Practical Flow Cytometry in Haematology: 100 Worked Examples

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Library of Congress Cataloging-in-Publication Data

Leach, Richard M. (Haematologist), author.

Practical flow cytometry in haematology : 100 worked examples / Mike Leach [and 5 others].

p. ; cm.

Includes index.

ISBN 978-1-118-74703-2 (hardback)

I. Title.

[DNLM: 1. Hematologic Diseases–diagnosis–Case Reports. 2. Hematologic Neoplasms–diagnosis–Case Reports. 3. Flow Cytometry–methods–Case Reports. 4. Hematology–methods–Case Reports. WH 120]

RC636

616.1′5075—dc23

2015007734

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.

Preface

In our first publication ‘Practical Flow Cytometry in Haematology Diagnosis’ we presented an outline approach to the use and applications of flow cytometric immunophenotyping in the diagnostic haematology laboratory. We showed how this technique could be used to study blood, bone marrow and tissue fluid samples in a variety of clinical scenarios to achieve a diagnosis, taking into account important features from the clinical history and examination alongside haematology, morphology, biochemistry, immunology, cytogenetic, histopathology and molecular data. This text was illustrated with a series of ‘worked examples’ from real clinical cases presenting to our institution. These cases have proven to be very popular and so a companion publication dedicated to 100 new ‘worked examples’ seemed justified and is presented here.

The principles used in the approach to each case are exactly the same as used in the first publication and cases are illustrated with tissue pathology and cytogenetic and molecular data, which are integrated to generate, where appropriate, a diagnosis based on the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. We present a spectrum of clinical cases encountered in our department from both adult and paediatric patients and of course, if the title is to be justified, flow cytometry plays a role in every case. Furthermore, we present both neoplastic and reactive disorders and the cases appear in no particular order so that the reader should have no pre-conceived idea as to the nature of the diagnosis in any case. May−Grünwald−Giemsa (MGG)-stained films of peripheral blood and bone marrow aspirates are presented with flow cytometry data alongside haematoxylin and eosin (H&E)-stained bone marrow and tissue biopsy sections. Immunohistochemistry is used to further clarify the tissue lineage and cell differentiation. Cytogenetic studies using metaphase preparations are used to identify translocations and chromosome gains and losses whilst interphase fluorescence in situ hybridisation (FISH) studies and polymerase chain reaction (PCR) are used to identify gene fusions, break-aparts and deletions. The presentation is brought to a conclusion and the particular features that are important in making a diagnosis are highlighted and discussed. The cases are also listed according to disease classification toward the end (page 390) so that the text can also be used as a reference manual.

The analysis of blood, bone marrow and tissue fluid specimens requires a multi-faceted approach with the integration of scientific data from a number of disciplines. No single discipline can operate in isolation or errors will occur. Flow cytometry technology is in a privileged position in that it can provide rapid analysis of specimens; it is often the first definitive investigation to produce results and help formulate a working diagnosis. The results from flow cytometry can help to structure investigative algorithms to ensure that the appropriate histopathological, cytogenetic and molecular studies are performed in each case. Tissue samples are often limited in volume and difficult to acquire so it is important to stratify investigations accordingly and to get the most from the material available. It is not good scientific or economic practice to run a large series of poorly focussed analyses on every case. Appropriate studies need to be executed in defined circumstances and flow cytometry can guide subsequent investigations in a logical fashion. In some situations a rapid succinct diagnosis can be achieved; immunophenotyping excels in the identification of acute leukaemia. Cytogenetic studies and molecular data give important prognostic information in these patients. But of course the recognised genetic aberrations need to be demonstrated if the diagnosis is to be substantiated. Acute promyelocytic leukaemia can often be confidently diagnosed using morphology alongside immunophenotyping data, but a PML translocation to the RARA fusion partner, needs to be shown. Flow cytometry cannot operate in isolation; despite having the ‘first bite of the cherry’ the differential diagnosis can still be wide open. There are a good number of worked examples illustrated here where immunophenotyping was not able to indicate a specific diagnosis. The disease entities with anaplastic or ‘minimalistic’ phenotypes frequently cause difficulty. Appropriate histopathology and FISH, performed on the basis of flow cytometric findings, highlighting abnormal protein expression and gene rearrangement respectively, can make a major contribution to diagnosis and disease classification. Only when a specific diagnosis is made and prognostic parameters are assessed can the optimal management plan be considered for each individual patient. Finally, the goal posts are constantly moving and developments in the molecular basis of disease, refining disease classification, are evolving rapidly. Whether we are considering eosinophilic proliferations, the myriad of myeloproliferative neoplasms, lymphoproliferative disorders or acute leukaemias we are constantly noting developments and adjusting diagnosis and prognosis accordingly. This is an era of evolving diagnostic challenge and rapid molecular evolution where the practising clinician needs to keep abreast of the significant developments in all areas of haematopathology.

