30
Practical Flow Cytometryin Haematology: 100Worked Examples
Practical Flow Cytometryin Haematology: 100Worked ExamplesMike Leach FRCP, FRCPathConsultant Haematologist and Honorary Senior LecturerHaematology Laboratories and West of Scotland Cancer CentreGartnavel General HospitalGlasgow, UK
Mark Drummond PhD, FRCPathConsultant Haematologist and Honorary Senior LecturerHaematology Laboratories and West of Scotland Cancer CentreGartnavel General HospitalGlasgow, UK
Allyson Doig MSc, FIBMSHaemato-Oncology Laboratory ManagerHaematology LaboratoriesGartnavel General HospitalGlasgow, UK
Pam McKayConsultant HaematologistHaematology Laboratories and West of Scotland Cancer CentreGartnavel General HospitalGlasgow, UK
Bob JacksonConsultant PathologistDepartment of PathologySouthern General HospitalGlasgow, UK
Barbara J. Bain MBBS, FRACP, FRCPathProfessor of Diagnostic HaematologySt Mary’s Hospital Campus of Imperial CollegeFaculty of Medicine, Londonand Honorary Consultant Haematologist,St Mary’s Hospital, London, UK
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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]RC636616.1′5075–dc23
2015007734
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Contents
Preface, vii
Acknowledgement, ix
List of Abbreviations, xi
Technical Notes, xv
Laboratory Values, xix
Case 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Case 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Case 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Case 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Case 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Case 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Case 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Case 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Case 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Case 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Case 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Case 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Case 13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Case 14 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Case 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Case 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Case 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Case 18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Case 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Case 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Case 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Case 22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Case 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Case 24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Case 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Case 26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Case 27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Case 28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Case 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Case 30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Case 31 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Case 32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Case 33 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Case 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Case 35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Case 36 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Case 37 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Case 38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Case 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Case 40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Case 41 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Case 42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Case 43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Case 44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Case 45 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Case 46 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Case 47 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Case 48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Case 49 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Case 50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
v
vi Contents
Case 51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Case 52 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Case 53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Case 54 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Case 55 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Case 56 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Case 57 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Case 58 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Case 59 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Case 60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Case 61 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Case 62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
Case 63 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Case 64 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Case 65 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Case 66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Case 67 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Case 68 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Case 69 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Case 70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Case 71 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Case 72 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Case 73 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Case 74 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Case 75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Case 76 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Case 77 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Case 78 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Case 79 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Case 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Case 81 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Case 82 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Case 83 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Case 84 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
Case 85 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Case 86 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Case 87 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Case 88 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Case 89 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Case 90 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Case 91 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Case 92 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
Case 93 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Case 94 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Case 95 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Case 96 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Case 97 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Case 98 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Case 99 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
Case 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Antibodies Used in ImmunohistochemistryStudies, 381
Flow Cytometry Antibodies, 386
Molecular Terminology, 389
Classification of Cases According to Diagnosis, 390
Index, 391
Preface
In our first publication ‘Practical Flow Cytometry in Haema-tology Diagnosis’ we presented an outline approach to theuse and applications of flow cytometric immunophenotypingin the diagnostic haematology laboratory. We showed howthis technique could be used to study blood, bone marrowand tissue fluid samples in a variety of clinical scenariosto achieve a diagnosis, taking into account importantfeatures from the clinical history and examination alongsidehaematology, morphology, biochemistry, immunology,cytogenetic, histopathology and molecular data. This textwas illustrated with a series of ‘worked examples’ from realclinical cases presenting to our institution. These cases haveproven to be very popular and so a companion publicationdedicated to 100 new ‘worked examples’ seemed justifiedand is presented here.
