Laboratory Hematology Practice · 2013. 7. 23. · Carol Briggs and Samuel J. Machin 6 Method...

Laboratory Hematology Practice

This book is dedicated to the memory of Dr Berend Houwen and Dr Noriyuki Tatsumi

Laboratory Hematology PracticeEDITED BY

Kandice Kottke-Marchant MD, PhDChair, Pathology & Laboratory Medicine InstituteProfessor and Chair, Department of Pathology, Cleveland Clinic Lerner College of MedicineSection Head, Hemostasis and ThrombosisDepartment of Clinical PathologyCleveland ClinicCleveland, OH, USA

WITH

Bruce H. Davis MDTrillium Diagnostics, LLC,Bangor, ME, USA

A John Wiley & Sons, Ltd., Publication

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Library of Congress Cataloging-in-Publication Data

Laboratory hematology practice / edited by Kandice Kottke-Marchant, Bruce H. Davis.

p. ; cm.

Includes bibliographical references and index.

ISBN-13: 978-1-4051-6218-0 (hard cover : alk. paper)

ISBN-10: 1-4051-6218-X (hard cover : alk. paper)

1. Hematology. 2. Blood–Analysis. I. Kottke-Marchant, Kandice. II. Davis, Bruce H., M.D.

[DNLM: 1. Hematologic Tests–methods. 2. Blood Chemical Analysis–methods. 3. Cytological

Techniques. 4. Hematologic Diseases–diagnosis. QY 400]

RB45.L235 2012

616.07'561–dc23

2011023990

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print

may not be available in electronic books.

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v

Contributors, viii

Preface, xiii

Abbreviations, xv

I Cellular Analysis

1 Historical Perspective on Cellular Analysis, 3Elkin Simson

2 Cellular Morphologic Analysis of Peripheral Blood, 10Powers Peterson, Sheila McNeill, and Gene Gulati

3 Automated Cell Analysis: Principles, 26Bruce H. Davis and Patrick W. Barnes

4 Differential Leukocyte Analysis, 33Marie-Christine Béné and Francis Lacombe

5 Automated Platelet Analysis, 48Carol Briggs and Samuel J. Machin

6 Method Standardization in Cellular Analysis, 59Ian Giles and Richard Kendall

7 Spurious Counts and Spurious Results on Hematology Analyzers: Platelets, 66Marc Zandecki, Franck Genevieve, Jérémie Gérard, and Alban Godon

8 Spurious Counts and Spurious Results on Hematology Analyzers: White Blood Cells, Red Blood Cells, Hemoglobin, Red Cell Indices, and Reticulocytes, 79Marc Zandecki, Franck Genevieve, Jérémie Gérard, and Alban Godon

9 Evaluation of Hematology Analyzers, 96Carol Briggs

II Flow Cytometry in Hematologic Diagnosis

10 Flow Cytometric Specimen Collection, Processing, and Reporting, 105Maryalice Stetler-Stevenson, Bruce Greig, and Constance Yuan

11 Validation and Quality Control in Clinical Flow Cytometry, 115Norman B. Purvis and Teri Oldaker

12 A New Paradigm for Cytometric Analysis, 131C. Bruce Bagwell

III Molecular Diagnostics

13 Sample Collection, Processing, and Storage for Molecular Genetic Testing, 145Bobby L. Boyanton Jr. and Domnita Crisan

14 Development of Amplification-Based Molecular Genetic Testing in Hematology, 155Raymond R. Tubbs

15 Single Nucleotide Polymorphisms in Molecular Diagnostics, 168Gurunathan Murugesan, Seongsoo Jang, and Jin-Yeong Han

16 Molecular Cytogenetic (FISH) Analysis of Hematolymphoid Disorders, 181Mark A. Micale and Anwar N. Mohamed

17 Immunoglobulin and T-Cell Receptor Gene Rearrangement Analysis for Diagnosis of Hematologic Malignancies, 199Jacques J. M. van Dongen, Anton W. Langerak, and Tomasz Szczepański

18 Minimal Residual Disease Detection in Acute Myeloid Leukemia by RQ-PCR and Multiparameter Flow Cytometry, 220Wolfgang Kern and Susanne Schnittger

19 Engraftment Analysis Using Short Tandem Repeats Following Allogeneic Hematopoietic Cell Transplantation, 234Ilka Warshawsky and Hyun-Sook Chi

20 Molecular Profiling Methods in the Diagnosis of Hematologic Disorders, 244Annette S. Kim, Stephen R. Master, and Cherie H. Dunphy

Contents

Contents

vi

21 The Role of Classical Cytogenetics in Hematologic Diagnosis, 267Kathleen Richkind

IV Bone Marrow Analysis

22 Bone Marrow Processing and Normal Morphology, 281Karl S. Theil

23 Cytochemical, Immunocytochemical, Histochemical, and Immunohistochemical Staining of Bone Marrow and Peripheral Blood, 300Alexandra Rice and Barbara J. Bain

24 The Myeloproliferative Neoplasms, 315Sindhu Cherian and Brent L. Wood

25 Myelodysplastic Disorders, 331Maryalice Stetler-Stevenson, Joseph A. DiGiuseppe, and Diane C. Arthur

26 Acute Leukemias, 345Joseph A. DiGiuseppe and Laila Mnayer

27 Lymphoproliferative Disorders, 364Robert W. Allan, Samer Z. Al-Quran, Ying Li, and Raul C. Braylan

28 Plasma Cell Disorders, 381Bill G. Richendollar and James R. Cook

29 Diagnosis of Adult Mastocytosis: Role for Bone Marrow Analysis, 388Luis Escribano, Andrés Garcia-Montero, Laura Sanchez-Muñoz, Cristina Teodosio, Ivan Alvarez-Twose, Maria Jara-Acevedo, José Mario Morgado, Julia Almeida, Alberto Orfao for the Spanish Network on Mastocytosis (REMA)

30 Paroxysmal Nocturnal Hemoglobinuria (PNH) Analysis, 399Stephen J. Richards

V Hemostasis and Thrombosis

31 Sample Collection and Processing in Hemostasis, 409Dorothy M. Adcock

32 Performance and Interpretation of Routine Coagulation Assays, 420Kandice Kottke-Marchant

33 Clotting Factors and Inhibitors: Assays and Interpretation, 435Piet Meijer, H. W. Verbruggen, and Michael Spannagl

34 Diagnosis of von Willebrand Disease, 447Emmanuel J. Favaloro and Jerry Koutts

35 Diagnosis of Fibrinolytic Disorders, 460Wayne Chandler

36 Laboratory Evaluation of Acquired Coagulation Disorders, 468Richard A. Marlar and Katerine Seywerd

37 Platelet Assays and Platelet Dysfunction, 480Paul Harrison and David Keeling

38 Thrombophilia: Assays and Interpretation, 492Sophia Yohe and John Olson

39 Lupus Anticoagulants, Antiphospholipid Antibodies, and Antiphospholipid Syndrome, 509William L. Nichols, Kandice Kottke-Marchant, Marlies R. Ledford-Kraemer, Henry A. Homburger, and Layna K. Cardel

40 Heparin-Induced Thrombocytopenia, 526Sixten Selleng and Andreas Greinacher

41 Anticoagulant, Antiplatelet, and Thrombolytic Drugs, 535Laura Green and Samuel J. Machin

42 Guidelines for Evaluation of Coagulation Analyzers and Coagulation Testing, 543Chris Gardiner

VI Special Hematology Techniques

43 Essentials of Immunohematology, 555A. Bradley Eisenbrey

44 Analysis of Hemoglobinopathies, Hemoglobin Variants and Thalassemias, 562Monica V. E. Gallivan and Piero C. Giordano

45 Nutritional Anemia, 586Ralph Green

46 Hemolytic Anemias Associated with Disorders of Erythrocyte Membrane and Cytoskeleton Proteins, 602May-Jean King, Lesley J. Bruce, and Didier Dhermy

47 Body Fluid Analysis, 615David J. Blomberg, Katherine Galagan, and Eric F. Glassy

48 Malaria Analysis, 626Qigui Li, Peter J. Weina, and R. Scott Miller

49 Erythrocyte Sedimentation Rate (ESR), 638Josep M. Jou

VII Point-of-Care Testing

50 Needs Analysis and Selection of Point-of-Care Testing Analyzers, 649Albert Huisman

Contents

vii

51 Point-of-Care Testing in Hemostasis, 655Steve Kitchen and Dianne Kitchen

52 Point-of-Care Hematology Assays, 668Albert Huisman

VIII Management of Information

53 Laboratory Management, 675Stefanie L. McFadden and Linda M. Sandhaus

54 Hematology Quality Practices, 686George S. Cembrowski

55 Digital Imaging in Hematology, 707Danny Hsu and Szu-Hee Lee

Index, 719

viii

Dorothy M. Adcock MDMedical Director

Esoterix Inc.