The flow cytometric principles applied to each case have been described in detail in ‘Practical Flow Cytometry in Haematology Diagnosis’ and some working knowledge is required to interpret the cases described. We also anticipate a reasonable ability in morphological assessment and a capacity to identify morphological variations seen in various disease states. In spite of this we do endeavour to describe the diagnostic logic that we have applied to each worked example and demonstrate how cellular immunophenotypes have helped determine the nature of the disorder.

This text will be of interest to all practicing haematologists and to histopathologists with an interest in haematopathology but it is particularly directed at trainee haematologists and scientists preparing for FRCPath examinations.

Acknowledgement

We are grateful for the substantial assistance of Dr Avril Morris DipRCPath, Principal Clinical Scientist, West of Scotland Genetic Services, Southern General Hospital, Glasgow with regard to the provision of the cytogenetic data and images relevant to the clinical cases presented here.

List of Abbreviations

ADP adenosine diphosphate

AITL angioimmunoblastic T-cell lymphoma

AL acute leukaemia

ALCL anaplastic large cell lymphoma

ALL acute lymphoblastic leukaemia

ALP alkaline phosphatase

ALT alanine transaminase

AML acute myeloid leukaemia

AML-MRC acute myeloid leukaemia with myelodysplasia-related changes

ANA antinuclear antibody

APC allophycocyanin

APL acute promyelocytic leukaemia

APTT activated partial thromboplastin time

ASM aggressive systemic mastocytosis

AST aspartate transaminase

ATLL adult T-cell leukaemia/lymphoma

ATRA all-trans-retinoic acid

AUL acute undifferentiated leukaemia

B-ALL B-lineage acute lymphoblastic leukaemia

BCLU B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

BEAM carmustine (BCNU), etoposide, cytarabine (cytosine arabinoside) and melphalan

BL Burkitt lymphoma

BP blast phase

BPDCN blastic plasmacytoid dendritic cell neoplasm

c cytoplasmic

CD cluster of differentiation

CHOP cyclophosphamide, doxorubicin, vincristine and prednisolone

CLL chronic lymphocytic leukaemia

CML chronic myeloid leukaemia

CMML chronic myelomonocytic leukaemia

CMV cytomegalovirus

CNS central nervous system

CODOX M/IVAC cyclophosphamide, vincristine, doxorubicin, methotrexate/ ifosphamide, mesna, etoposide, cytarabine

CR complete remission

CRAB calcium (elevated), renal failure, anaemia, bone lesions

CSF cerebrospinal fluid

CT computed tomography

CTCL cutaneous T-cell lymphoma

CTD cyclophosphamide, thalidomide and dexamethasone

CXR chest X-ray

cyt, cyto cytoplasmic

DEXA scanning dual energy X-ray absorptiometry scanning

DIC disseminated intravascular coagulation

DKC dyskeratosis congenita

DLBCL diffuse large B-cell lymphoma

DM double marking

EBER EBV-encoded small RNAs

EBV Epstein-Barr virus

EBV LMP Epstein-Barr virus latent membrane protein

EDTA ethylene diamine tetra-acetic acid

eGFR estimated glomerular filtration rate

EMA eosin-5-maleimide

EORTC European Organization for Research and Treatment of Cancer

ESHAP etoposide, methyl prednisolone, cytarabine, cisplatin

ESR erythrocyte sedimentation rate

ET essential thrombocythaemia

ETP-ALL early T-cell precursor acute lymphoblastic leukaemia

FAB French−American−British (leukaemia classification)