The principles used in the approach to each case areexactly the same as used in the first publication and casesare illustrated with tissue pathology and cytogenetic andmolecular data, which are integrated to generate, whereappropriate, a diagnosis based on the WHO Classificationof Tumours of Haematopoietic and Lymphoid Tissues.We present a spectrum of clinical cases encountered inour department from both adult and paediatric patientsand of course, if the title is to be justified, flow cytometryplays a role in every case. Furthermore, we present bothneoplastic and reactive disorders and the cases appearin no particular order so that the reader should have nopre-conceived idea as to the nature of the diagnosis in anycase. May−Grünwald−Giemsa (MGG)-stained films ofperipheral blood and bone marrow aspirates are presentedwith flow cytometry data alongside haematoxylin and eosin(H&E)-stained bone marrow and tissue biopsy sections.Immunohistochemistry is used to further clarify the tissuelineage and cell differentiation. Cytogenetic studies usingmetaphase preparations are used to identify translocationsand chromosome gains and losses whilst interphase fluores-cence in situ hybridisation (FISH) studies and polymerasechain reaction (PCR) are used to identify gene fusions,break-aparts and deletions. The presentation is brought to aconclusion and the particular features that are important inmaking a diagnosis are highlighted and discussed. The cases
are also listed according to disease classification toward theend (page 390) so that the text can also be used as a referencemanual.
The analysis of blood, bone marrow and tissue fluidspecimens requires a multi-faceted approach with theintegration of scientific data from a number of disciplines.No single discipline can operate in isolation or errors willoccur. Flow cytometry technology is in a privileged positionin that it can provide rapid analysis of specimens; it isoften the first definitive investigation to produce results andhelp formulate a working diagnosis. The results from flowcytometry can help to structure investigative algorithms toensure that the appropriate histopathological, cytogeneticand molecular studies are performed in each case. Tissuesamples are often limited in volume and difficult to acquireso it is important to stratify investigations accordingly andto get the most from the material available. It is not goodscientific or economic practice to run a large series of poorlyfocussed analyses on every case. Appropriate studies need tobe executed in defined circumstances and flow cytometrycan guide subsequent investigations in a logical fashion. Insome situations a rapid succinct diagnosis can be achieved;immunophenotyping excels in the identification of acuteleukaemia. Cytogenetic studies and molecular data giveimportant prognostic information in these patients. Butof course the recognised genetic aberrations need to bedemonstrated if the diagnosis is to be substantiated. Acutepromyelocytic leukaemia can often be confidently diagnosedusing morphology alongside immunophenotyping data, buta PML translocation to the RARA fusion partner, needsto be shown. Flow cytometry cannot operate in isolation;despite having the ‘first bite of the cherry’ the differentialdiagnosis can still be wide open. There are a good numberof worked examples illustrated here where immunophe-notyping was not able to indicate a specific diagnosis. Thedisease entities with anaplastic or ‘minimalistic’ phenotypesfrequently cause difficulty. Appropriate histopathology andFISH, performed on the basis of flow cytometric findings,highlighting abnormal protein expression and gene rear-rangement respectively, can make a major contribution todiagnosis and disease classification. Only when a specific
vii
viii Preface
diagnosis is made and prognostic parameters are assessedcan the optimal management plan be considered for eachindividual patient. Finally, the goal posts are constantlymoving and developments in the molecular basis of disease,refining disease classification, are evolving rapidly. Whetherwe are considering eosinophilic proliferations, the myriad ofmyeloproliferative neoplasms, lymphoproliferative disordersor acute leukaemias we are constantly noting develop-ments and adjusting diagnosis and prognosis accordingly.This is an era of evolving diagnostic challenge and rapidmolecular evolution where the practising clinician needs tokeep abreast of the significant developments in all areas ofhaematopathology.
The flow cytometric principles applied to each case havebeen described in detail in ‘Practical Flow Cytometry in
Haematology Diagnosis’ and some working knowledge isrequired to interpret the cases described. We also anticipatea reasonable ability in morphological assessment and acapacity to identify morphological variations seen in variousdisease states. In spite of this we do endeavour to describethe diagnostic logic that we have applied to each workedexample and demonstrate how cellular immunophenotypeshave helped determine the nature of the disorder.
This text will be of interest to all practicing haematologistsand to histopathologists with an interest in haematopathol-ogy but it is particularly directed at trainee haematologistsand scientists preparing for FRCPath examinations.
