Table Of Content

Flow Cytometry in Neoplastic Hematology This fourth edition presents an updated and expanded text and illustrations to reflect continued morphologic, immunophenotypic, and especially molecular advances in the field of neoplastic hematology, mostly due to the rapidly expanding application of next-generation sequencing. Those advances not only allow a more reliable diagnosis of the majority of tumors and identification of early changes such as monoclonal B-cell lymphocytosis or clonal hematopoiesis of indeterminate potential (CHIP), but also in many cases identify mutations or phenotypic changes in tumors that can be targeted by mutation-specific or antigen-specific drugs. This edition incorporates the updated WHO classification of hematopoietic tumors and new immunophenotypic and molecular markers to provide a thorough pathologic overview of hematologic neoplasms while focusing on flow cytometric features. Special emphasis has been put on hematological neoplasms with crucial clinical significance such as acute promyelocytic leukemia, other acute leukemias, and difficult areas in flow cytometry. Flow cytometric features in AML, MDS, CMML, CLL, and measurable residual disease were sig- nificantly expanded. There are many new comparative tables, illustrations, and diagrams of algorithmic approaches. Wojciech Gorczyca, MD, PhD, is Director of Flow Cytometry/Hematopathology at Bioreference Laboratories, OPKO Health Compa- nies, Elmwood Park, New Jersey, USA. His many publications include Atlas of Differential Diagnosis in Neoplastic Hematopathology, Fourth Edition, and Cytogenetics, FISH and Molecular Testing in Hematologic Malignancies. From the reviews of the previous edition: “The third edition of this book is a valuable addition… comprehensive… clearly written, well illustrated and supported by extensive references… Since there is a great deal of detailed information, it will serve as a useful reference for immunophenotyping laboratories as well as being an educational tool for others.” —Barbara J. Bain, British Journal of Haematology Flow Cytometry in Neoplastic Hematology Morphologic-Immunophenotypic-Genetic Correlation Fourth Edition Wojciech Gorczyca, MD, PhD Director of Flow Cytometry/Hematopathology Bioreference Laboratories, OPKO Health Companies Elmwood Park, New Jersey, USA Fourth edition published 2023 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2023 Wojciech Gorczyca This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufac- turer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the relevant national drug formulary and the drug companies’ and device or material manufactur- ers’ printed instructions, and their websites, before administering or utilizing any of the drugs, devices or materials mentioned in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including pho- tocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑in‑Publication Data Names: Gorczyca, Wojciech, 1959- author. Title: Flow cytometry in neoplastic hematology : morphologic-immunophenotypic-genetic correlation / Wojciech Gorczyca. Description: Fourth edition. | Boca Raton : CRC Press, 2023. | Includes bibliographical references and index. Identifiers: LCCN 2022032059 (print) | LCCN 2022032060 (ebook) | ISBN 9781032055251 (hardback) | ISBN 9781032055268 (paperback) | ISBN 9781003197935 (ebook) Subjects: MESH: Hematologic Neoplasms--diagnosis | Flow Cytometry | Leukemia, Lymphoid--diagnosis | Leukemia, Myeloid, Acute--diagnosis Classification: LCC RC280.H47 (print) | LCC RC280.H47 (ebook) | NLM WH 525 | DDC 616.99/418--dc23/eng/20220817 LC record available at https://lccn.loc.gov/2022032059 LC ebook record available at https://lccn.loc.gov/2022032060 ISBN: 978-1-032-05525-1 (hbk) ISBN: 978-1-032-05526-8 (pbk) ISBN: 978-1-003-19793-5 (ebk) DOI: 10.1201/9781003197935 Typeset in Warnock Pro by KnowledgeWorks Global Ltd. To my wife Elżbieta, my daughters, Marta, Ewa, and Małgorzata, and my grandchildren, Emily and Kaiden, with love. To my teachers in pathology, cytopathology, flow cytometry, and hematopathology, Late Professor S. Woyke, Professor W. Olszewski, Late Dr. Z. Darzynkiewicz, and Dr. J. Weisberger. CONTENTS Preface ................................................................................................................................................................................................................................xii Abbreviations ..................................................................................................................................................................................................................xiii 1. Introduction to Flow Cytometry ............................................................................................................................................................................1 The major roles of flow cytometry .........................................................................................................................................................................1 Flow cytometry in hematological emergencies ...................................................................................................................................................6 10-color flow cytometry analysis ...........................................................................................................................................................................7 Automated flow cytometry data analysis ...........................................................................................................................................................11 Flow cytometry parameters...................................................................................................................................................................................11 Limitations of flow cytometry ..............................................................................................................................................................................19 Classification of hematopoietic neoplasms ........................................................................................................................................................27 2. Flow Cytometry Analysis of Blood and Bone Marrow ....................................................................................................................................35 Bone marrow: Normal structure and hematopoiesis ......................................................................................................................................35 Antigen expression during hematopoietic maturation ...................................................................................................................................36 Flow cytometry of blood and bone marrow: Gating strategies .....................................................................................................................43 Blood: Normal flow cytometry pattern ...............................................................................................................................................................50 Bone marrow: Normal flow cytometry pattern ................................................................................................................................................51 3. Identification of Clonal B-Cell Population ........................................................................................................................................................55 Polyclonal versus monoclonal B-cells .................................................................................................................................................................55 Monoclonal B-cell lymphocytosis .......................................................................................................................................................................61 B-cells versus plasma cells .....................................................................................................................................................................................68 Mature versus immature B-cells ..........................................................................................................................................................................71 4. Identification of Abnormal T-Cell Population ..................................................................................................................................................75 Introduction ..............................................................................................................................................................................................................75 Aberrant T-cell phenotype by flow cytometry ..................................................................................................................................................75 Peripheral (mature) disorders versus T-ALL .....................................................................................................................................................89 T-cell markers expressed by non-T-cell processes ............................................................................................................................................89 5. Identification of Myeloblasts .................................................................................................................................................................................91 Introduction ..............................................................................................................................................................................................................91 Identification of myeloblasts by flow cytometry ...............................................................................................................................................96 Flow cytometric pattern in major AML subtypes ..........................................................................................................................................101 Leukemia-associated immunophenotype (LAIP) ...........................................................................................................................................109 Differential diagnosis of AML based on flow cytometry pattern ...............................................................................................................109 6. Identification of Lymphoblasts ...........................................................................................................................................................................127 Identification of B-lymphoblasts ........................................................................................................................................................................127 Identification of T-lymphoblasts ........................................................................................................................................................................138 7. Immunophenotypic Markers in Flow Cytometry ..........................................................................................................................................148 BCL2 (B-cell lymphoma/leukemia 2) ................................................................................................................................................................148 CD1a (cluster of differentiation 1a) ....................................................................................................................................................................148 CD2 ...........................................................................................................................................................................................................................148 CD3 ...........................................................................................................................................................................................................................151 CD4 and CD8 ..........................................................................................................................................................................................................151 CD5 ...........................................................................................................................................................................................................................153 CD7 ...........................................................................................................................................................................................................................153 CD10 .........................................................................................................................................................................................................................153 CD11b .......................................................................................................................................................................................................................155 CD11c........................................................................................................................................................................................................................155 CD13 and CD33 ......................................................................................................................................................................................................157 CD14 .........................................................................................................................................................................................................................157 CD15.........................................................................................................................................................................................................................158 vii viii Contents CD16 .........................................................................................................................................................................................................................159 CD19 .........................................................................................................................................................................................................................160 CD20 .........................................................................................................................................................................................................................160 CD22 .........................................................................................................................................................................................................................160 CD23 .........................................................................................................................................................................................................................161 CD24 .........................................................................................................................................................................................................................162 CD25 .........................................................................................................................................................................................................................162 CD26 .........................................................................................................................................................................................................................163 CD27 .........................................................................................................................................................................................................................163 CD30 .........................................................................................................................................................................................................................163 CD34 .........................................................................................................................................................................................................................163 CD36 .........................................................................................................................................................................................................................165 CD38 .........................................................................................................................................................................................................................165 CD43 .........................................................................................................................................................................................................................166 CD45 .........................................................................................................................................................................................................................166 CD49d.......................................................................................................................................................................................................................168 CD52 .........................................................................................................................................................................................................................168 CD56 .........................................................................................................................................................................................................................168 CD57 .........................................................................................................................................................................................................................168 CD64 .........................................................................................................................................................................................................................168 CD65 .........................................................................................................................................................................................................................171 CD71 (transferrin receptor) .................................................................................................................................................................................171 CD79 .........................................................................................................................................................................................................................171 CD81 .........................................................................................................................................................................................................................171 CD103 .......................................................................................................................................................................................................................172 CD117 (c-Kit) ..........................................................................................................................................................................................................172 CD123 .......................................................................................................................................................................................................................174 CD133 .......................................................................................................................................................................................................................174 CD138 .......................................................................................................................................................................................................................174 CD157 .......................................................................................................................................................................................................................174 CD200 .......................................................................................................................................................................................................................174 CD235a/GPHA (glycophorin A) .........................................................................................................................................................................175 FLAER ......................................................................................................................................................................................................................175 HLA-DR (human leukocyte antigen-DR isotype) ..........................................................................................................................................175 LILRB1 (leukocyte immunoglobulin-like receptor B1) .................................................................................................................................175 TCR beta F1 (T-cell receptor beta F1) ...............................................................................................................................................................175 TCR gamma/delta ..................................................................................................................................................................................................175 TdT (terminal deoxynucleotidyl transferase) ..................................................................................................................................................175 8. Morphologic-Flow Cytometric Correlation in Blood ....................................................................................................................................179 Normal blood ..........................................................................................................................................................................................................179 Pancytopenia/cytopenia .......................................................................................................................................................................................179 Neutrophilia ............................................................................................................................................................................................................179 Eosinophilia.............................................................................................................................................................................................................180 Lymphocytosis ........................................................................................................................................................................................................182 Erythrocytosis/polycythemia ..............................................................................................................................................................................184 Basophilia ................................................................................................................................................................................................................186 Thrombocytosis......................................................................................................................................................................................................186 Circulating blasts ...................................................................................................................................................................................................186 Monocytosis ............................................................................................................................................................................................................187 Leukoerythroblastosis ..........................................................................................................................................................................................190 Circulating plasma cells .......................................................................................................................................................................................191 9. Morphologic-Flow Cytometric Correlation in Bone Marrow .....................................................................................................................194 