Multiparameter flow cytometry in the diagnosis of hematologic malignancies PDF
Preview Multiparameter flow cytometry in the diagnosis of hematologic malignancies
Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit Lund University, Sweden Marie-Christine Béné University of Nantes, France © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information University Printing House, Cambridge CB2 8BS, United Kingdom One Liberty Plaza, 20th Floor, New York, NY 10006, USA 477 Williamstown Road, Port Melbourne, VIC 3207, Australia 314–321, 3rd Floor, Plot 3, Splendor Forum, Jasola District Centre, New Delhi – 110025, India 79 Anson Road, #06-04/06, Singapore 079906 Cambridge University Press is part of the University of Cambridge. It furthers the University’s mission by disseminating knowledge in the pursuit of education, learning and research at the highest international levels of excellence. www.cambridge.org Information on this title: www.cambridge.org/9781107503830 DOI: 10.1017/9781316218549 © Cambridge University Press 2018 his publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 2018 Printed in the United Kingdom by Clays, St Ives plc A catalogue record for this publication is available from the British Library. Library of Congress Cataloging-in-Publication Data Names: Porwit, Anna, editor. | Bene, Marie-Christine, editor. Title: Multiparameter low cytometry in the diagnosis of hematologic malignancies / edited by Anna Porwit, Marie Christine Bene. Description: Cambridge, United Kingdom ; New York, NY : Cambridge University Press, 2018. | Includes bibliographical references and index. Identiiers: LCCN 2017042275 | ISBN 9781107503830 (paperback) Subjects: | MESH: Hematologic Neoplasms—diagnosis | Flow Cytometry—methods Classiication: LCC RC280.H47 | NLM WH 525 | DDC 616.99/418—dc23 LC record available at https://lccn.loc.gov/2017042275 ISBN 978-1-107-50383-0 Paperback Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. Every efort has been made in preparing this book to provide accurate and up-to-date information that is in accord with accepted standards and practice at the time of publication. Although case histories are drawn from actual cases, every efort has been made to disguise the identities of the individuals involved. Nevertheless, the authors, editors and publishers can make no warranties that the information contained herein is totally free from error, not least because clinical standards are constantly changing through research and regulation. he authors, editors and publishers therefore disclaim all liability for direct or consequential damages resulting from the use of material contained in this book. Readers are strongly advised to pay careful attention to information provided by the manufacturer of any drugs or equipment that they plan to use. © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Contents List of Contributors vii Preface ix List of Abbreviations xi 1 Flow Cytometry in Clinical Haematopathology: 9 Mature T-Cell Neoplasms and Natural Basic Principles and Data Analysis of Killer-Cell Malignancies 140 Multiparameter Data Sets 1 Anne Tierens Francis Lacombe and Marie-Christine Béné 10 Flow Cytometric Diagnosis of Hodgkin’s 2 Antigens 14 Lymphoma in Lymph Nodes 161 Marie-Christine Béné and Anna Porwit Lori Soma, Brent L. Wood and Jonathan R. Fromm 3 Flow Cytometry of Normal Blood, Bone Marrow and Lymphatic Tissue 36 11 Minimal Residual Disease in Acute Myeloid Anna Porwit and Marie-Christine Béné Leukaemia 171 Gerrit J. Schuurhuis, Angèle Kelder, 4 Reactive Conditions and Other Diseases Gert J. Ossenkoppele, Jacqueline Cloos and Where Flow Cytometric Findings May Mimic Wendelien Zeijlemaker Haematological Malignancies 61 Wolfgang Kern, Marie-Christine Béné 12 Ambiguous Lineage and Mixed Phenotype and Anna Porwit Acute Leukaemia 191 Anna Porwit and Marie-Christine Béné 5 Examples of Immunophenotypic Features in Various Categories of Acute Leukaemia 75 13 Flow Cytometry in Myelodysplastic Marie-Christine Béné and Anna Porwit Syndromes 199 heresia M. Westers and Arjan A. van de 6 Acute Lymphoid Leukaemias (All) and Minimal Loosdrecht Residual Disease in All 89 Giuseppe Basso, Barbara Buldini 14 Future Applications of Flow Cytometry