Englewood, CO, USA

Robert W. Allan MDClinical Associate Professor

Department of Pathology and Laboratory

Medicine

University of Florida College of Medicine

Gainesville, FL, USA

Julia Almeida MD, PhDAssociate Professor

Servicio General de Citometría and

Departamento de Medicina

Instituto de Biología Celular y Molecular del

Cáncer

Centro de Investigación del Cáncer/IBMCC

(CSIC-USAL)

Universidad de Salamanca

Salamanca, Spain

Samer Z. Al-Quran MDAssociate Professor

Department of Pathology, Immunology and

Laboratory Medicine



Ivan Alvarez-Twose MDClinical Associate Professor

Instituto de Estudios de Mastocitosis de

Castilla La Mancha

Hospital Virgen del Valle

Toledo, Spain

Diane C. Arthur MDStaff Clinician

Head, Clinical Cytogenetics Section

Laboratory of Pathology, CCR, NCI, NIH

Bethesda, MD, USA

C. Bruce Bagwell MD, PhDPresident

Verity Software House

Topsham, ME, USA

Barbara J. Bain MBBS, FRACP, FRCPathProfessor of Diagnostic Haematology

Imperial College Faculty of Medicine

St Mary’s Hospital

London, UK

Patrick W. Barnes MA MT(ASCP)Laboratory Manager—Hematology

Barnes-Jewish Hospital

St. Louis, MO, USA

Marie-Christine Béné PharmSciD, PhDImmunology Professor and Biologist

Centre Hospitalier Universitaire et Faculté de

Médecine de Nancy

Nancy Université

Nancy, France

David J. Blomberg MDFormerly Associate Professor, Pathology and

Laboratory Medicine, Univ. of MN, Duluth,

School of Medicine.

Formerly of Arrowhead Pathologists, P.A.

Duluth, MN, USA

Bobby L. Boyanton Jr. MDAssociate Professor of Pathology

Oakland University William Beaumont School

of Medicine

Medical Director, Clinical Microbiology

Associate Medical Director, Molecular

Pathology

Beaumont Hospital

Department of Clinical Pathology

Royal Oak, MI, USA

Raul C. Braylan MDProfessor Emeritus


Medicine



Contributors

Carol Briggs BSc, FIBMSChief Biomedical Scientist and Researcher

Department of Haematology

University College London Hospitals

London, UK

Lesley J. Bruce PhDSenior Research Scientist

Bristol Institute for Transfusion Sciences

NHS Blood & Transplant

Bristol, UK

Layna K. Cardel BS, MT(ASCP)Education Specialist

Mayo Special Coagulation Laboratory

Department of Laboratory Medicine and

Pathology

Mayo Clinic Rochester;

Instructor, Laboratory Medicine

Mayo Clinic College of Medicine

Rochester, MN, USA

George S. Cembrowski MD, PhDDirector, Medical Biochemistry,

University Hospital

Associate Professor,


Pathology

University of Alberta

Edmonton, Alberta, Canada

Wayne Chandler MDVice Chair

Clinical Coagulation and Chemistry


Medicine

The Methodist Hospital

Houston, TX, USA

Sindhu Cherian MDAssistant Professor

Department of Laboratory Medicine

University of Washington

Seattle, WA, USA

Contributors

ix

Hyun-Sook Chi MD, PhDEmeritus Professor

University of Ulsan

College of Medicine and Asan Medical

Center

Seoul, Korea

James R. Cook MD, PhDAssociate Professor of Pathology

Cleveland Clinic Lerner College of Medicine

Molecular Hematopathology Section Head

Pathology and Laboratory Medicine Institute

Cleveland Clinic

Cleveland, OH, USA

Domnita Crisan MD, PhDProfessor of Pathology


of Medicine

Medical Director, Molecular Pathology

William Beaumont Hospital


Royal Oak, MI, USA

Bruce H. Davis MDTrillium Diagnostics, LLC,

Bangor, ME, USA

Didier Dhermy MD, PhDDirecteur de Recherche au CNRS

Institut National Transfusion Sanguine

Paris, France

Joseph A. DiGiuseppe MD, PhDDirector, Special Hematology Laboratory

Department of Pathology & Laboratory

Medicine

Hartford Hospital

Hartford, CT, USA

Cherie H. Dunphy MD, FCAP, FASCPProfessor of Pathology and Laboratory

Medicine


Medicine

University of North Carolina

Chapel Hill, NC, USA

A. Bradley Eisenbrey MD, PhDLaboratory Director

Gift of Life Michigan

Ann Arbor, MI, USA

Luis Escribano MD, PhDDirector


Castilla La Mancha


Toledo, Spain

Emmanuel J. Favaloro PhD FFSc (RCPA)Principle Hospital Scientist


Institute of Clinical Pathology and Medical

Research (ICPMR)

Westmead Hospital

Westmead, NSW, Australia

Katherine Galagan MDDirector of Clinical Laboratories and Section

Head

Virginia Mason Medical Center

Seattle, WA, USA

Monica V. E. Gallivan MDMedical Director, Hematology

Quest Diagnostics Nichols Institute

Chantilly, VA, USA

Andrés Garcia-Montero PhDResearcher

Servicio General de Citometria and



Cáncer


(CSIC-USAL)


Salamanca, Spain

Chris Gardiner PhD, MSc, FIBMSPostdoctoral Scientist

Nuffield Department of Obstetrics and

Gynaecology

University of Oxford

Oxford, UK

Franck Geneviève MDLaboratoire d’Hématologie

Centre Hospitalier Universitaire

Angers, France

Jérémie Gérard PhDDoctor of Pharmacy

Laboratoire d’Hématologie


Angers, France

Ian Giles MDDirector of Scientific Affairs

Sysmex America Inc

Mundelein, IL, USA

Piero C. Giordano PhDAssociated Professor

Clinical biochemical molecular geneticist

Center for Human and Clinical Genetics

Leiden Academic Hospital (LUMC)

Leiden, The Netherlands

Eric F. GlassDc

Contributors

x

Henry A. Homburger MDConsultant (Emeritus), Laboratory Medicine &

Pathology

Former Director, Mayo Antibody Immunology

Laboratory

Division of Clinical Biochemistry and

Immunology


Pathology


Professor (Emeritus), Laboratory Medicine and

Pathology


Rochester, MN, USA

Danny Hsu MBBS, FRACP, FRCPASenior Registrar in Haematology


St George Hospital

Sydney, NSW, Australia

Albert Huisman PhDClinical Chemist

University Medical Center Utrecht

Department of Clinical Chemistry and

Haematology

Utrecht, The Netherlands

Seongsoo Jang MD, PhDAssistant Professor

Asan Medical Center

University of Ulsan College of Medicine

Seoul, Korea

Maria Jara-Acevedo MScPhD student, Servicio General de Citometría

and Departamento de Medicina,


Cáncer


(CSIC-USAL)


Salamanca, Spain

Josep M. Jou MD, PhDSenior Consultant

Servei Hemoteràpia i Hemostàsia

Hospital Clinic, University of Barcelona

Barcelona, Spain

David Keeling BSc MD FRCP FRCPathOxford Haemophilia and Thrombosis Centre

Churchill Hospital

Oxford, UK

Richard Kendall PhDDirector of Global Scientific Affairs

Commercial Director Future Solutions

Abbott Hematology

Santa Clara, CA, USA

Wolfgang Kern MDHead Immunophenotyping

MLL Munich Leukemia Laboratory

Munich, Germany

Annette S. Kim MD, PhDAssistant Professor of Pathology, Microbiology

and Immunology

Department of Pathology, Microbiology and

Immunology

Vanderbilt University Medical Center

Nashville, TN, USA

May-Jean King PhDSenior Research Biochemist/

Clinical Scientist

Membrane Biochemistry

International Blood Group Reference

Laboratory

NHS Blood & Transplant

Bristol, UK

Steve Kitchen PhDClinical Scientist

Sheffield Haemostasis and

Thrombosis Centre

Royal Hallamshire Hospital

Sheffield, UK

Dianne Kitchen PhDSenior BMS

UK NEQAS for Blood Coagulation

Sheffield, UK

Kandice Kottke-Marchant MD, PhDChair, Pathology & Laboratory Medicine

Institute

Professor and Chair, Department

of Pathology, Cleveland Clinic Lerner

College of Medicine

Section Head, Hemostasis and

Thrombosis

Dept. of Clinical Pathology

Cleveland Clinic,

Cleveland, OH, USA

Jerry Koutts MD (Syd) BS FRACP FRCPADepartment of Haematology

Institute of Clinical Pathology and Medical

Research (ICPMR)

Westmead Hospital

Westmead, NSW, Australia

Francis Lacombe MD, PhDHematology Laboratory

Hôpital Haut-Lévêque

Pessac, France

Anton W. Langerak PhDAssociate Professor

Medical Immunologist

Department of Immunology

Erasmus MC

University Medical Center Rotterdam

Rotterdam, The Netherlands

Marlies R. Ledford-Kraemer MBA, BS, MT(ASCP)SHPresident, CLOT-ED, Inc.