Acknowledgement
We are grateful for the substantial assistance of Dr AvrilMorris DipRCPath, Principal Clinical Scientist, West ofScotland Genetic Services, Southern General Hospital,
Glasgow with regard to the provision of the cytogenetic dataand images relevant to the clinical cases presented here.
ix
List of Abbreviations
ADP adenosine diphosphateAITL angioimmunoblastic T-cell lymphomaAL acute leukaemiaALCL anaplastic large cell lymphomaALL acute lymphoblastic leukaemiaALP alkaline phosphataseALT alanine transaminaseAML acute myeloid leukaemiaAML-MRC acute myeloid leukaemia with
myelodysplasia-related changesANA antinuclear antibodyAPC allophycocyaninAPL acute promyelocytic leukaemiaAPTT activated partial thromboplastin timeASM aggressive systemic mastocytosisAST aspartate transaminaseATLL adult T-cell leukaemia/lymphomaATRA all-trans-retinoic acidAUL acute undifferentiated leukaemiaB-ALL B-lineage acute lymphoblastic
leukaemiaBCLU B-cell lymphoma, unclassifiable, with
features intermediate between diffuselarge B-cell lymphoma and Burkittlymphoma
BEAM carmustine (BCNU), etoposide,cytarabine (cytosine arabinoside) andmelphalan
BL Burkitt lymphomaBP blast phaseBPDCN blastic plasmacytoid dendritic cell
neoplasmc cytoplasmicCD cluster of differentiationCHOP cyclophosphamide, doxorubicin,
vincristine and prednisoloneCLL chronic lymphocytic leukaemia
CML chronic myeloid leukaemiaCMML chronic myelomonocytic leukaemiaCMV cytomegalovirusCNS central nervous systemCODOX M/IVAC cyclophosphamide, vincristine,
doxorubicin, methotrexate/ifosphamide, mesna, etoposide,cytarabine
CR complete remissionCRAB calcium (elevated), renal failure,
anaemia, bone lesionsCSF cerebrospinal fluidCT computed tomographyCTCL cutaneous T-cell lymphomaCTD cyclophosphamide, thalidomide and
dexamethasoneCXR chest X-raycyt, cyto cytoplasmicDEXA scanning dual energy X-ray absorptiometry
scanningDIC disseminated intravascular
coagulationDKC dyskeratosis congenitaDLBCL diffuse large B-cell lymphomaDM double markingEBER EBV-encoded small RNAsEBV Epstein-Barr virusEBV LMP Epstein-Barr virus latent membrane
proteinEDTA ethylene diamine tetra-acetic acideGFR estimated glomerular filtration rateEMA eosin-5-maleimideEORTC European Organization for Research
and Treatment of CancerESHAP etoposide, methyl prednisolone,
cytarabine, cisplatinESR erythrocyte sedimentation rate
xi
xii List of Abbreviations
ET essential thrombocythaemiaETP-ALL early T-cell precursor acute
lymphoblastic leukaemiaFAB French−American−British (leukaemia
classification)FBC full blood countFDG fluorodeoxyglucoseFISH fluorescence in situ hydridisationFITC fluorescein isothocyanateFL follicular lymphomaFLAER fluorescein-conjugated proaereolysinFLAG fludarabine, cytarabine, granulocyte
colony-stimulating factorFLAG-IDA fludarabine, cytarabine, granulocyte
colony-stimulating factor, idarubicinFSC forward scatterGGT gamma glutamyl transferaseGI gastrointestinalGp glycoproteinGP general practitionerGPI glycosylphosphatidylinositolH&E haematoxylin and eosinHb haemoglobin concentrationHCL hairy cell leukaemiaHCL-V hairy cell leukaemia variantHHV human herpesvirusHIV human immunodeficiency virusHL Hodgkin lymphomaHLA-DR human leucocyte antigen DRHS hereditary spherocytosisHTLV-1 human T-cell lymphotropic virus-1ICC immunocytochemistryIg immunoglobulinIgA immunoglobulin AIgG immunoglobulin GIgM immunoglobulin MIHC immunohistochemistryIPSS International Prognostic Scoring SystemISCL International Society for Cutaneous
LymphomasISH in situ hybridisationISM indolent systemic mastocytosisITD internal tandem duplicationITP ‘idiopathic’ (autoimmune)