FBC full blood count

FDG fluorodeoxyglucose

FISH fluorescence in situ hydridisation

FITC fluorescein isothocyanate

FL follicular lymphoma

FLAER fluorescein-conjugated proaereolysin

FLAG fludarabine, cytarabine, granulocyte colony-stimulating factor

FLAG-IDA fludarabine, cytarabine, granulocyte colony-stimulating factor, idarubicin

FSC forward scatter

GGT gamma glutamyl transferase

GI gastrointestinal

Gp glycoprotein

GP general practitioner

GPI glycosylphosphatidylinositol

H&E haematoxylin and eosin

Hb haemoglobin concentration

HCL hairy cell leukaemia

HCL-V hairy cell leukaemia variant

HHV human herpesvirus

HIV human immunodeficiency virus

HL Hodgkin lymphoma

HLA-DR human leucocyte antigen DR

HS hereditary spherocytosis

HTLV-1 human T-cell lymphotropic virus-1

ICC immunocytochemistry

Ig immunoglobulin

IgA immunoglobulin A

IgG immunoglobulin G

IgM immunoglobulin M

IHC immunohistochemistry

IPSS International Prognostic Scoring System

ISCL International Society for Cutaneous Lymphomas

ISH in situ hybridisation

ISM indolent systemic mastocytosis

ITD internal tandem duplication

ITP ‘idiopathic’ (autoimmune) thrombocytopenia purpura

IVLBCL intravascular large B-cell lymphoma

LAP leukaemia-associated phenotype

LBL lymphoblastic lymphoma

LDH lactate dehydrogenase

LFTs liver function tests

LGL large granular lymphocyte

LPD lymphoproliferative disorder

MCH mean cell haemoglobin

MCL mantle cell lymphoma

MCV mean cell volume

MDS myelodysplastic syndrome/s

MDS/MPN myelodysplastic/myeloproliferative neoplasm

MF mycosis fungoides

MGG May−Grünwald−Giemsa

MGUS monoclonal gammopathy of undetermined significance

MM multiple myeloma

mod moderate fluorescence

MPAL mixed phenotype acute leukaemia

MPN myeloproliferative neoplasm

MPO myeloperoxidase

MRD minimal residual disease

MRI magnetic resonance imaging

MZL marginal zone lymphoma

NLPHL nodular lymphocyte-predominant Hodgkin lymphoma

NOS not otherwise specified

NR normal range

PAS periodic acid-Schiff

PCR polymerase chain reaction

PD-1 an antigen, programmed death 1(CD279)

PE phycoerythrin

PEL primary effusion lymphoma

PET positron-emission tomography

Ph Philadelphia (chromosome)

PMF primary myelofibrosis

PNET primitive neuroectodermal tumour

PNH paroxysmal nocturnal haemoglobinuria

PRCA pure red cell aplasia

PT prothrombin time

PTCL-NOS peripheral T-cell lymphoma, not otherwise specified

PTLD post-transplant lymphoproliferative disorder

PV polycythaemia vera

RBC red blood cell (count)

R-CHOP rituximab, doxorubicin, vincristine and prednisolone

R-CVP rituximab, cyclophosphamide, vincristine and prednisolone

RNA ribonucleic acid

RQ-PCR real-time quantitative polymerase chain reaction

RS Reed–Sternberg

RT-PCR reverse transcriptase polymerase chain reaction

SAA severe aplastic anaemia

Sig surface membrane immunoglobulin

SLE systemic lupus erythematosus

SM systemic mastocytosis

SM-AHNMD systemic mastocytosis with associated clonal haematological non-mast cell disease