Emeritus Executive Secretary, NASCOLA

Islamorada, FL, USA

Szu-Hee Lee MBBChir, PhD, FRACP, FRCPAProfessor (Conjoint) and Senior Staff

Haematologist


St George Hospital

Sydney, NSW, Australia

Qigui Li MD, PhDChief of Pharmacokinetics/Pharmacodynamics

Department of Pharmacology

Division of Experimental Therapeutics

Walter Reed Army Institute of Research

Silver Spring, MD, USA

Ying Li MD, PhDClinical Associate Professor


Medicine



Samuel J. Machin MD, FRCP, FRCPathProfessor of Haematology, University College

London

Consultant Haematologist and Clinical Lead

for Laboratory Services

University College London Hospitals

London, UK

Richard A. Marlar PhDProfessor of Pathology

Laboratory Services

Oklahoma City Veterans Administration

Medical Center

University of Oklahoma Health Sciences

Center

Oklahoma City, OK, USA

Stephen R. Master MD, PhDAssistant Professor of Pathology and

Laboratory Medicine


Medicine

University of Pennsylvania School of Medicine

Philadelphia, PA, USA

Contributors

xi

Stefanie L. McFadden MT(ASCP)SHClinical Laboratory Consultant

McFadden Laboratory Consulting

Columbus, OH, USA

Sheila McNeill BSMT(ASCP)SHClinical Specialist Flow Cytometry

Sentara Norfolk General Hospital

Norfolk, VA, USA

Piet Meijer PhDDirector, ECAT Foundation

Leiden, The Netherlands

Mark A. Micale PhD, FACMGAssociate Professor of Patholgy and Laboratory

Medicine


of Medicine

Medical Director

Clinical Cytogenomics Laboratory

Beaumont Health System

Beaumont Laboratory

Royal Oak, MI, USA

R. Scott Miller MD, FIDSADivision of Experimental Therapeutics



Laila Mnayer PhD, FACMGDirector of Molecular Pathology and

Cytogenetics


Medicine

Hartford Hospital

Hartford, CT, USA

Anwar N. Mohamed MD, FACMGProfessor of Pathology

Department of Pathology/Cytogenetic

Laboratory

Wayne State University School of Medicine/

Detroit Medical Center

Detroit, MI, USA

José Mario Morgado MScMaster in Science, Biology Researcher


Castilla La Mancha


Toledo, Spain

Gurunathan Murugesan PhDPathology and Laboratory Medicine Institute

Cleveland Clinic

Cleveland, OH, USA

William L. Nichols MDConsultant, Hematology & Internal Medicine,

and Laboratory Medicine & Pathology

Mayo Special Coagulation Laboratory and

Coagulation Clinical Centers

Divisions of Hematopathology and Hematology


Associate Professor, Medicine and Laboratory

Medicine


Rochester, MN, USA

Teri Oldaker MLS (ASCP), QCYMSenior Director of Flow Cytometry

Genoptix Medical Laboratory

Carlsbad, CA, USA

John Olson MD, PhDProfessor and Vice Chair for Clinical Affairs

Department of Pathology

The University of Texas Health Science Center

Director of Clinical Laboratories

University Health System

San Antonio, TX, USA

Alberto Orfao MD, PhDProfessor, Servicio General de Citometría, and



Cáncer


(CSIC-USAL)


Salamanca, Spain

Powers Peterson MDFormerly: Associate Professor of Pathology &

Laboratory Medicine

Weill Cornell Medical College in Qatar

New York, NY, USA;

Consultant in Laboratory Medicine and

Pathology

Hamad Medical Corporation

Doha, Qatar

Currently: Medical Director

Quest Diagnostics Nichols Institute

Valencia, CA, USA

Norman B. Purvis, Jr. PhDSenior Director, Development

Nodality, Inc.

South San Francisco, CA, USA

Alexandra Rice FRCPathConsultant Histopathologist

Royal Brompton Hospital

London, UK

Stephen J. Richards PhD FRCPathConsultant Clinical Scientist

Haematological Malignancy Diagnostic Service


St James’s Institute of Oncology

Leeds Teaching Hospitals NHS Trust

Leeds, UK

Bill G. Richendollar MDStaff Pathologist

De Kalb Medical

Decatur, GA, USA

Kathleen Richkind PhD, FACMGClinical Cytogeneticist

Genzyme Genetics

Santa Fe, NM, USA

Laura Sanchez-Muñoz MD, PhDInstituto de Estudios de Mastocitosis de

Castilla La Mancha


Toledo, Spain

Linda M. Sandhaus MD, MSAssociate Professor of Pathology

University Hospitals Case Medical Center

Cleveland, OH, USA

Susanne Schnittger PhDHead of Molecular Genetics

MLL Munich Leukemia Laboratory

Munich, Germany

Sixten Selleng MDConsultant in Anaesthesia

Klinik für Anasthesiologie und

Intensivmedizin

Ernst-Moritz-Arndt Universität

Greifswald, Germany

Katerine Seywerd MDPathology Resident

Department of Pathology

University of Oklahoma Health Sciences

Center

Oklahoma City, OK, USA

Elkin Simson MD, FCAP, FASCPConsultant in Hematology, Laboratory

Automation and Laboratory Informatics

Clinical Associate Professor, Mount Sinai

School of Medicine

New York, NY, USA

Michael Spannagl MD, PhDHemostasis and Transfusion Medicine

University Hospital

Munich, Germany

Contributors

xii

Maryalice Stetler-Stevenson MD, PhDChief, Flow Cytometry Unit

Laboratory of Pathology, CCR, NCI, NIH

Bethesda, MD, USA

Tomasz Szczepanski MD, PhDProfessor, Head of the Department

Department of Pediatric Hematology and

Oncology

Medical University of Silesia

Zabrze, Poland

Cristina Teodosio MScPhD student, Servicio General de Citometria

and Departamento de Medicina


Cáncer


(CSIC-USAL)


Salamanca, Spain

Karl S. Theil MDStaff Pathologist


Cleveland Clinic

Cleveland, OH, USA

Raymond R. Tubbs DOHead, Section of Molecular Oncologic

Pathology

Department of Molecular Pathology

Pathology and Laboratory Medicine Institute

Cleveland Clinic;

Professor of Pathology

Cleveland Clinic Lerner College of Medicine

Cleveland, OH, USA

Jacques J. M. van Dongen MD, PhDProfessor of Immunology

Department of Immunology

Erasmus MC

University Medical Center Rotterdam

Rotterdam, The Netherlands

H. W. Verbruggen PhDUniversity Medical Centre St. Radboud

Central Laboratory for Hematology

Nijmegen

The Netherlands

Ilka Warshawsky MD, PhDStaff Pathologist

Department of Molecular Pathology

Cleveland Clinic Foundation

Cleveland, OH, USA

Peter J. Weina PhD, MD, FACP, FIDSAHeadquarters



Brent L. Wood MD, PhDProfessor, Hematopathology Laboratory

Department of Laboratory Medicine

University of Washington

Seattle, WA, USA

Sophia Yohe MDAssistant Professor


Pathology

Divisions of Hematopathology and Molecular

Diagnostic Pathology

University of Minnesota Medical Center

Minneapolis, MN, USA

Constance Yuan MD, PhDStaff Clinician

Laboratory of Pathology, NCI, NIH

Bethesda, MD, USA

Marc Zandecki PhDProfessor of Hematology

Laboratoire d’Hématologie


Angers, France

xiii

Preface

Hematologic disorders range from some of the most common medical conditions, like anemia and leukocytosis, to some of the most rare and perplexing conditions, like inherited thrombocytopenia and mast cell disorders. Yet, due to the relatively easy accessibility of blood samples, hematology is a rapidly evolving area of medical knowledge that has seen a virtual explosion of diagnostic laboratory testing and ther-apeutics in the past decade. Common diagnostic methods range from visual peripheral blood and bone marrow mor-phology to flow cytometric analysis of erythrocytes, leuko-cytes and platelets, to functional testing of hemostasis proteins and platelets, to electrophoretic hemoglobin analy-sis and molecular DNA and RNA analysis of leukemias and lymphomas.

Laboratory Hematology Practice is a new textbook focused on a practical approach to understanding the diagnostic utility of new hematology laboratory testing, how to critically assess new testing technology and how to implement, inter-pret and manage new assays and new technology in the laboratory. Most hematology textbooks focus on disease pathophysiology and patient management, but gloss over the details of laboratory diagnosis, an important aspect of patient care. Laboratory testing is complex and there are many details of accurate laboratory testing, from method selection to assay validation, quality control, results report-ing and interpretation. Thus we chose to focus on diagnostic methodologies for this book and structured it along technol-ogy lines instead of by disease category. Technologies covered include cellular analysis, flow cytometry, molecular diagnos-tics, bone marrow analysis, hemostasis and thrombosis testing, special hematology techniques, point-of-care testing and information management. Emphasis is placed upon the use of established and emerging technologies in the diagno-sis of hematologic disorders, but also includes operational aspects of laboratory testing, such as proper sample collec-tion, quality control, informatics and laboratory manage-ment. Unique features of Laboratory Hematology Practice are incorporation of international standards for laboratory prac-tice, methods for evaluation of testing technology, and on-line access to book content and figures. We hope that Laboratory Hematology Practice will provide practical informa-

tion and fill a niche in the field as it will help laboratorians critically understand, assess, implement and manage a wide variety of hematologic technologies ranging from cellular analysis to hemostasis testing and molecular diagnostics. We hope this text will become a welcome addition to the library of every practicing hematologist, hematopathologist, labora-tory scientist in hematology, and trainees in these areas of medical and laboratory practice.