and Andrea Zangrando and Related Techniques 215 Marie-Christine Béné and Francis Lacombe 7 Immunophenotyping of Mature B-Cell Lymphomas 105 Olof Axler and Anna Porwit 8 Plasma Cell Myeloma and Related Index 231 Disorders 128 Ruth M. de Tute, Andrew C. Rawstron and Roger G. Owen v © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Contributors Olof Axler Gert J. Ossenkoppele Lund University, Sweden VU University Medical Center, Amsterdam, he Netherlands Giuseppe Basso University of Padova, Italy Roger G. Owen Leeds University, UK Barbara Buldini University of Padova, Italy Andrew C. Rawstron Leeds University, UK Jacqueline Cloos VU University Medical Center, Gerrit J. Schuurhuis Amsterdam, he Netherlands VU University Medical Center, Amsterdam, he Netherlands Ruth M. de Tute Leeds University, UK Lori Soma University of Washington, Seattle, Washington, USA Jonathan R. Fromm University of Washington, Seattle, Washington, USA Anne Tierens University of Toronto, Canada Angèle Kelder VU University Medical Center, Theresia M. Westers Amsterdam, he Netherlands VU University Medical Center, Amsterdam, he Netherlands Wolfgang Kern MLL Munich Leukemia Laboratory, Brent L. Wood Munich, Germany University of Washington, Seattle, Washington, USA Francis Lacombe Andrea Zangrando Bordeaux University, France University of Padova, Italy Arjan A. van de Loosdrecht Wendelien Zeijlemaker VU University Medical Center, VU University Medical Center, Amsterdam, he Netherlands Amsterdam, he Netherlands vii © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Preface Together with cytological examination, low cytom- We then collected pertinent information about the etry is oten the irst exploration step in patients with structure, function and expression of a large number clinical symptoms suggesting haematological malig- of the antigens investigated in low cytometry, all men- nancy or with fortuitously discovered anomalies in a tioned somewhere in this book, together with a brief whole blood cell count. Depending on the healthcare history of the way they were characterised or discov- organisation, low cytometry results will stand alone ered (Chapter 2). and be discussed later during a diagnostic conference, Before tackling pathological issues, we thought or be integrated in a comprehensive set of investiga- that readers would appreciate some information about tions including bone marrow biopsy morphology, what to expect when low cytometry is applied to nor- cytogenetics and sophisticated molecular studies. mal samples of blood, bone marrow or lymphatic tissue Over the years, knowledge and skills have devel- (Chapter 3). We also listed a series of non-malignant oped so that in many cases the subtleties of the sets of conditions where the hypothesis of malignancy is plau- markers, as well as their expression of absence, have sible and must be ruled out (Chapter 4). become familiar to clinicians expecting a diagnosis. In Chapter 5, we present a collection of typical low Yet, the thousands of references in the literature, deal- cytometry graphs characteristic for various categories ing with this speciic part of laboratory haematology, of acute leukaemia. provide a good idea of the puzzlement that may over- From Chapters 6 to 13, the authors considered spe- whelm any novice in the ield. ciic sets of diseases and their idiosyncratic low cytom- hinking about the outlines of this book, we placed etry features. ourselves in the position of a young laboratory haema- Finally, Chapter 14 provides a glimpse at what lays tologist or haematopathologist and wondered which ahead, in the already foreseeable developments of the questions would need an answer likely to be found in a versatile and powerful technology of cell analysis. single document. We then asked Expert Friends to work We built this book, not only as a manual that may be with us with this aim in mind. Moreover, we wanted to read through while starting to work with low cytom- focus on the new 8- and 10-colour methodologies. etry diagnostics, but also as a reference document to We decided to start with basic characteristics of the consult when interested in any aspect of low cytom- structure and functions of low cytometers, trying to etry