SMILE dexamethasone, methotrexate, ifosfamide, L-asparaginase and etoposide

SSC side scatter

T-ALL T-lineage acute lymphoblastic leukaemia

TBI total body irradiation

TdT terminal deoxynucleotidyl transferase

TIA T-cell intracellular antigen

TKI tyrosine kinase inhibitor

T-LBL T-lymphoblastic lymphoma

t-MDS therapy-related myelodysplastic syndrome

TRAP tartrate-resistant acid phosphatase

TT thrombin time

TTP thrombotic thrombocytopenic purpura

U&Es urea, electrolytes and creatinine

USS ultrasound

WAS Wiskott−Aldrich syndrome

WASp Wiskott−Aldrich syndrome protein

WBC white blood cell (count)

WM Waldenström macroglobulinaemia

Technical Notes

The patients presented in 100 Worked Examples were all real cases encountered and investigated in a regional flow cytometry laboratory serving a population of approximately 2.5 million over a period of 18 months. These are individually presented with a history that reflects the actual events for each patient, commencing with the presenting clinical features and the initial basic laboratory tests and then proceeding to flow cytometry, bone marrow aspirate morphology, bone marrow trephine biopsy histology with immunohistochemistry studies and other specialised cytogenetic and molecular analyses.

Full blood counts

The full blood counts and marrow counts (for appropriate dilutions in relation to antibody) were performed on a Sysmex XN analyser. The differential leucocyte counts are automated counts from the analyser. It should be noted that sometimes, in an automated count, abnormal cells are misidentified and the leucocyte sub-populations differ from a manual differential performed on a blood film. Such misidentifications are indicated by inverted commas.

Biochemistry and immunology studies

All relevant biochemistry and immunology data is given in relation to the context of each patient presentation and in terms of investigations that were thought to be relevant to the case as the clinical diagnosis evolved. Some retrospectively relevant data may be missing but this reflects the true nature of these actual patient scenarios and the investigations that were considered necessary at that time. Serum calcium values given are all corrected in accordance with serum albumin level.

Flow cytometry analysis

Flow cytometry studies were all performed using a Becton Dickinson FACS Canto II analyser. The findings are presented as a list of positive and negative results in relation to the antigen and target cell population and the gating strategies applied to each case are explained. A series of scatter plots and histograms are presented to illustrate specific informative points. The expression of most membrane antigens is graded as positive when more than 20% of gated events are positive; the exceptions being CD34, CD117 and cytoplasmic antigens where a threshold of 10% has been used. Where the percentage positivity for a given membrane antigen in the gated target population is borderline positive so that some cells appear negative and some positive we have used the term ‘partial’ to describe antigen expression. Cytoplasmic expression of an antigen is indicated with the prefix ‘c’ (cytoplasmic expression of CD3 being cCD3) but on some scatter plots ‘cyt’ or ‘cyto’ has been used. The intensity of antigen expression in terms of median fluorescence intensity is graded as dim, moderate or bright compared to our laboratory reference ranges for normal cells of each relevant lineage. See Figures 1.1a–g for a schematic representation of these principles.

Figure 1.1 Visual representation of strength of fluorescence in flow cytometry (not actual patient specimens), showing an isotype control and eight CD19-positive samples which show fluorescence intensity with CD20 varying from negative to bright. (a) Isotype control, used to set thresholds. (b) Negative (consistent with a CD19-positive, CD20-negative B-cell precursor neoplasm). (c) Partial positive, indicating that CD20 antigen expression varies from negative to positive (consistent with a precursor B-cell neoplasm). (c adjusted) Indicating that the threshold for positivity might be reduced by the cytometrist where a discrete dim positive population is identified. (d) Dim CD20 antigen expression (consistent with chronic lymphocytic leukaemia). (e) Moderate intensity, indicating medium strength of CD20 antigen expression (consistent with B-cell non-Hodgkin lymphoma). (f) bright, indicating strong CD20 antigen expression (consistent with hairy cell leukaemia). (g) Two distinct populations, one partial and dim and one bright (could indicate two unrelated B-lineage neoplasms or transformation of a low grade lymphoma). (h) Contrasting with (g), a heterogeneous single population with fluorescence intensity varying from negative to moderate with a minority being bright.