This project was sponsored by the International Society for Laboratory Hematology (ISLH). The ISLH arose out of a series of symposia on “Technological Innovations in Laboratory Hematology” organized by Dr Berend Houwen that started in Lake Louise, Canada in 1983. Dr Houwen and Dr Bruce Davis then led an international group of laboratory professionals in the formation of ISLH in 1992. In addition to Drs Houwen and Davis, the original founding ISLH Directors were Dr Brian Bull, Dr Ralph Green, Professor Samuel Machin, Dr Robert Pierre, Dr Elkin Simson and Dr Noriyuki Tatsumi. The goal of ISLH is “to further the interests of laboratory hematology as a professional activity and to provide a forum for its members for the exchange of new ideas and information on the subject of laboratory hematology”. It is the only international society specifically focused on the technological and laboratory aspects of hematology. Since 1992, the ISLH has grown to an interna-tional organization that holds annual meetings around the globe, with over 700 members from over 50 countries. The ISLH has developed strong affiliations with many national hematology societies. The ISLH originated as a society focused on aspects of cellular analysis and has grown to incorporate dissemination of knowledge in hemostasis and thrombosis, hematologic molecular diagnosis, bone marrow morphology, standards and guidelines, hematology infor-matics, hemoglobinopathies and point-of-care testing. The ISLH publishes a scientific journal, the International Journal of Laboratory Hematology. Given the focus on technological advances in laboratory hematology, it is truly fitting that this textbook, Laboratory Hematology Practice, should be sponsored by the ISLH.

It is with gratitude and respect that we pay tribute and dedicate this book to two of the original ISLH Directors who

Preface

xiv

have passed away, Dr Berend Houwen and Dr Noriyuki Tatsumi. Dr Berend Houwen (1941–2004), the co-founder of ISLH, was most recently Corporate Medical Director of Beckman Coulter, preceded by Associate Professor of Pathology at Loma Linda University and Associate Professor of Pathology & Medicine at the University of Calgary. Berend was a man of vision and determination, who was a cham-pion for laboratory hematology and published widely on many aspects of laboratory hematology testing, most notably flow cytometry, microcytic anemia, hematology testing errors, red cell volume distribution and red cell indices. Dr Noriyuki Tatsumi (1937–2010) was most recently Professor of Clinical and Laboratory Medicine at Osaka City

University. Dr Tatsumi was a leader in standardization and quality assurance of hematological testing and carried out a wide range of investigation in the field of laboratory hema-tology. In addition to many years of dedication to ISLH, he founded the Japanese Society for Laboratory Hematology (JSLH) in 2000. These two original ISLH Directors exemplify the passion for laboratory hematology and for lifelong research and education, which we hope pervades every page of this textbook.

Kandice Kottke-Marchant, MD, PhDBruce H. Davis, MD

7/1/11

xv

1P oneparameter2P twoparameter2D-DIGE two-dimensionaldifferencegel

electrophoresisA adenine(deoxyadenylate)AABB AmericanAssociationofBloodBanks7-AAD 7-aminoactinomycinDABC activatedB-cellAC AlternatingcurrentAC/DC Alternatingcurrent/directcurrentACD acidcitratedextroseaCGH arraycomparativegenomichybridizationaCL anticardiolipinantibodiesACMG AmericanCollegeofMedicalGeneticsaCML atypicalchronicmyelogenousleukemiaACS acutecoronarysyndromeACT activatedclottingtimeAct activityADAMTS-13 adisintegrinandmetalloproteinasewitha

thrombospondintype1motif,member13ADP adenosinediphosphateAg antigenAIDS acquiredimmunedeficiencysyndromeAILT angioimmunoblasticT-celllymphomaALCL anaplasticlargecelllymphomaALK anaplasticlymphomakinaseALL acutelymphoblasticleukemiaAML acutemyeloidleukemiaAMP AssociationforMolecularPathologyANAE α-naphthylacetateesteraseANBE α-naphthylbutyrateesteraseANSI AmericanNaitonalStandardsInstituteAP antiplasminAPC activatedproteinC;allophycocyaninAPCR activatedproteinCresistanceaPL antiphospholipidantibodiesAPS antiphospholipidsyndromeAPTT activatedpartialthromboplastintimeARMS amplificationrefractorymutationsystemASM aggressivesystemicmastocytosisaSNP arraysinglenucleotidepolymorphisms

ASO allelespecificoligonucleotideASPCR allele-specificpolymerasechainreactionAT antithrombinATLL adultTcellleukemia/lymphomaATM mutatedinataxiatelangiectasiaATP adenosinetriphosphateATRA all-trans-retinoicacidAVK anti-vitaminKtherapyAZF aceticacid-zinc-formalinBAC bacterialartificialchromosomeB-ALL B-cellacutelymphoblasticleukemiaBCP-ALL B-cellprecursoracutelymphoblastic

leukemiaBCR breakpointclusterregionBCSH BritishCommitteeforStandardsin

Haematologyβ2-GPI β2-glycoproteinIbHLH basichelix-loop-helixBL BurkittlymphomaB-LBL B-lymphoblasticlymphomaBM bonemarrowBMT bonemarrowtransplantationbp basepairs2,3-BPG 2,3-bisphosphoglycericacidB-PLL B-cellprolymphocyticleukemiaBREC B-cellreceptorexcisioncircleC constantregion;cytosine(deoxy

cytidylate)C4b-BP C4bbindingproteinCa calciumCaCl2 calciumchlorideCADP collagenplusadenosinediphosphateCAE chloroacetateesterasecAMP cyclicadenosinemonophosphateCAP CollegeofAmericanPathologistsCAPRIE ClopidogrelversusAspirininPatientsat

RiskofIschaemicEventsCB collagenbindingCBC completebloodcountCBF core-bindingfactorCBFβ corebindingfactorbeta

Abbreviations

Abbreviations

xvi

CBS cystathionine-β-synthaseCCD charge-coupleddeviceCD clusterofdifferentiationCDAII congenitaldyserythropoieticanemia

typeIIcDNA codingDNA;complementaryDNACDR complementaritydeterminingregionCE ComformitéEuropéenneCEBPA CCAAT/enhancerbindingprotein-αCEL chroniceosinophilicleukemiaCEP centromere-enumerationprobesCEPI collagenplusepinephrineCF:C clottingfactorcoagulantactivityCGH comparativegenomichybridizationcGMP cyclicguanosinemonophosphateCH3 methylgroupCHAMP CHatAMPCHARISMA ClopidogrelforHighAtherothrombotic

RiskandIschemicStabilization,ManagementandAvoidance

CHARM comprehensivehigh-throughputarraysforrelativemethylation

CHCM cellularhemoglobinconcentrationmeanChIP chromatinimmunoprecipitationcHL classicalHodgkinlymphomaCHOP cyclophosphamide,doxorubicin,vincristine

andprednisoloneCISH chromogenicin situhybridizationCLIA ClinicalLaboratoryImprovement

AmendmentsCLL chroniclymphocyticleukemiaCLL/PL chroniclymphocyticleukemiawith

increasedprolymphocytesCLSI ClinicalandLaboratoryStandardsInstituteCML chronicmyelogenousleukemiaCMML chronicmyelomonocyticleukemiaCMOS complementarymetal-oxide

semiconductorCMS CenterforMedicareServicesCMV cytomegalovirusCNL chronicneutrophilicleukemiaCNLOH copy-neutrallossofheterozygosityCNS CentralnervoussystemCO2 carbondioxideCODIS CombinedDNAIndexSystemCOG Children’sOncologyGroupCOSHH controlofsubstanceshazardoustohealthCOX-1 cyclooxygenase-1CP cancerprocoagulantCPDA citrate-phosphate-dextrose-adenineCpG cytosine–phosphate–guaninedinucleotideCPT currentproceduralterminologyCR completeremissionCR1 complementreceptortype1

cRNA complementaryRNACRP C-reactiveproteinCRTH2 chemo-attractanthomologousreceptor

expressedonT-helper2cellsCSA CanadianStandardsAssociationCSF cerebrospinalfluidCSR class-switchrecombinationCT computerizedtomographyCT cyclethresholdCTAD citrate,theophylline,adenosine,

dipyridamoleCTCL cutaneousT-celllymphomaCV coefficientofvariationCy3 cyanine3Cy5 cyanine5Cy7 cyanine7CYP2C9 cytochromeP4502C9CZE capillaryzoneelectrophoresisD diversityregion;diagnosisDAB 3,3′-diaminobenzidineDAF decayaccelerationfactorDAPI 4′,6-diamidino-2-phenylindoledAPTT diluteAPTTDAT directantiglobulintestDC dualcolorDCBA dual-colorbreak-apartDCDF dual-colordual-fusionDCFDA dichlorodihydrofluoresceindiacetateDCIP dichlorophenolindophenolddNTP dideoxynucleosidetriphosphateDEPC diethylpyrocarbonateD-FISH double-fusionfluorescencein situ

hybridizationDGGE denaturinggradientgelelectrophoresisdhfr dihydrofolatereductasedhps dihydropteroatesynthaseDI deformabilityindexDIC disseminatedintravascularcoagulationDICOM DigitalImagingandCommunicationsin