thrombocytopenia purpuraIVLBCL intravascular large B-cell lymphomaLAP leukaemia-associated phenotypeLBL lymphoblastic lymphomaLDH lactate dehydrogenaseLFTs liver function testsLGL large granular lymphocyte
LPD lymphoproliferative disorderMCH mean cell haemoglobinMCL mantle cell lymphomaMCV mean cell volumeMDS myelodysplastic syndrome/sMDS/MPN myelodysplastic/myeloproliferative
neoplasmMF mycosis fungoidesMGG May−Grünwald−GiemsaMGUS monoclonal gammopathy of
undetermined significanceMM multiple myelomamod moderate fluorescenceMPAL mixed phenotype acute leukaemiaMPN myeloproliferative neoplasmMPO myeloperoxidaseMRD minimal residual diseaseMRI magnetic resonance imagingMZL marginal zone lymphomaNLPHL nodular lymphocyte-predominant
Hodgkin lymphomaNOS not otherwise specifiedNR normal rangePAS periodic acid-SchiffPCR polymerase chain reactionPD-1 an antigen, programmed death
1(CD279)PE phycoerythrinPEL primary effusion lymphomaPET positron-emission tomographyPh Philadelphia (chromosome)PMF primary myelofibrosisPNET primitive neuroectodermal tumourPNH paroxysmal nocturnal haemoglobinuriaPRCA pure red cell aplasiaPT prothrombin timePTCL-NOS peripheral T-cell lymphoma, not
otherwise specifiedPTLD post-transplant lymphoproliferative
disorderPV polycythaemia veraRBC red blood cell (count)R-CHOP rituximab, doxorubicin, vincristine and
prednisoloneR-CVP rituximab, cyclophosphamide,
vincristine and prednisoloneRNA ribonucleic acidRQ-PCR real-time quantitative polymerase chain
reactionRS Reed–Sternberg
List of Abbreviations xiii
RT-PCR reverse transcriptase polymerase chainreaction
SAA severe aplastic anaemiaSig surface membrane immunoglobulinSLE systemic lupus erythematosusSM systemic mastocytosisSM-AHNMD systemic mastocytosis with associated
clonal haematological non-mast celldisease
SMILE dexamethasone, methotrexate,ifosfamide, L-asparaginase andetoposide
SSC side scatterT-ALL T-lineage acute lymphoblastic
leukaemiaTBI total body irradiation
TdT terminal deoxynucleotidyl transferaseTIA T-cell intracellular antigenTKI tyrosine kinase inhibitorT-LBL T-lymphoblastic lymphomat-MDS therapy-related myelodysplastic
syndromeTRAP tartrate-resistant acid phosphataseTT thrombin timeTTP thrombotic thrombocytopenic purpuraU&Es urea, electrolytes and creatinineUSS ultrasoundWAS Wiskott−Aldrich syndromeWASp Wiskott−Aldrich syndrome proteinWBC white blood cell (count)WM Waldenström macroglobulinaemia
Technical Notes
The patients presented in 100 Worked Examples were allreal cases encountered and investigated in a regional flowcytometry laboratory serving a population of approximately2.5 million over a period of 18 months. These are indi-vidually presented with a history that reflects the actualevents for each patient, commencing with the presentingclinical features and the initial basic laboratory tests andthen proceeding to flow cytometry, bone marrow aspi-rate morphology, bone marrow trephine biopsy histologywith immunohistochemistry studies and other specialisedcytogenetic and molecular analyses.
Full blood counts
The full blood counts and marrow counts (for appropriatedilutions in relation to antibody) were performed on a Sys-mex XN analyser. The differential leucocyte counts are auto-mated counts from the analyser. It should be noted that some-times, in an automated count, abnormal cells are misidenti-fied and the leucocyte sub-populations differ from a manualdifferential performed on a blood film. Such misidentifica-tions are indicated by inverted commas.