Immunohistochemistry in paraffin-embedded formalin fixed tissue

In the following section a list is presented of the immunohistochemical reagents used in assessing the paraffin embedded material (bone marrow trephine and lymph node biopsies) in the worked examples described. It should be pointed out that specificities and sensitivities may differ from the antibodies used in flow cytometry due to the effects of formalin fixation and decalcification resulting in antigen loss or masking. For example, CD5 may be detected by flow cytometry in a peripheral blood B-cell lymphocytosis but immunocytochemistry may on occasion be negative for the same marker in the trephine specimen. CD56 is aberrantly expressed by plasma cells in myeloma yet immunoreactivity for this antibody within plasma cells in paraffin sections is seen in only a minority of cases. The opposite situation may also occur where an antigen such as TdT is strongly positive by immunohistochemistry on the fixed tissue but is negative on the flow sample. Reticulin fibrosis is reported as per the WHO classification as grade 0, 1, 2 or 3.

These specific features of different techniques need to be appreciated when formulating the combined pathology report and an understanding of the strengths and weaknesses of each approach is essential when establishing a final diagnosis. Cytogenetic and molecular studies have a major influence on disease classification. Specific findings can carry diagnostic significance way in excess of any other single investigative modality e.g. BCR-ABL1, PML-RARA, FIP1L1-PDGRFA. Metaphase cytogenetic studies not infrequently fail, either reflecting the quality of the specimen or the disease entity being studied. Informed FISH and PCR studies can carry great diagnostic importance in certain clinical circumstances and molecular diagnostics will continue to inform disease classification with increasing power and specificity over the decades ahead.

Laboratory Values

Abbreviations and Normal Ranges.

Case 1

An 11-year-old boy was admitted with a short history of fever, sweats, dyspnoea and left chest discomfort. There was no past history of note. Examination identified features of a left pleural effusion. There was also a tender swelling of the left anterior chest in the upper pectoral region and palpable cervical lymphadenopathy. The liver and spleen were not palpable.

Laboratory investigations

FBC and blood film: normal

U&Es, LFTs: normal. LDH was 1460 U/L.

Imaging

The CXR showed opacification and loss of aeration of the left hemithorax in keeping with a pleural effusion (Figure 1.1). CT imaging confirmed this but in addition identified a left pleural-based mass, abnormal soft tissue in the left pectoral muscles (arrows, Figure 1.2) and cervical lymphadenopathy. In addition, there was collapse/consolidation of the lower left lung, creating the appearance of an air bronchogram. A core biopsy of a cervical node was taken and the pleural effusion was aspirated for analysis.

Figure 1.1 CXR.

Figure 1.2 CT.

Flow cytometry

The pleural fluid cell count was 0.98 × 10⁹/L. A cytospin preparation showed three distinct cell types: a small mature lymphoid population in keeping with reactive lymphocytes, an intermediate sized/large sized lymphoid population and a large cell population with pleomorphic morphology and blue cytoplasm (Figures 1.3–1.6). The cells with the abundant cytoplasm (Figures 1.3 and 1.4) and the single binucleate cell (Figure 1.6) are reactive mesothelial cells. The cells with the cytoplasmic blebs (Figures 1.4–1.6) are the disease cells, which were the subsequent focus for immunophenotyping studies.

Figure 1.3 MGG, ×500.

Figure 1.4 MGG, ×500.

Figure 1.5 MGG, ×500.

Figure 1.6 MGG, ×500.

By applying a blast gate to the suspected malignant cells in the FSC/SSC analysis (Figure 1.7), they were shown to express CD45bright (Figure 1.8), CD2 (Figure 1.9), cCD3 [whilst surface CD3 was negative apart from a few reactive T cells (Figure 1.8)], partial CD7 (Figure 1.10) and CD13. Other T-lineage markers were negative.

Figure 1.7 FSC/SSC.

Figure 1.8 CD3/CD45.

Figure 1.9 CD2/CD19.

Figure 1.10 CD7/CD16.

