31 g

oe Sodium dihydrogen phosphate dihydrate 4.86 g
Patient OS mOS 0.5 0.5 ae ;
as oe Distilled wate qns ieee
to2 L

Control OomnO San 05" Graduated pipets

serum 0.01-1 mL
*heat inactivated 0.1-10 mL

5. Mix all the tubes by shaking well, incubate at 37°C for Volumetric pipet
| hour, and centrifuge at 2000 rpm for 5 minutes. Examine 10 mL
the supernatants for hemolysis.
P y Volumetric flask

Interpretation 100 mL
A positive test is indicated by hemolysis in the tubes contain- Spectrophotometer
ing the patient’s cells with acidified
; serum (tubes
wy 2 and 3), the Heparinized venous blood
other tubes are unaffected (Fig. 31-33). A positive test is usu-
ally diagnostic of PNH. Procedure
1. Make a 1% saline from the stock by diluting 10 mL to
Limitations 100 mL with distilled water.
From 10% to 50% lysis is usually obtained in a positive test, 2. Make serial dilutions of the 1% working solution as
when lysis is measured quantitatively on the basis of liberated depicted below:
hemoglobin. The test can be made more sensitive by using a 1% Working Distilled % Final
young red cell population (reticulocytes) such as the upper saline (mL) water (mL) saline
red cell layer obtained by centrifugation.** 2.0 3.0 0.20
Dee) Tes 0.25
Reporting Results 3.0 7.0 0.30
Hemolysis is reported as a percentage. Qe 6.5 0.35
SD 625 O75
Method 23. Osmotic Fragility 4.0 6.0 0.40
4.25 2p //) 0.425
Principle 4.5 SS 0.45
The test determines the resistance of the red cells to hemoly- 4.75 D9) 0.475
sis In varying concentrations of hypotonic saline at room 5.0 5.0 0.50
temperature. ae) 4.5 05
6.0 4.0 0.60
Specimen 6.5 SD 0.65
Fresh heparinized blood 7.0 3.0 0.70
fie) De) O75
Reagents/Equipment 8.0 2.0 0.80
Stock 10% sodium chloride, buffered (pH 7.4) 8.5 IES 0.85

Sodium chloride 180 g 3. Mix the saline dilutions well and add 0.1 mL of heparinized
whole blood to each tube.
4. Stopper the tubes and mix by inversion. Allow the tubes to
stand for 2 hours at room temperature and then remix the
same way.
5. Centrifuge the suspensions for 5 minutes at 2000 rpm. Cal-
culate the percent hemolysis in each of the supernatants us-
ing a spectrophotometer at 540 nm. Set up a 100% standard
by adding 0.1 mL of blood to 10 mL of distilled water.
6. Calculate % hemolysis as follows:

OD of test dilutions < 100

= % hemolysis
OD of the standard

7. Plot the % hemolysis from each tube against the % saline

and compare against the known normal range.

Interpretation
When red cells are placed in a hypertonic solution, they lose
Figure 31-35 @ Results of a Ham’s test. fluid until an equilibrium is set up with the surrounding fluid,
786. Chapter 31 Hematology Methods