diagnostics of haematological malignancies. provide a clear explanation of what sometimes seems We hope that, together with our co-authors, we to be very complex. We also depicted the analysis have reached that goal. tools available in current sotware to make the most of acquired data (Chapter 1). Anna Porwit and Marie-Christine Béné ix © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Abbreviations 7-AAD 7-aminoactinomycin D ECD Energy coupled dye AIHA Auto immune haemolytic anaemia ( phycoerythrine-Texas red AITL Angio-immunoblastic T-cell conjugate) lymphoma EDTA Ethylene diamine tetraacetic acid ALL Acute lymphoblastic leukaemia ETP-ALL Early T-cell precursor acute AML Acute myeloid (or myeloblastic) lymphoblastic leukaemia leukaemia FCM Flow cytometry APC Allophycocyanin FITC Fluorescein isothiocyanate APL Acute promyelocytic leukemia FNA Fine-needle aspirate ATLL Adult T-cell leukemia/lymphoma FL Follicular lymphoma BCP ALL B-cell progenitor acute FLAER Fluorescein-labelled proaerolysin lymphoblastic leukaemia FLT3-ITD FMS-like tyrosine kinase-3 inversion BCR B-cell receptor tandem duplication mutation BCR-ABL Breakpoint cluster region-abelson FSC Forward scatter [t(9;22) also called Philadelphia HCL Hairy cell leukaemia chromosome] GFP Green luorescent protein BDCA Blood-derived dendritic cell antigens GvL Grat versus leukaemia BF Body luid HIV Human immunodeiciency virus BM Bone marrow HL Hodgkin lymphoma BPDCN Blastic plasmacytoid dendritic cell HLA-DR Human leukocyte antigen – antigen neoplasm D related BR Blast region HSL Hepatosplenic lymphoma CALLA Common acute lymphoblastic HSCT Haematopoeitic stem cell leukemia antigen transplantation CAR T-cell Chimeric antigen receptor T-cell HTLV-1 Human T-cell lymphotropic virus-1 CBF Core binding factor ICOS Inducible costimulatory CCR Chemokine receptor Ig Immunoglobulin CD Cluster of diferentiation IL Interleukin ChIP Chromatin immunoprecipitation JAK Janus kinase CLL Chronic lymphocytic leukemia KIR Killer immunoglobulin-like CLPD Chronic lymphoproliferative receptors disorders KrO Krome orange CML Chronic myeloid leukaemia LAIP Leukemia associated CMML Chronic myelomonocytic leukaemia immunophenotype CRLF2 Cytokine receptor-like factor LCA Leucocyte common antigen Cy Cyanin LGL Large granular lymphocyte DAPI 4′,6-diamidino-2-phenylindole LSC Leukemic stem cell DC Dendritic cell Lin Lineage DLBCL Difuse large B-cell lymphoma LPD Lymphoproliferative disorder xi DNA Deoxyribonucleic acid MAPK Mitogen activated protein kinase © in this web service Cambridge University Press www.cambridge.org Cambridge University Press 978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies Edited by Anna Porwit , Marie Christine Béné Frontmatter More Information Abbreviations MBL Monoclonal B-cell lymphocytosis PCR Polymerase chain reaction MCL Mantle cell lymphoma PD-1 Programmed death-1 MDS Myelodysplastic syndrome Percp Peridinin chlorophyll-A protein MF Mycosis fungoïdes PE R- Phycoerythrin MFI Mean luorescence intensity PI Propidium iodide MHC Major histocompatibility PI3K Phosphoinositide 3 kinase complex PML-RARA Promyelocytic leukemia/ MLL/KMT2A Mixed lineage leukaemia/lysine retinoic acid receptor A [t(15;17) methyl transferase 2A translocation] MRD Minimal residual disease PMT Photomultiplier m-TOR mammalian transporter of RNA Ribonucleic acid rapamycin RBC Red blood cell MZL Marginal zone lymphoma SC Sézary cell MPAL Mixed phenotype acute leukemia SLL Small lymphocytic lymphoma MRD Minimal residual disease SS Sézary syndrome NF Nuclear factor-kappa B SSC Side scatter NGS Next generation sequencing STAT Signal transducer and activator of NK Natural killer transcription NHL Non-hodgkin lymphoma TCR T-cell receptor NPM Nucleophosmin TdT Terminal deoxynucleotidyl PB Peripheral blood transferase Pbl Paciic blue TK Tyrosine kinase PC Plasma cell Tregs Regulatory T-cells PCA Principal component analysis WBC White blood cell PCM Plasma cell myeloma WHO World Health Organization xii © in this web service Cambridge University Press www.cambridge.org Chapter Flow Cytometry in Clinical 1 Haematopathology: Basic Principles and Data Analysis of Multiparameter Data Sets Francis Lacombe and Marie-Christine Béné Introduction