Biochemistry and immunologystudies
All relevant biochemistry and immunology data is given inrelation to the context of each patient presentation and interms of investigations that were thought to be relevant to the
case as the clinical diagnosis evolved. Some retrospectivelyrelevant data may be missing but this reflects the true natureof these actual patient scenarios and the investigations thatwere considered necessary at that time. Serum calciumvalues given are all corrected in accordance with serumalbumin level.
Flow cytometry analysis
Flow cytometry studies were all performed using a BectonDickinson FACS Canto II analyser. The findings are pre-sented as a list of positive and negative results in relationto the antigen and target cell population and the gatingstrategies applied to each case are explained. A series ofscatter plots and histograms are presented to illustratespecific informative points. The expression of most mem-brane 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 of10% has been used. Where the percentage positivity for agiven membrane antigen in the gated target population isborderline positive so that some cells appear negative andsome positive we have used the term ‘partial’ to describeantigen expression. Cytoplasmic expression of an antigen isindicated with the prefix ‘c’ (cytoplasmic expression of CD3being cCD3) but on some scatter plots ‘cyt’ or ‘cyto’ has beenused. The intensity of antigen expression in terms of medianfluorescence intensity is graded as dim, moderate or brightcompared to our laboratory reference ranges for normal cellsof each relevant lineage. See Figures 1.1a–g for a schematicrepresentation of these principles.
xv
xvi Technical Notes
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CD19
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CD19
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CD19
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(a) (b)
(c)
Figure 1.1 Visual representation of strength of fluorescence in flow cytometry (not actual patient specimens), showing an isotype control andeight CD19-positive samples which show fluorescence intensity with CD20 varying from negative to bright. (a) Isotype control, used to setthresholds. (b) Negative (consistent with a CD19-positive, CD20-negative B-cell precursor neoplasm). (c) Partial positive, indicating that CD20antigen expression varies from negative to positive (consistent with a precursor B-cell neoplasm). (c adjusted) Indicating that the thresholdfor 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 (consistentwith B-cell non-Hodgkin lymphoma). (f) bright, indicating strong CD20 antigen expression (consistent with hairy cell leukaemia). (g) Two distinctpopulations, 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 minoritybeing bright.
Technical Notes xvii
(f) (g)
(h)
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CD19
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(d) (e)
Figure 1.1 (Continued)
xviii Technical Notes
Immunohistochemistryin paraffin-embedded formalin fixedtissue
In the following section a list is presented of the immunohis-tochemical reagents used in assessing the paraffin embeddedmaterial (bone marrow trephine and lymph node biopsies)in the worked examples described. It should be pointed outthat specificities and sensitivities may differ from the anti-bodies used in flow cytometry due to the effects of formalinfixation and decalcification resulting in antigen loss or mask-ing. For example, CD5 may be detected by flow cytometryin a peripheral blood B-cell lymphocytosis but immunocyto-chemistry may on occasion be negative for the same markerin the trephine specimen. CD56 is aberrantly expressed byplasma cells in myeloma yet immunoreactivity for this anti-body within plasma cells in paraffin sections is seen in onlya minority of cases. The opposite situation may also occurwhere an antigen such asTdT is strongly positive by immuno-histochemistry on the fixed tissue but is negative on the flowsample. Reticulin fibrosis is reported as per the WHO classi-fication as grade 0, 1, 2 or 3.
These specific features of different techniques need tobe appreciated when formulating the combined pathologyreport and an understanding of the strengths and weak-nesses of each approach is essential when establishing afinal diagnosis. Cytogenetic and molecular studies have amajor influence on disease classification. Specific findingscan carry diagnostic significance way in excess of any othersingle investigative modality e.g. BCR-ABL1, PML-RARA,FIP1L1-PDGRFA. Metaphase cytogenetic studies not infre-quently fail, either reflecting the quality of the specimen orthe disease entity being studied. Informed FISH and PCRstudies can carry great diagnostic importance in certain clin-ical circumstances and molecular diagnostics will continueto inform disease classification with increasing power andspecificity over the decades ahead.
Laboratory Values
Abbreviations and Normal Ranges.