and they crenate. In hypotonic solutions, the cells take up Reagents/Equipment

fluid and swell until either an equilibrium is attained or the Sterile ATP: Dissolve 1 g of ATP in 4 mL of sterile
cell ruptures. Increased osmotic fragility of red cells to saline normal saline
solution is related to cell shape. The more spherical the Sterile 10% glucose in normal saline
cell, the greater its fragility to concentrations of saline. The
Sterile 0.85% sodium chloride
osmotic fragility is increased in hereditary spherocytosis and
decreased in sickle cell anemia™ (Fig. 31-34). Heparinized blood
13 X 100 sterile tubes
Limitations Drabkin’s reagent
Oxalate, EDTA, or citrate should not be used because of
Spectrophotometer
additional salts present. This test should be performed imme-
diately because shape change and osmotic conditions change Centrifuge
with time. If the patient has a low hemoglobin level, wash the
patient and control cells once with isotonic saline and resus- Procedure
pend with equal volumes of RBCs and saline. This will cor- 1. Heparinize 20 mL of blood and divide equally into three
rect for anemia. sterile tubes.
2. Add 0.2 mL of ATP solution and 0.2 mL of glucose solu-
Normal Values tion to the first and second tubes, respectively.
Initial hemolysis 0.45% 3. Add 0.2 mL of sterile saline to the third tube. This is used
Complete hemolysis 0.30—0.35% as the blank control tube.
4. Gently invert all three tubes and incubate for 24 hours
ates 7e@.
Method 24. Autohemolysis Test 5. At the end of this time, gently invert again and reincubate
Principle for an additional 24 hours.
The amount of spontaneous lysis in sterile heparinized blood 6. Centrifuge all tubes at 2000 rpm for 10 minutes.
is estimated after incubation with and without glucose and 7. Perform routine hemoglobin estimations on all three super-
ATP. Though rarely used in today’s hematology laboratory, natants, using the cyanmethemoglobin reagent as the zero
this test is being mentioned for historical reasons. blank.
8. Resuspend the heparinized blood in the third tube and
Specimen determine a whole blood hemoglobin.
EDTA-anticoagulated blood 9. Obtain the % hemolysis of the 3 tubes by dividing each by
the whole blood hemoglobin in g/dL.

Interpretation
100
Normal blood undergoes little spontaneous hemolysis when
incubated under sterile conditions for 48 hours. The addition
of either glucose or ATP reduces the degree of hemolysis. In
hereditary spherocytosis, the addition of glucose reduces the
degree of hemolysis, while in pyruvate kinase deficiency the
addition of glucose does not affect the degree of hemolysis
(Fig. 31-35).

Ooio)

Limitations
hemolysis
% The autohemolysis test is no longer used in the differential
diagnosis of nonspherocytic congenital hemolytic anemia,
because specific enzymatic assays are now available that pro-
vide better accuracy.

Reporting Results
Hemolysis is reported as a percentage.
140) Ok) CH O:7/ OG O5 Off 08 @2 @al ©
Sodium chloride (g/100 mL)
Normal Values
Figure 351-54 = Comparative osmotic fragility curve (blue line, Lysis at 48 hours: without added dextrose 0.2%
sickle cell anemia; purple line, hereditary spherocytosis). Normal
range is shaded. 7, normal biconcave disc; 2, disc-to-sphere trans- with added dextrose 0-0.9%
formation; 3, disc-to-sphere transformation; 4, lysis. with added ATP 0-0.8%
 Chapter 31 Hematology Methods 787

INCUBATION HEMOLYSIS Disposable pipets

50 0.5 mL of sodium citrate or sodium chloride in
puncture-ready vials

Procedure
1. Collect whole blood anticoagulated with EDTA.
10 Pyruvate
kinase 2. Dilute whole blood with 0.5 mL of 3.8% sodium citrate or
0.85% sodium chloride.
[oS). Mix the blood—diluent solution by inversion.

4. Insert the Westergren tube into the plungeable vial cap of the
diluent mixture. Let blood draw to the top of the tube by cap-
illary action. Place the tube in the Westergren rack in a ver-
(%)
glucose
with
Lysis !
tical position and leave undisturbed for | hour (Fig. 31-36).
I
I Hereditary 5. After | hour has passed, record the distance in mm from the
Normal I spherocytosis top of the column of red cells to the plasma-red cell interface.
range
1
The buffy coat should not be included in the measurement.
i
Interpretation
ieee
15) e010 20 eas
Various diseases can yield an increased ESR. These are listed
Lysis without glucose (%)
in Table 31-6. An increased ESR can also be seen in cases of
Figure 31-55 @ Incubation hemolysis test. This test provides a hyperfibrinogenemia, patients who are able to form rouleaux,
further measure of cell resistance to hemolysis. Pyruvate kinase— and patients with hypergammaglobulinemia. Abnormal red
deficient blood demonstrates an abnormal rate of hemolysis that is cell morphology may be associated with a decreased ESR’
independent of the presence or absence of glucose in the incuba-
(Table 31-7).
tion media. In contrast, the blood from a patient with hereditary
spherocytosis shows more marked hemolysis when glucose is
absent. (From Hillman, RS, and Finch, CA: Red Cell Manual, ed 7.
Limitations
FA Davis, Philadelphia, 1996, p 124, with permission.) Low results can occur when the collected specimen is
allowed to stand for more than 3 hours. The Westergren