MedicineDLBCL diffuselargeB-celllymphomaDLCL diffuselargecelllymphomaDMSO dimethylsulfoxideDNA deoxyribonucleicacidDNase deoxyribonucleaseDOT DepartmentofTransportationdpi dotsperinchdPT dilutePTdRVVT diluteRussell’sViperVenomTimedsDNA double-strandedDNADTI directthrombininhibitorDVT deepveinthrombosisEBV Epstein–BarrvirusEC endothelialcell

Abbreviations

xvii

ECD electroncoupleddyeECM extracellularmatrixECTA EuropeanConcertedActionon

ThrombophiliaEDP EDTA-dependentpseudothrombocytopeniaEDTA ethylenediaminetetraaceticacidED-WORTY EDTAwithwortmaninandtyrphostinEIA enzymeimmunoassayELISA enzyme-linkedimmunosorbentassayELN EuropeanLeukemiaNetEMA eosin-5-maleimide;epithelialmembrane

antigenEORTC EuropeanOrganisationforResearchand

TreatmentofCancerEPCR endothelialproteinCreceptorEPI epinephrineERG v-etserythroblastosisvirusE26oncogene

homologERIC EuropeanResearchInitiativeonCLLES extrasignalESI electrosprayionizationESR erythrocytesedimentationrateET essentialthrombocythemiaFAB French–American–BritishFACS fluorescence-activatedcellsortingFBI FederalBureauofInvestigationFcεRI high-affinityimmunoglobulinEFc

receptorFcγRI high-affinityimmunoglobulinGFc

receptorFcγRIIa immunoglobulinGFcreceptorIIaFCS fetalcalfserumFDA FoodandDrugAdministrationFDP fibrindegradationproductFEU fibrinogenequivalentunitFFP freshfrozenplasmaFGFR fibroblastgrowthfactorreceptorFICTION fluorescenceimmunophenotypingand

interphasecytogeneticsasatoolfortheinvestigationofneoplasms

FII:C factorIIcoagulantactivityFISH fluorescencein situhybridizationFITC fluoresceinisothiocyanateFIX factorIXFIX:C factorIXcoagulantactivityfL femtoliterFL follicularlymphomaFLAER FLuorescentAERolysinFLT3 fms-liketyrosinekinase3FRET fluorescenceresonanceenergytransferFSC forwardscatterFSP fibrinsplitproductsFV:C factorVcoagulantactivityFVII:C factorVIIcoagulantactivity

FVIIIB factorVIIIbindingFVIII:C factorVIIIcoagulantactivityFVL factorVLeidenFXa activatedfactorXFX:C factorXcoagulantactivityFXI:C factorXIcoagulantactivityFXII:C factorXIIcoagulantactivityg relativecentrifugalforceG guanosine;gauge(deoxyguanylatl)GB gigabyteG-CSF granulocytecolonystimulatingfactorGCB germinalcenterB-cellGCT giantcelltumorGe GerbichGGCX γ-glutamylcarboxylaseGGS GlobinGeneServerGM-CSF granulocyte-macrophagecolonystimulat-

ingfactorG6PD glucose-6-phosphatedehydrogenaseGP glycoproteinGPI glycosylphosphatidylinositolGPIBA glycoproteinIbαGS GepriiftesrchorhoitG-TBF GiemsastainedthickbloodfilmGTG G-bandingwithtrypsintreatmentand

GiemsastainGTLLF GroupedeTravailsurlesLeucémieset

LymphomesFrancophoneGVHD graft-versus-hostdiseaseGVT graft-versus-tumorHb hemoglobinconcentrationHCDD heavychaindepositiondiseaseHCT hematocritHE hereditaryelliptocytosisH&E hematoxylinandeosinHEP HumanEpigenomeProjectHES hypereosinophilicsyndromeHFE hemochromatosisgeneHGP HumanGenomeProjectHGVS HumanGenomeVariationSocietyHHV8 humanherpesvirus8HiCN hemiglobincyanideHIPA heparin-inducedactivationassayHIPAA HealthInsurancePortabilityand

AccountabilityActHIT heparin-inducedthrombocytopeniaHIV humanimmunodeficiencyvirusHL HodgkinLymphomaHLA humanleukocyteantigenHLDA humanleukocytedifferentialantigenHMW highmolecularweightHMWK highmolecularweightkininogenHPC hematopoieticprecursorand

progenitorcells

Abbreviations

xviii

HPF high-powerfieldHPFH hereditarypersistenceoffetalhemoglobinHPLC high-performanceliquidchromatographyHPPK hereditarypyropoikilocytosisHRP horseradishperoxidaseHRP2 histidine-richprotein2HRT hormonereplacementtherapyHS hereditaryspherocytosisHSP heatshockproteinHSTCL hepatosplenicT-celllymphomaHSV herpessimplexvirusHTLV-I humanT-lymphotrophicvirustypeIHUMARA humanandrogenreceptorHUPO HumanProteomeOrganizationHUS hemolyticuremicsyndromeIAT indirectantiglobulintestICA indexofcirculatinganticoagulantsICC InternationalColorConsortiumICSH InternationalCouncilforStandardization

inHaematologyIEC InternationalElectrotechnical

CommissionIEF isoelectricfocusingiFISH interphasefluorescencein situ

hybridizationIg immunoglobulinIgA immunoglobulinAIgG immunoglobulinGIgH immunoglobulinheavychainIGHV immunoglobulinheavy-chainvariable

regionIgκ kappalightchainIgλ lambdalightchainIgM immunoglobulinMIL-1b interleukin-1bIL2 interleukin-2IL4 interleukin-4IL-8 interleukin-8ILD immunophenotypicleukocytedifferentialIMGT ImMunoGeneTicsIMW intermediatemolecularweightINR internationalnormalizedratioIP intellectualpropertyIP3 inositoltriphosphateIPF immatureplateletfractionIPI InternationalPrognosticIndexI-PIG InternationalPNHInterestGroupIPSS InternationalPrognosticScoringSystemIQ installationqualificationIRF immaturereticulocytefractionIRIS InternationalRandomizedStudyof

InterferonandSTI571IRP internationalreferencepreparationIS internationalstandard

ISCN InternationalSystemofCytogeneticNomenclature

ISI internationalsensitivityindexISLH InternationalSocietyforLaboratory

HematologyISM indolentsystemicmastocytosisISO InternationalOrganizationfor

StandardizationISTH InternationalSocietyonThrombosisand

HemostasisITD internaltandemduplicationITP idiopathicthrombocytopenicpurpuraIU internationalunitIVD in vitrodiagnosticJ joiningregionJAK2 Januskinase2JCAHO JointCommissionontheAccreditationof

HealthcareOrganizationsJCI JointCommissionInternationalJMML juvenilemyelomonocyticleukemiaJPEG JointPhotographicExpertsGroupK2-EDTA dipotassiumethylenediaminetetraacetic

acidK3-EDTA tripotassiumethylenediaminetetraacetic

acidKbp kilobasepairsKCl potassiumchlorideKCT kaolinclottingtimekDa kiloDaltonKde kappa-deletingelementKIR killinginhibitoryreceptorLA lupusanticoagulantLAIP leukemia-associatedaberrant

immunophenotypeLAN localareanetworkLBL lymphoblasticlymphomaLCDD lightchaindepositiondiseaseLCR locuscontrolregionLDH lactatedehydrogenaseLD-PCR long-distancepolymerasechainreactionLDT laboratorydevelopedtestLELY lowexpressionalleleLyonLEPRA lowexpressionallelePragueLGL largegranularlymphocyte;largegranular

lymphocytosisLIS lowionicstrengthLL lymphoplasmacyticlymphomaLM lengthmutationLMW lowmolecularweightLMWH lowmolecularweightheparinLOQ LimitofQuantitationLp(a) lipoprotein(a)LPD lymphoproliferativedisorderLPS lipopolysaccharide

