Multiparameter flow cytometry in the diagnosis of hematologic malignancies

242 Pages·2018·41.375 MB·English

by  Anna Porwit, Marie-Christine Béné

Cambridge University Press
978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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Multiparameter Flow
Cytometry in the Diagnosis of
Hematologic Malignancies
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Cambridge University Press
978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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Multiparameter Flow
Cytometry in the Diagnosis of
Hematologic Malignancies
Edited by
Anna Porwit
Lund University, Sweden
Marie-Christine Béné
University of Nantes, France
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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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
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ISBN 978-1-107-50383-0 Paperback
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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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
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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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
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
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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
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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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
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978-1-107-50383-0 — Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies
Edited by Anna Porwit , Marie Christine Béné
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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
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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