Nonspecific Tests of Inflammation

Method 25. The Westergren Erythrocyte
Sedimentation Rate
Principle
The erythrocyte sedimentation rate (ESR) is a nonspecific test
used to detect illnesses associated with acute and chronic
infection, inflammation and tissue necrosis or infarction.' The
ESR measures the settling of erythrocytes in diluted human
plasma over a specified time period. This numeric value is
determined (in millimeters) by measuring the distance from the
bottom of the surface meniscus to the top of erythrocyte sedi-
mentation in a vertical column containing diluted whole blood
that has remained perpendicular to its base for 60 minutes. Vari-
ous factors affect the ESR, such as RBC size and shape, plasma
fibrinogen, and globulin levels, as well as mechanical and tech-
nical factors. The ESR is directly proportional to the RBC mass
and inversely proportional to plasma viscosity. In normal whole
blood, RBCs do not form rouleaux; the RBC mass is small and
therefore the ESR is decreased (cells settle out slowly). In abnor-
mal conditions, when RBCs can form rouleaux, the RBC mass is POL, YMEDCO
CONTLAN DY 1
HOT ARMOR MEW Yong
greater, thus increasing the ESR (cells settle out faster). The
Westergren tube is 30 cm long, 2.5 mm in diameter, and cali-
brated in mm from 0 to 200.

Reagents/Equipment
Westergren tubes Figure 31-56 ® Westergren methodology for ESR. (Courtesy of
Westergren rack Polymedco, Inc, Cortlandt Manor, NY.)
788 — Chapter 31 Hematology Methods

as fibrinogen and gamma-globulins, and the decrease in

table 51-6 Diseases Associated retarding proteins, such as albumin.
with an Elevated Esha Sis
Specimen
Rheumatoid arthritis In 2.0-mL ESR-vacuum tubes, containing sodium citrate,
Multiple myeloma
Cryoglobulinemia
mix the blood with the sodium citrate solution immediately
Temporal arteritis after drawing the patient sample by inverting the tube at
Inflammatory diseases least 6 to 8 times. The air bubble in the tube must reach the
Pregnancy opposite end of the tube between every inversion. Hold the
Anemia tube at an angle of about 35_degrees to enhance mixing.
Malignant neoplasms
Patient samples may also be drawn directly into an EDTA
Paraproteinemias
Macroglobulinemia vacuum tube. When using an EDTA vacuum tube, transfer
Hyperfibrinogenemia the well-mixed patient sample into the ESR-vacuum tube.
Chronic infections Fill the ESR-vacuum tube to the marked fill line on
Collagen disease the label. Do not remove the sodium citrate in the ESR-
Polymyalgia rheumatica
vacuum tube.

Reagents
tube must be situated perfectly vertical as any tilt can cause ESR-vacuum tube, 2.0 mL, containing 3.2% sodium
invalid results. citrate