the cells/particles suspension with the sheath liquid. The latter can therefore very well be pure water, its Flow cytometry (FCM), as indicated by its name, is a main characteristic being to be devoid of any particle. semi-automated method, which combines two basic The flux of cells/particles is guided in a specific device approaches, cytometry and flow, respectively. called a flow cell, through which a source of light will Cytometry is by essence the measurement of cell illuminate each cell as it passes in front of it. characteristics. More broadly, it can be applied to vari- The major advantages of FCM, compared to the ous types of particles. In fact, the very first application methods briefly mentioned above, are that larger num- of automated cytometry was invented by Wallace and bers of cells will be counted and that many parameters Joseph Coulter with the first objective of counting paint including immunological characterisation of cells will particles [1]. Before that era, cell counts were performed be examined. Moreover, all results will be electroni- manually under a microscope with specific calibrated cally stored, remaining available for analysis at any slides called haemocytometers usually bearing the names time after data acquisition. of their inventors (Malassez, Thoma, Neubauer, Nageotte and others) [2]. Such devices also allow us to recognise Cell Counting in FCM some cell types based on their size and granularity, detect- able without any staining, by simple phase contrast. Haemocytometers allow us to count cells in a well- Other properties of cells can be examined with an defined chamber of 0.1 mm3 using unmanipulated sus- optical bright field microscope after preparing smears pensions (i.e. cerebrospinal fluid) or diluted samples or cytospins where the cells are spread in a thin mono- where red blood cells have been lysed. Data are then con- layer fixed on a slide. Such preparations are then verted by calculation in the usual measurement units of stained, most frequently with May Grünwald Giemsa events per mm3 or per litre. For stained cells, typically, (MGG) or Wright stains [3]. These panoptic stains con- between 100 and 500 cells are counted manually when tain eosin and methylene blue, plus azure for MGG, performing cell differentials. Both these methods are and thus make acidic components appear blue and prone to errors linked to the small number of events basic components appear orange-red to violet. Smears actually taken into account and thus lack precision. This or cell suspensions can also be labelled with antibodies has been well established by Rümke, who designed a conjugated to fluorochromes and examined under UV table displaying the decreasing level of incertitude asso- light in specially equipped microscopes. ciated with larger numbers of events counted [4]. The flow component of FCM is a liquid sheath that Flow cytometry, that examines several thousands allows to convert a cell suspension in a narrow linear of events in a few minutes, provides a high level of sen- flux individualizing cells/particles. The flow part of sitivity and exactitude. The relative numbers (propor- the instrument performs what is called hydrofocus- tions) of each cell subsets acquired will therefore be ing, i.e. single cell alignment. Hydrodynamic focus- accurate. However, for exact absolute counting, flow ing is achieved by injecting the cell suspension in the cytometers require the use of standardised bead sus- core of sheath fluid at a slightly higher pressure and at a pensions with a known number of beads per microlitre point when the channel becomes smaller. The accelera- mixed volume/volume with the cells/particles prepa- tion of the fluids through this narrow channel and the ration. When this known number of beads has been different speed of the sheath and the cell suspension recorded by the instrument, it can therefore be con- 1 result in cell alignment. Of note, there is no mixing of cluded that 1 μL of sample has been examined. This can 19:40:10 02 Chapter 1: Flow Cytometry in Clinical Haematopathology then be extrapolated to the other cells recorded during the same time. Another possibility is to use calibrated volumetric systems built in the