Abbreviations

xix

LSI locus-specificidentifierLSRB lengthsedimentationreactioninbloodLUC largeunstainedcellsM monitoringtherapyM molarMAC membraneattackcomplexMAGE MicroArrayandGeneExpressionMALDI matrix-assistedlaserdesorption/ionizationMALDI-TOF timeoffightMALT mucosa-associatedlymphoidtissueMAQC MicroArrayQualityControlMASS methylationassessmentofsinglesamplesMb megabasepairmBAND multicolor-bandingfluorescencein situ

hybridizationM-BCR majorbreakpointclusterregionm-BCR minorbreakpointclusterregionMCF meanchannelfluorescenceMCH meancellhemoglobinMCHC meancellhemoglobinconcentrationMCL mantlecelllymphomaMCV meancellvolumeMD medicaldoctorMDS myelodysplasticsyndromeMDS-U myelodysplasticsyndrome-unclassifiedMeDIP methylatedDNAimmunoprecipitationMELD modelforendstageliverdiseaseMESF moleculesofequivalentsoluble

fluorochromeMFI meanfluorescentintensity;median

fluorescentintensityM-FISH multicolorfluorescencein situ

hybridizationMGG May–Grünwald–GiemsaMgSO4 magnesiumsulfateMGUS monoclonalgammopathyofundetermined

significanceMHC majorhistocompatibilitycomplexMHOP molecularoncologyforparaffinMIAME MinimumInformationAboutaMicroarray

ExperimentMIRL membraneinhibitorofreactivelysismL milliliterMLD morphologicalleukocytedifferentiationMLL myeloidlymphoidlineage;mixedlineage

leukemiaMLPA multipleligation-dependentprobe

amplificationMNDA

myeloidcellnucleardifferentiationantigen

MO molecularoncologyMoAb monoclonalautibodyMPC meanplateletcomponentMPCM maculopapularcutaneousmastocytosis

MPN myeloproliferativeneoplasmMPO myeloperoxidaseMPV meanplateletvolumeMRD minimalresidualdiseaseMRE majorregulatoryelementMRI magneticresonanceimagingmRNA messengerRNAMS massspectrometry;methionine

synthetaseMS/MS tandemmassspectrometryMTC majortranslocationclusterMTHF methylenetetrahydrofolateMTHFR methylenetetrahydrofolate

reductaseμ-BCR microbreakpointclusterregionμL microliterMYH9 nonmusclemyosinheavychainMYH11 smoothmusclemyosinheavychainMZL marginalzoneB-celllymphomaNADH nicotinamideadeninedinucleotideNAIT neonatalalloimmunethrombocytopeniaNAP neutrophilalkalinephosphataseNAP-2 neutrophilactivatingprotein-2NASA naphtholASacetateesteraseNASCOLA NorthAmericanSpecializedCoagulation

LaboratoryAssociationNASDA naphtholAS-DacetateesteraseNBF neutralbufferedformalinNBU Nijmegen-BethesdaunitNCCLS NationalCommitteeforClinicalLaboratory

StandardsNCI NationalCancerInstituteNEMA NationalElectricalManufacturers

AssociationNEQAS NationalExternalQualityAssessment

Serviceng nanogramNHANES NationalHealthandNutritionExamination

SurveyNHL non-HodgkinlymphomaNIBSC NationalInstituteforBiologicStandards

andControlNIH NationalInstitutesofHealthNINDS NationalInstituteofNeurologicalDisorders

andStrokeNK naturalkillerNL normalnm nanometerNO nitrousoxideNPM1 nucleophosminNPP normalpooledplasmaNRBC nucleatedredbloodcellNSE nonspecificesteraseNSTEMI non-ST-elevationmyocardialinfarction

Abbreviations

xx

OASIS OrganizationfortheAssessmentofStrategiesforIschemicSyndromes

OB obstetricOD opticaldensityOS overallsurvivalOQ operationqualificationP prognosisPACS PictureArchivingandCommunication

SystemsPAI-1 plasminogenactivatorinhibitor-1PAP plasmin/antiplasmincomplexPAR protease-activatedreceptorPAS periodicacid–SchiffPB peripheralbloodPBS phosphatebufferedsalinePBSC peripheralbloodstemcellPC proteinC;plasmacellPCA principlecomponentanalysisPCL plasmacellleukemiaPCM plasmacellmyelomapCO2 partialpressureofcarbondioxidePCR polymerasechainreactionPCT platelet-critPCV packedcellvolumePDGFR platelet-derivedgrowthfactorreceptorPDW plateletdistributionwidthPE phycoerythrin;pulmonaryembolismPerCP peridininchlorophyllproteinPET polyethyleneterephthalatePf Plasmodium falciparumPF1.2 prothrombinfragment1.2PF4 plateletfactor4PFA plateletfunctionanalyzerPfATPase Plasmodium falciparumadenosine

triphosphatasePfMSP Plasmodium falciparummerozoitesurface

proteinpg picogramPGE1 prostaglandinE1PGR progressionPHA phytohemagglutininPhD DoctorofPhilosophyPI propidiumiodidePIG-A phosphatidylinositolglycancomplementa-

tionclassAPlgn plasminogenPK prekallikreinPL phospholipid;prolymphocytePLA plateletantigenPLATO PlateletInhibitionandPatientOutcomesPLC phospholipaseCP-LCR plateletslargecellratiopLDH PlasmodiumlactatedehydrogenasePLL prolymphocyticleukemia

PLT plateletPMBL primarymediastinallargeB-cell

lymphomaPMF primarymyelofibrosisPML promyelocyticleukemiaPMN polymorphonuclearneutrophilPMP plateletmicroparticlePMT photomultipliertubePNA peptidenucleicacidpNA paranitroanilinePNET primitiveneuroectodermaltumorPNG PortableNetworkGraphicsPNH paroxysmalnocturnalhemoglobinuriaPNP plateletneutralizationprocedurepO2 partialpressureofoxygenPOC point-of-carePPACK D-phenylalanine-proline-arginine

-chloromethylketonePPi pyrophosphatePPP plateletpoorplasmaPQ performancequalificationPS proteinSPSM probabilitystatemodelPT prothrombintime;prothrombinPTCL-US peripheralT-celllymphoma,unspecifiedPTD partialtandemduplicationPTLD post-transplantlymphoproliferative

disorderPTT partialthromboplastintimePT-VWD platelet-typevonWillebranddiseasePV polycythemiaveraPv Plasmodium vivaxPvMSP1 Plasmodium vivaxmerozoitesurface

protein-1QBC quantitativebuffycoatQC qualitycontrolr correlationcoefficientRA refractoryanemiaRAEB refractoryanemiawithexcessblastsRAEB-t refractoryanemiawithexcessblastsin

transformationRAG recombinase-activatinggeneRARS refractoryanemiawithringedsideroblastsRBC redbloodcellcountRCMD refractorycytopeniawithmultilineage

dysplasiaRCo ristocetincofactorRCPA RoyalCollegeofPathologistsofAustralasiaRCUD refractorycytopeniawithunilineage

dysplasiaRDW redcelldistributionwidthRECORD RegulationofCoagulationinOrthopedic

SurgerytoPreventDeepVenousThrombosisandPulmonaryEmbolism

Abbreviations

xxi

RE-LY RandomizedEvaluationofLongTermAnticoagulantTherapy

RFLP restrictionfragmentlengthpolymorphismRFS relapsefreesurvivalRGB red,greenandblueRh RhesusRHAG Rhesus-associatedglycoproteinRI refractiveindexRIA radioimmunoassayRIPA ristocetin-inducedplateletagglutinationRNA ribonucleicacidRNase ribonucleaseROC receiveroperatingcharacteristicROTEM rotationalthromboelastometryRPMI RoswellParkMemorialInstituteRQ-PCR real-timequantitativepolymerasechain

reactionRR relapseriskrRNA ribosomalRNARSS recombinationsignalsequenceRT reptilasetimeRT-PCR reversetranscriptionpolymerasechain

reactionS switchregionSAH S-adenosylhomocysteineSAM S-adenosylmethioninesb between-runimprecisionSBB SudanblackBSBPE singlebaseprimerextensionSC single-colorSCD sicklecelldiseaseSCT stemcelltransplantationSD standarddeviationSDI standarddeviationindexSDS-PAGE sodiumdodecylsulfatepolyacrylamidegel

electrophoresisSELDI surface-enhancedlaserdesorption/

ionizationS-FISH suspensionfluorescencein situ

hybridizationSHM somatichypermutationSKY spectralkaryotypingSLE systemiclupuserythematosusSLL smalllymphocyticlymphomaSLVL spleniclymphomawithvillous

lymphocytesSM systemicmastocytosisSmIg surfacemembrane-boundimmunoglobulinSNP singlenucleotidepolymorphismSOM self-organizingmapSRA serotoninreleaseassaySSC ScienceandStandardizationCommittee;

sidescatterSSCP single-strandconformationpolymorphism

SSOP sequence-specificoligonucleotideprobest totalimprecisionSTEMI ST-elevationmyocardialinfarctionSTR shorttandemrepeatsw within-runimprecisionsy/x standarddeviationoftheregressionlineT (deoxythymidylate)thymineTAFI thrombin-activatablefibrinolysisinhibitorTA-GVHD transfusion-associatedgraft-versus-host

diseaseT-ALL T-cellacutelymphoblasticleukemiat-AML therapy-relatedacutemyeloidleukemiaTAR thrombocytopeniawithabsentradiiTAT thrombinantithrombincomplexTBNK T,BandNKcellTC triple-colorTCR T-cellreceptorTD-FISH tricolordouble-fusionfluorescencein situ

hybridizationTDI template-directeddye-terminator

incorporationTdT terminaldeoxynucleotidyltransferaseTEG thromboelastographyTEM transmissionelectronmicroscopyTF tissuefactorT-FISH targetfluorescencein situhybridizationTFPI tissuefactorpathwayinhibitorTHF tetrahydrofolateTIFF taggedimagefileformatTKR TotalkneereplacementT-LBL T-acutelymphoblasticlymphomaTM thrombomodulinTm meltingtemperaturet-MDS therapy-relatedmyelodysplasticsyndromeTMEP telangiectasiamaculariseruptivaperstansTNFα tumornecrosisfactorαTOF time-of-flightTPA 12-O-tetradecanoylphorbol-13-acetatetPA tissueplasminogenactivatorT-PLL T-cellprolymphocyticleukemiaTPO thrombopoietinTRAP tartrate-resistantacidphosphataseTRITON-TIMI TRialtoAssessImprovementin