Benign bone marrow .............................................................................................................................................................................................194 Increased blasts ......................................................................................................................................................................................................194 Diffuse small cell infiltrate ..................................................................................................................................................................................194 Diffuse intermediate/large cell infiltrate ..........................................................................................................................................................198 Diffuse blastoid (“high-grade”) infiltrate..........................................................................................................................................................201 Increased number of scattered large cells (other than megakaryocytes) ..................................................................................................201 Paratrabecular lymphoid infiltrate ....................................................................................................................................................................201 Intrasinusoidal infiltrate ......................................................................................................................................................................................201 Contents ix Lymphoid aggregates (nodular lymphoid infiltrate) .....................................................................................................................................202 Pleomorphic lymphoid infiltrate .......................................................................................................................................................................204 Megakaryocytosis .................................................................................................................................................................................................204 Eosinophilia............................................................................................................................................................................................................204 Erythroid hyperplasia with dyserythropoiesis ...............................................................................................................................................206 Marrow infiltrate with fibrosis ..........................................................................................................................................................................207 Plasmacytosis .........................................................................................................................................................................................................207 Metastatic tumors and other infiltrative processes ......................................................................................................................................207 10. Morphologic-Flow Cytometric Correlation in Lymph Nodes .....................................................................................................................210 Reactive follicular hyperplasia ............................................................................................................................................................................210 Diffuse pattern with mostly small cells ............................................................................................................................................................210 Diffuse pattern with medium-sized and/or large cells ..................................................................................................................................212 Mixed (pleomorphic) infiltrate ...........................................................................................................................................................................214 Blastoid and/or “high-grade” infiltrate .............................................................................................................................................................218 Nodular pattern......................................................................................................................................................................................................219 Paracortical (interfollicular; T-zone) pattern ..................................................................................................................................................221 Intrasinusoidal pattern .........................................................................................................................................................................................222 Anaplastic infiltrate ...............................................................................................................................................................................................222 Histiocyte-rich infiltrate ......................................................................................................................................................................................223 11. Molecular-Flow Cytometric Correlation ..........................................................................................................................................................226 Introduction – Overview of genetic testing .....................................................................................................................................................226 Molecular-flow cytometric correlation ............................................................................................................................................................230 12. Phenotypic Classification of Mature B-Cell Neoplasms ..............................................................................................................................242 Introduction ...........................................................................................................................................................................................................242 Classification of B-cell neoplasms based on CD5 and CD10 expression .................................................................................................242 Additional phenotypic markers in B-cell neoplasms .....................................................................................................................................251 Classification of B-cell neoplasms based on forward scatter (grading) ....................................................................................................252 B-cell neoplasms with plasmacytic differentiation ........................................................................................................................................255 13. Mature B-Cell Lymphoproliferations................................................................................................................................................................258 Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) .............................................................................................258 B-cell prolymphocytic leukemia (B-PLL)/splenic B-cell lymphoma/leukemia with prominent nucleoli (SBLPN) ........................268 Nodal marginal zone lymphoma (MZL) ...........................................................................................................................................................271 Gastric MZL (MALT lymphoma) ......................................................................................................................................................................279 Splenic marginal zone lymphoma (SMZL).......................................................................................................................................................281 Marginal zone lymphoma with blood and/or BM involvement ..................................................................................................................285 Splenic B-cell lymphoma/leukemia with prominent nucleoli/hairy cell leukemia variant (SBLPN/HCL-v) ...................................285 Hairy cell leukemia (HCL) ..................................................................................................................................................................................286 Splenic diffuse red pulp small B-cell lymphoma (SDRPL) ...........................................................................................................................294 Lymphoplasmacytic lymphoma (LPL) ..............................................................................................................................................................295 Follicular lymphoma ............................................................................................................................................................................................303 Mantle cell lymphoma (MCL) .............................................................................................................................................................................320 Diffuse large B-cell lymphoma (DLBCL) ..........................................................................................................................................................333 Specific variants of DLBCL .................................................................................................................................................................................342 Burkitt lymphoma (BL) ........................................................................................................................................................................................345 High-grade B-cell lymphomas ............................................................................................................................................................................352 Plasmablastic lymphoma (PBL) ..........................................................................................................................................................................358 Primary effusion lymphoma (PEL) .....................................................................................................................................................................363 14. Plasma Cell Neoplasms ........................................................................................................................................................................................379 Plasma cell myeloma (PCM) ................................................................................................................................................................................379 Introduction ............................................................................................................................................................................................................379 Morphology .............................................................................................................................................................................................................379 Immunohistochemistry .......................................................................................................................................................................................381 Flow cytometry.......................................................................................................................................................................................................382 Measurable (minimal) residual disease (MRD) ..............................................................................................................................................390 Differential diagnosis of PCM .............................................................................................................................................................................392 Plasmacytoma .........................................................................................................................................................................................................396

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