flow cell. Because of these large numbers, it also allows to identify minute populations, likely to be missed with smaller counts. Photodiode Laser No cell: no signal FCM and Light (a) Besides counting particles, FCM allows to appreciate their physical, chemical or biological properties. The major physical properties of particles/cells exploited by FCM are their ability to diffract, reflect and refract the coherent monochromatic light of a laser beam. Different types of lasers are available. Flow cytometers were initially equipped with gas lasers using argon, Laser Photodiode krypton, helium-neon or helium-cadmium, some Cell: light diffraction → signal requiring a cooling system. Solid-state lasers are crys- (b) tals [ruby, yttrium aluminium garnet (YAG)] or ions Figure 1.1 Forward scatter measurement of cell size. (a) The laser such as titanium or chromium. More recent instru- light is blocked by the mask when no cell passes through the beam. ments use laser diodes based on semi-conductors and (b) Each cell, by diffracting light, allows for a signal proportional to its size to be collected on the detector around the mask. similar to light-emitting diodes (LEDs) [5]. When the narrow and focused coherent light of the laser encounters a cell/particle (so-called event), its diffraction intensity is proportional to the size of type of cells investigated, i.e. it will have to be higher the ‘event’. Flow cytometers are equipped with photo- to see small particles such as platelets and lower to see diodes collecting the light diffracted by the cell in the larger cells such as granulocytes. path of the laser beam, of forward scatter (FSC). The Chemical parameters can also be measured by instruments are also equipped with a device (mask) flow cytometers, typically based on the properties of blocking the laser light from the FSC photodiode when fluorochromes. The latter are chemical substances able no particle crosses it. The beam is widened proportion- to absorb light at a defined wavelength and re-emit it ally to the size of the cell as one enters the laser’s path at a higher and defined wavelength. This is based on and the FSC photodiode can collect and transform it in the fact that, in these molecules, absorption of a pho- an electronic signal proportional to the size of the cell/ ton will result in a modification of electrons, moving particle (Figure 1.1). from a ground state to an excited state. When electrons Concomitantly, a second detector (photodiode or return to their ground state, they restore the energy by photomultiplier tube (PMT)) collects the light reflected going through transition stages resulting in the emis- by the surface of the cell/particle as well as by any sur- sion of a quantum of light with a higher energy and face inside it (i.e. organelles, vesicles, granules, etc.) at a thus higher wavelength than the emission light [6]. defined angle, lateral to the path of the laser beam. The Basically, in FCM, lasers provide excitation light and intensity of this side scatter (SSC) signal will thus be fluorochromes emission light. To collect emitted light proportional to the granularity of the cell. Typically, in from each fluorochrome, flow cytometers are equipped a blood sample, the small erythrocytes with no nuclei with dichroic mirrors and bandpass filters before each will provide very small signals while those generated signal is registered by a dedicated PMT. Dichroic mir- by the larger granulocytes will be more intense. The rors reflect light at a specific wavelength while letting pattern of scatter signals will differ slightly between all other light pass through. They are positioned at an instruments, depending on the angle of the SSC detec- angle from the emission source so that reflected beams tor and the number of display channels (see below in make a 90° angle to the mirror and get directed towards signal acquisition). the relevant PMT. Just before PMTs, filters of a specific 2 The voltage applied to the detector will also modify wavelength will narrow the beam of light collected, the intensity of light collected. It must be adapted to the ideally at the level of peak fluorescence. 19:40:10 02
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