This is therefore a T-lymphoid neoplasm, indicated by positivity for cCD3 expression, with limited lineage-specific markers and an aberrant myeloid marker. The tumour has medium sized/large cell morphology. It was showing aggressive clinical behaviour with extranodal tissue invasion in this 11-year-old patient. An anaplastic large cell lymphoma had to be considered and the medium sized/large cells in the pleural fluid were shown to be strongly expressing CD30 (not shown).

Histopathology

An H&E-stained core biopsy of a cervical node is shown in Figure 1.11. The node is replaced by an infiltrate of undifferentiated pleomorphic large cells with prominent nucleoli.

Figure 1.11 H&E, ×400.

Immunohistochemistry showed the large cells to express CD45, epithelial membrane antigen (EMA), CD2 focally (Figure 1.12), CD7, granzyme B and CD30 (Figure 1.13). In addition, there was strong nuclear and cytoplasmic staining for anaplastic lymphoma kinase (ALK) protein (Figure 1.14).

Figure 1.12 CD2, ×400.

Figure 1.13 CD30, ×400.

Figure 1.14 ALK, ×400.

The CD30 staining was particularly useful in demonstrating lymphatic invasion within the capsule of the node (Figure 1.15).

Figure 1.15 CD30, ×100.

FISH studies

A t(2;5)(p23;q35) translocation, rearranging the ALK and NPM1 (nucleophosmin) genes, was shown by FISH studies on paraffin-embedded lymph node tissue. The presence of this specific translocation is highly associated with both nuclear and cytoplasmic positivity for ALK.

Discussion

Anaplastic large cell lymphoma (ALCL) is an aggressive mature T-cell neoplasm with pleomorphic, often large cell, morphology. It frequently fails to show surface expression of T-lineage-specific markers and to potentially further mislead may express aberrant myeloid antigens. This is an important condition to recognise; it frequently shows rapid progression with extranodal tissue involvement and it can rarely appear in the blood. Treatment of ALK+ ALCL is usually rewarding, particularly in paediatric patients, with prompt response to chemotherapy and frequent durable remissions.

Final diagnosis

Anaplastic large cell lymphoma (ALK+)

Case 2

A 72-year-old woman presented with a few months' history of fatigue and the more recent onset of breathlessness and night sweats. On clinical examination she had a large right-sided pleural effusion but no palpable lymphadenopathy.

Laboratory results

FBC: Hb 158 g/L, WBC 16.6 × 10⁹/L (neutrophilia and monocytosis) and platelets 502 × 10⁹/L.

U&Es: normal. LFTs were mildly deranged (ALT 52 U/L, alkaline phosphatase 173 U/L). Albumin was low at 29 g/L and serum LDH was raised at 584 U/L.

Imaging

A CT scan demonstrated a large right-sided pleural effusion with collapse of the right middle and lower lobes and partial collapse of the upper lobe (Figure 2.1). In addition, there were large volume, confluent, necrotic nodal masses in the right hilar, mediastinal, retrocrural, paracardiac and para-aortic areas (not shown) as well as pleural deposits (arrow, Figure 2.1).

Figure 2.1 CT.

Pleural fluid biochemistry and cytology

The pleural fluid LDH was markedly elevated at 2171 U/L with relatively low glucose at 7.2 mmol/L (patient diabetic) and protein of 43 g/L.

Microscopy of the pleural fluid showed lymphoid cells admixed with neutrophils, histiocytes and mesothelial cells. Most of the lymphoid cells were small but an admixed population of medium-sized cells with slightly irregular nuclei was also present. On morphology alone, the lymphoid cells were thought likely to be reactive but the reporting pathologist suggested that a fresh pleural fluid specimen should be assessed using flow cytometry.

Morphology (pleural fluid)

A specimen of pleural fluid was received by our laboratory. The WBC was found to be 6.3 × 10⁹/L. A cytospin preparation showed a cellular specimen with notable macrophages, neutrophils and small lymphocytes. In addition, some large blastoid lymphoid cells were seen (Figures 2.2–2.5).

Figure 2.2 MGG, ×500.

Figure 2.3 MGG,