TherapeuticOutcomesbyOptimizingPlateletInhibitioNwithPrasugrel–ThrombolysisInMyocardialInfarction

TT thrombintimeTTI tissuethromboplastininhibitionTTP thromboticthrombocytopenicpurpuraTxA2 thromboxaneUFH unfractionatedheparinUK UnitedKingdomUL UnderwritersLaboratoriesUMP uridinemonophosphate

Abbreviations

xxii

uPA urokinaseplasminogenactivatorUPD uniparentaldisomyUPGMA unweightedpair-groupmethodaverageURCP UltraRainbowCalibrationParticlesUS ultrasoundUTR UntranslatedregionsUV ultravioletV variableregionVa activatedfactorVVCS volume,conductivity,andlightscatterVEGF vascularendothelialgrowthfactorVEGFR vascularendothelialgrowthfactorreceptorVKOR vitaminKepoxidereductaseVKORC1 vitaminKepoxidereductasecomplex

subunit1

VNTR variablenumberoftandemrepeatVTE venousthromboembolismVWD vonWillebranddiseaseVWF vonWillebrandfactorWAS Wiskott–AldrichsyndromeWBC whitebloodcellcountWCP whole-chromosomepaintWHO WorldHealthOrganizationWSI wholeslideimageXa activatedfactorXXY-FISH sexchromosomefluorescencein situ

hybridizationYAC yeastartificialchromosome

Cellular AnalysisI

3

Laboratory Hematology Practice, First Edition. Edited by Kandice Kottke-Marchant, Bruce H. Davis.

© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

Introduction

Cellular analysis in hematology has a fascinating history spanning more than three centuries. Blood cell analysis is noteworthy for a very high degree of technologic ingenuity, sometimes shown by rather unlikely people. Throughout, the inventions have been characterized by extremely careful observation, meticulous attention to detail, and the applica-tion of techniques advanced for their time. Throughout the centuries, analysis of cells, whether by the observational skills required for microscopy, the manual dexterity required for manual analytical techniques, or the advanced technical knowledge required to operate modern multiparameter ana-lyzers, has always required a high degree of skill from the practitioners of the art and science.

Much of the material for this chapter has been obtained from the publications detailed in the reference section, within which the references to the original papers of the named individuals are detailed.

Microscopy

The origin of cellular analysis through the medium of micro-scopy is widely associated with Antonie van Leeuwenhoek (1632–1723) from Delft, in the Netherlands, although similar observations of blood cells were in fact first documented in 1668 in the personal documents of his fellow Dutchman Jan Swammerdam [1]. van Leeuwenhoek was however the first to publish his observations in a scientific journal [2].

van Leeuwenhoek (see Figure 1.1) was an unlikely scien-tist, a draper (fabric merchant), who came from a family of tradesmen, had no fortune, received no higher education or university degree, and knew no languages other than his native Dutch. This would have been enough to completely exclude him from the scientific community of his time; yet

with skill, diligence, an endless curiosity, and an open mind free of the scientific dogma of his day, Leeuwenhoek suc-ceeded in making some of the most important discoveries in the history of biology. His microscope consisted of a small lens that was essentially a bead of glass fixed to a brass plate, which was held close to the eye. Solid specimens were fixed to the point of an adjustable pin, while liquids were placed in tiny glass tubes. With this simple lens he discovered bac-teria, amoebae, rotifers, and protozoa. He first observed the cells of blood in 1675, when he observed that his own blood was composed of “small red globules, driven through a crys-talline humidity of water.” His estimates of red cell size were remarkably accurate [1,2].

The compound microscope, which consists of an eyepiece lens as well as an objective lens, was actually invented in 1590, well before Leeuwenhoek’s birth, by two Dutch eye-glass makers, Zaccharias Janssen and his son Hans Janssen. However, van Leeuwenhoek was able to achieve greater magnification and better resolution by skillful grinding of the lens of his simple microscope than by using the crude compound microscopes of his time. In the early 18th century compound microscopes were improved by the use of lenses that combined two types of glass, which were found to reduce the chromatic effect, the disturbing halos that result from differences in the refraction of light. A further signifi-cant advance in the mid-19th century was the development of achromatic microscopes, with objectives comprised of multiple lenses. The resultant images were sharp and well defined, with far better resolution than was possible with even the best simple microscope, and enabled further dis-coveries to be made. In 1842 Alfred Donné described plate-lets as the third cellular element in blood, and in 1875 Hayem introduced a method for counting them. Gulliver, in 1846, was able to differentiate between lymphocytes and granulocytes by size alone.

Paul Ehrlich, a man who contributed greatly in many spheres to the health of humanity, has been called the

1 Historical Perspective on Cellular Analysis

Elkin SimsonMount Sinai School of Medicine, New York, NY, USA

PART I Cellular Analysis

4

eral blood. Heilmeyer divided the reticulocytes into four groups, plus a group 0 for normoblasts that contained a nucleus as well as a dense perinuclear reticulum.

Manual cell analysis

The addition of quantitation to microscopic observation was a very important step in the analysis of blood cells. Manual methods of cell counting and cell characterization were all highly dependent on the quality of microscopes. Leeuwenhoek himself developed a method for counting the number of erythrocytes pulled into a glass capillary tube with graduation marks. In 1851 Karl Vierordt published a procedure for cell counting that required 3 hours or more to complete. He used a capillary pipette that was calibrated in diameter and length, the contents of which were expelled onto a flat slide where they were mixed with diluting and preserving fluid. The entire spread was then counted with the aid of a finely squared micrometer in the eyepiece of the microscope.

During the next 60 years many modifications of this basic procedure were introduced. In 1874 Malassez reported counting of white blood cells using an instrument called a hemocytometer, a shallow, graduated, rectangular chamber into which diluted blood was injected. To measure and mix the blood, pipettes were designed that sampled a fixed volume of blood and accurately diluted this before counting. A variety of hemocytometers and various diluting solutions were introduced. Red cells, white cells, and platelets were counted using this technique. The Neubauer hemocytome-ter, which consists of two chambers, each of which has finely ruled squares, has become the standard method for the performance of manual cell counts. This basic design is still employed when manual microscopic cell counts are done today.

Measurement of cell size was also initiated by van Leeuwenhoek. However, it was not until 1718 that Jurin accurately established the diameter of the human red cell. As with cell counting, measurement of cell size was per-formed visually until the 20th century. The magnified images of cells (usually flattened in a dried film of blood) were compared to a known dimension by calibrating the microscope.

Early in the 20th century, Wintrobe applied centrifugation techniques to whole blood, which enabled quantitation of the cellular fraction of the blood by measurement of the packed cell volume (PCV). Dividing this result by the RBC provided an indirect measurement of average red cell size. Although now largely replaced by automated methods that calculate the hematocrit (HCT) from the red blood cell count (RBC) and directly measure the size of red cells to obtain the mean cell volume (MCV; see Table 1.1), this manual centrifugation method is still, on occasions, used today.

Father of Hematology, of Immunology, and of Chemotherapy. While still a medical student in 1877, he began to use aniline dyes to stain blood cells. He classified aniline dyes as acidic or basic and showed that one group of dyes preferentially stained the red blood corpuscles and eosinophil leukocyte granules, whereas the other group stained nuclei and lym-phocyte cytoplasm. In 1879 he developed a neutral stain that could stain both groups simultaneously. With this stain, he documented the violet granules of the neutrophil leuko-cytes. He went on to describe in detail the appearance of lymphocytes, neutrophils, eosinophils, and basophils; and initiated the white cell differential in the form still used today.