Normal Values Procedure

Adult men 0-15 mm/h 1. From Standby, scan the barcode on the test rack or press
Adult women 0-20 mm/h and hold NOT OK. The Main Menu will be shown.
2. Enter 5,RUN TEST RACK. You will be prompted:
RUN TEST RACK
Method 26. The Automated Streck ESR-100 Insert Rack (OK)
3. Insert the test rack in any free slot to start the verification
The ESR-100 is an automated 100-position ESR analyzer
procedure.
designed for clinical laboratories that run over 50 samples
4. After the test rack has been read, the results will be
daily. The instrument measures the sedimentation rate of ery-
reported on the printout.
throcytes in |.2-mL (pediatric) or 2.0-mL ESR-vacuum tubes.
5. If the printout indicates TEST OK, continue with the QC
Results are measured in mm/h and are available in 30 min-
procedure.
utes. Data can be downloaded to a Laboratory Information
6. Insert control samples in the desired ESR-100 rack.
system (LIS).
7. From Standby scan the barcode on the desired test rack or
enter the rack number on the keypad and press OK.
Principle
8. Enter an ID code for each control sample in the rack on
The instrument is designed to accurately and precisely mea-
the keypad. Press OK after each entry. Use the NOT OK
sure the sedimentation rate of erythrocytes in ESR-vacuum
key to delete any controls entered incorrectly. An ID-code
tubes. The results are recorded as mm per hour (Westergren
may contain a maximum of 16 characters.
method) by converting to the QuickMode Method, a scientif-
9. When prompted, insert the well-mixed rack into any
ically developed method for measuring ESR in only 30 min-
free slot in the ESR-100 (Fig. 31-37). The position light
utes. The instrument also makes a partial compensation for
will turn red and the ESR-100 will initiate testing. Con-
temperatures above 26°C. Red cell sedimentation is acceler-
trol results will automatically print when the test is
ated by an increase in the plasma concentration of acute phase
complete.
proteins, which are increased in acute tissue damage, chronic
10. Insert patient samples in the desired ESR-100 rack.
inflammation, chronic infection, and pregnancy. The ESR
11. From Standby scan the rack with the barcode scanner.
reflects both the increase in certain accelerating proteins, such
12. Scan each patient sample in the rack with the barcode
scanner to enter the patient ID codes. Use the NOT OK
key to delete any samples scanned incorrectly.
Table 51-7 Factors Affecting = Ee EES
13. When prompted, insert the well-mixed rack into any free
slot in the ESR-100. The position light will turn red and
the ESR-100 will initiate testing.
Increased ESR Decreased ESR
Interpretation
Rouleaux formation Microcytes
Fibrinogen (elevated) Sickle cells The ESR-100 will automatically scan the samples and print
Immunoglobulin (excess) Spherocytes the results when the sedimentation test is finished. Any error
codes will be printed on the ticket with the results.
 Chapter 31. Hematology Methods 789

Case Study
A 70-year-old woman diagnosed with multiple myeloma is
currently on melphalan and prednisone. Her CBC revealed
the following:

WBC 3.6 X 10°/L

RBC Bye) ee MOPe AE:
Heb 12.1 g/dL
Het 36%
MCV 100 fL
MCH 30 pg
MCHC 31%
Platelets ==
Manual differential:
Plasma cells >)
Figure 51-357 @ Streck ESR-100. A high-capacity erythrocyte sedi-
Lymphocytes 30
mentation rate (ESR) analyzer designed for laboratories that run
Segs 50
over 100 samples daily. (Courtesy of Streck Inc., Omaha, NE.).
Bands 5
Monocytes 7
Eosinophils B)
Limitations NRBCs 9/100 WBCs
If the sodium citrate in the tube is not properly mixed with the
blood, microscopic clots/aggregates may form and cause
A manual platelet count was done using Unopette
results to appear higher than they actually are. ESR-vacuum
methodology. Eight platelets were counted on one side and
tubes preserve the integrity of the patient sample for up to
10 platelets on the other side of the hemacytometer.
72 hours when transported or stored at 2 to 10°C. The
reportable range is 0-120 mm/h.?’
QUESTIONS
1. What is the platelet count (manual)?
2. What is the corrected WBC count?
3. What is the absolute number of plasma cells?

reer ome
1. Twenty microliters of blood are drawn into a Unopette 4. Which of the following factors affect the ESR?
system for a WBC count. Fifty cells are counted on one a. Increased fibrinogen
side and 52 on the other side of the hemacytometer b. Extreme poikilocytosis
(4 large squares). Calculate the WBC count. c. Use of heparin as an anticoagulant
dee. VOTE d. All of the above
Bel 2ae OL, 5. What type of hemoglobin electrophoresis would be best
Cole OC OWE to separate hemoglobins S and D?
dO L077 a. Cellulose acetate at alkaline pH
N . If a patient has a reticulocyte count of 8% with a hemat- b. Citrate agar at acid pH
ocrit of 18%, what is the corrected reticulocyte count? c. Either cellulose acetate or citrate agar may be used
a. 20% 6. What is the principle for the acid elution test for HbF?
be2 370 a. HbF is resistant to acid elution; it can be precipitated
Cas070 and stained.
d. 8% b. HbF is susceptible to acid elution; it can be dissolved
3. Which of the following errors will cause falsely elevated and measured photometrically.
results for a centrifugal microhematocrit? c. HbF is resistant to acid elution; it can be separated by
a. Reading of buffy coat with red cells aspirating the acid from the remaining hemoglobin.
b. Incomplete sealing of capillary tubes d. HbF is susceptible to acid elution; it can be destroyed
c. Allowing the centrifuge to