In the 1860s Erb noted granules in the red blood cells of humans and animals that had been made anemic by vene-section. These granules may have been reticulum or perhaps denatured hemoglobin similar to Heinz bodies. Ehrlich was probably the first investigator to describe the cells now regarded as reticulocytes, using methylene blue to stain the reticulum. In 1891 Smith described supravital-stained eryth-rocytes that contained reticulum in cattle with pernicious anemia. He felt that these cells represented not degenerative forms, but rather “embryonic corpuscles, sent into the cir-culation before their time to make good the losses going on” [3]. In the early 1930s Heilmeyer published descriptions of reticulocyte morphology at different stages of maturation, as well as the relative frequencies of these stages in the periph-

Figure 1.1 Antonie van Leeuwenhoek. (From http://

commons.wikimedia.org/wiki/File:Anton_van_Leeuwenhoek.png.)

http://commons.wikimedia.org/wiki/File:Anton_van_Leeuwenhoek.png

http://commons.wikimedia.org/wiki/File:Anton_van_Leeuwenhoek.png

CHAPTER 1 Historical Perspectives

5

1964. Wintrobe used the measurement of hemoglobin and the PCV combined with the RBC to obtain red cell indices that indirectly measured the properties of the red blood cells, and in 1934 he published a classification of anemia based on the hemoglobin content and volume of red cells. The study of hemoglobin has been a prototype for the study of genotypes manifested into specific cellular and clinical phe-notypes [4].

Single-channel analyzers

In the early 20th century, with advances in electronics and electro-optics, several attempts to simplify blood cell count-ing were made [5]. Moldavan, in 1934, described an appa-ratus in which a suspension of red blood cells was forced through a capillary glass tube on a microscope stage, each passing cell being registered and counted by a photoelectric apparatus attached to the ocular lens. He noted problems in standardizing the capillary tube, assuring proper focus, maintaining flow, and obtaining an appropriately sensitive photoelectric apparatus, and reported no further work himself. Around 1945 yet another instrument was described in which erythrocytes could be counted automatically by means of photoelectric spot-scanning of a thin layer of a diluted blood sample. This was an attempt to automate the manual counting chamber technique described above, in which the microscopist was replaced by a photomultiplier and an electronic counting unit, while the counting chamber was moved by a motor-driven system. This also failed.

Wallace Coulter’s discovery of an aperture impedance method, the Coulter principle, for counting and sizing cells, for which he obtained a patent in 1953, can be regarded as the origin of hematology automation. This principle made use of the lower conductivity of the erythrocytes compared with the diluting fluid. In Coulter’s instrument, blood cells suspended in an electrolyte solution were induced to flow through an electric field in a relatively short, small orifice drilled in a thin sapphire. The electric field in and surround-ing this orifice was the sensing portion of the instrument, also called the aperture. Because of the small dimensions, diluted blood cells were readily detected and counted more or less individually without a high frequency of clogging. Cells could be sized simultaneously because the magnitude of the electrical impulse was found to be proportional to the cell volume. The first analyzer was the Model A (see Figure 1.2) and was followed by an alphabetically named series of single-channel analyzers, each of which contained succes-sive improvements and additional features. An important feature of all these analyzers was that they aspirated, under mercury-manometer control, an accurate volume of blood. These analyzers were successfully used in thousands of labo-ratories worldwide and the Coulter principle provides the basis for most modern cell counters.

In 1949 Brecher described a method for quantitating retic-ulocytes by staining them with the supravital dye new meth-ylene blue, then counting them using a microscope, with due note of Heilmeyer’s maturity classification described above, and calculating a ratio to the number of red cells. Manual reticulocyte methods are inaccurate and imprecise because of the difficulty in identifying the more mature reticulocytes and the low number of cells counted. They remain in widespread use, but are progressively being replaced by more reliable automated methods.

Hemoglobin

The earliest attempts to determine the concentration of hemoglobin in the blood included the visual matching of dilutions of whole blood to a liquid color reference by Gowers (1878), Hoppe-Seyler (1883), Sahli (1895), and Haldane (1901). The method of Sahli, in which the blood sample is mixed with hydrochloric acid to obtain acid hematin, is still used. In developing this technique it was found to be simpler and more quantitative to determine the color through the use of colorimeters and/or spectropho-tometers; however, the spectral content of the various forms of hemoglobin precluded the choice of a good wavelength for measurement unless the hemoglobin was first converted to a single stable form. The determination of hemoglobin as cyanmethemoglobin or hemiglobincyanide (HiCN) was introduced by Stahe in 1920. The HiCN method has been studied extensively and was accepted by the International Council for Standardization in Haematology (ICSH) as the international reference method for hemoglobin analysis in

Table 1.1 Red cell measurements and indices by manual and

automated methods.

Manual Automated

MCV = PCV (measured) / RBC

(counted)

HCT = RBC (counted) × MCV

(measured)

HCT is equivalent to PCV

MCH = Hb (measured) / RBC

(counted)

MCH = Hb (measured) / RBC

(counted)

MCHC = Hb (measured) /

PCV(measured)

MCHC = Hb / (RBC × MCV)

Anisocytosis = visual

microscopic estimation of

red cell size distribution

RDW = size distribution of red

blood cells calculated from

the measurement of individual

red cell volumes

Hb, hemoglobin concentration; HCT, hematocrit; MCH, mean cell

hemoglobin; MCHC, mean cell hemoglobin concentration; MCV,

mean cell volume; PCV, packed cell volume; RBC, red blood cell

count; RDW, red cell distribution width.

PART I Cellular Analysis

6

reaction chambers reduced the need for frequent recalibra-tion. By the early 1970s the Model S had essentially revo-lutionized the hematology laboratory, consolidating routine testing into two workstations: the automated seven-parameter CBC and the remaining analysis. This second part of the analysis, typically called the leukocyte differential, still needed to be performed microscopically. Platelet counts were performed infrequently, using the microscopic manual method in which platelets were counted in a hemocytom-eter chamber, preferably using phase-contrast microscopy.

In 1970 Technicon introduced the Hemalog-8 instrument which added the platelet count to the seven-parameter automated CBC by counting platelets by photoelectric means in an additional parallel counting channel after hemolysis of the red blood cells. The PCV was obtained by automation of the centrifugal packing of red cells followed by a photoelec-tric scan that registered the cell/plasma interface. The system used an automated sampler, but because the continuous-flow dilution method was employed, periodic calibration was still required and it did not achieve widespread use.

In 1980 Coulter introduced the S Plus series, which added the platelet count to their automated CBC instrument. The platelet count was obtained simultaneously in the red blood cell counting aperture by discriminating between platelets and red blood cells on the basis of their signal size. A sub-sequent model, the S Plus II, added further parameters to the reported results of the automated CBC. These were the red cell distribution width (RDW), defined as the spread (coefficient of variation) of the red cell size distribution; the mean platelet volume (MPV), defined in analogy to the MCV of red cells by averaging the signal heights from the platelets; the lymphocyte percentage, which was defined by discriminating on the basis of signal size in the white cell counting aperture with the small cells labeled as lym-phocytes; and the lymphocyte count, which was obtained by multiplying the lymphocyte percentage by the total WBC.

The next major advance in consolidation of cell counting was the introduction of the Coulter S Plus IV system in 1983. In this system white cells were classified into three catego-ries: lymphocytes, monocytes (really mid-sized white cells), and granulocytes. In addition, the platelet distribution width (PDW) derived in analogy with the RDW was added. Three-part white cell differential counters remain in use to the present day, especially in smaller laboratories and physician offices.

Automation of the leukocyte differential count

Intense activity was initiated during the 1970s in attempts to automate the leukocyte differential count. Two different technologic approaches were pursued. In one, a direct attempt was made to automate the microscopic procedure

Early multiparameter cell analyzers

The first instrument to automate the performance of more than one cell count on a single sample was the SMA 4A-7A introduced by Technicon in 1965. In this instrument, each sample of blood was divided and diluted using continuous-flow technology that had been invented by Leonard Skeggs for chemistry analyzers. The cells in the sample were then counted individually with a photoelectric detector in two passes through a single, narrow flow cell, one without hemolysis of the red cells for the RBC, the other after hemo-lysis to obtain the white blood cell count (WBC). The hemo-globin content was determined in a separate and parallel channel after hemolysis of the red blood cells and conver-sion of the hemoglobin to cyanmethemoglobin. The instru-ment produced a seven-parameter complete blood count (CBC) on each specimen and operated at a throughput rate of 30 samples per hour. The instability of the dilution process using the continuous-flow method required the instrument to be recalibrated frequently, and it was not well accepted in laboratories.

The widespread use of a combined CBC analyzer was only achieved when Coulter introduced the Model S instrument in 1968. In this instrument, the sample was divided via a blood sampling valve and diluted into two glass reaction cham-bers, in one of which the red blood cells were hemolyzed. Samples from each chamber were passed through electrical counting apertures applying the Coulter principle to deter-mine the RBC and the WBC. Hemoglobin was determined by optical absorption in the WBC reaction chamber without conversion to cyanmethemoglobin. The MCV was calculated from the average signal size in the red blood cell counting aperture. The HCT, mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were then calcu-lated to produce a seven-parameter CBC. A maximum throughput rate of almost 100 samples per hour was possible by manually feeding samples into the analyzer, and the dilu-tion stability achieved with the blood sampling valve and

Figure 1.2 The Coulter counter model A. (Image provided by Beckman

Coulter.)

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