Blood
HaematologyHaemoglobin concentration (Hb) 130–180g/L (M)
125–170g/L (F)
Mean cell volume (MCV) 80–100 fl
Reticulocyte count 50–100 × 109/LWhite blood cell count (WBC) 4–11 × 109/LNeutrophils 2–7 × 109/LLymphocytes 1.5–4 ×109/LMonocytes 0.2–0.8 × 109/LEosinophils 0.04–0.4 × 109/LBasophils 0.01–0.1 × 109/L
Haematinics
Serum ferritin 10–275ng/mL
Serum folate 3.1–20 ng/mL
Serum vitamin B12 200–900pg/mL
Coagulation
Prothrombin time (PT) 9–13 s
Activated partial thromboplastin time
(APTT)
27–38 s
Thrombin time (TT) 11–15 s
Fibrinogen 1.5–4 g/L
D dimer 0–243ng/mL
BiochemistrySodium (Na) 135–145mmol/L
Potassium (K) 3.5–5.0mmol/L
Urea 2.5–7.5mmol/L
Creatinine 40–130 μmol/LBicarbonate 20–30mmol/L
Blood
Urate 0.2–0.43mmol/L
Lactate
1 Case 1
An11-year-old boywas admittedwith a short history of fever,sweats, dyspnoea and left chest discomfort.There was no pasthistory of note. Examination identified features of a left pleu-ral effusion. There was also a tender swelling of the left ante-rior chest in the upper pectoral region and palpable cervicallymphadenopathy. The liver and spleen were not palpable.
Laboratory investigations
FBC and blood film: normalU&Es, LFTs: normal. LDH was 1460U/L.
Imaging
TheCXR showed opacification and loss of aeration of the lefthemithorax in keeping with a pleural effusion (Figure 1.1).
Figure 1.1 CXR.
Practical Flow Cytometry in Haematology: 100 Worked Examples, First Edition. Mike Leach,Mark Drummond, Allyson Doig, PamMcKay, Bob Jackson and Barbara J. Bain.© 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.
Figure 1.2 CT.
CT imaging confirmed this but in addition identified a leftpleural-based mass, abnormal soft tissue in the left pectoralmuscles (arrows, Figure 1.2) and cervical lymphadenopathy.In addition, there was collapse/consolidation of the lower leftlung, creating the appearance of an air bronchogram. A corebiopsy of a cervical node was taken and the pleural effusionwas aspirated for analysis.
Flow cytometry
The pleural fluid cell count was 0.98 × 109/L. A cytospinpreparation showed three distinct cell types: a small maturelymphoid population in keeping with reactive lymphocytes,an intermediate sized/large sized lymphoid population and alarge cell population with pleomorphicmorphology and bluecytoplasm (Figures 1.3–1.6). The cells with the abundantcytoplasm (Figures 1.3 and 1.4) and the single binucleatecell (Figure 1.6) are reactive mesothelial cells. The cells withthe cytoplasmic blebs (Figures 1.4–1.6) are the disease cells,
1
2 Practical Flow Cytometry in Haematology
Figure 1.3 MGG, ×500.
Figure 1.4 MGG, ×500.
which were the subsequent focus for immunophenotypingstudies.
By applying a blast gate to the suspected malignant cellsin the FSC/SSC analysis (Figure 1.7), they were shown toexpress CD45bright (Figure 1.8), CD2 (Figure 1.9), cCD3[whilst surface CD3 was negative apart from a few reactive
Figure 1.5 MGG, ×500.
Figure 1.6 MGG, ×500.
T cells (Figure 1.8)], partial CD7 (Figure 1.10) and CD13.Other T-lineage markers were negative.
This is therefore a T-lymphoid neoplasm, indicated bypositivity for cCD3 expression, with limited lineage-specific
Case 1 3
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Figure 1.7 FSC/SSC.
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Figure 1.8 CD3/CD45.
markers and an aberrant myeloid marker. The tumour hasmedium sized/large cell morphology. It was showing aggres-sive clinical behaviour with extranodal tissue invasion in this11-year-old patient. An anaplastic large cell lymphomahad tobe considered and themedium sized/large cells in the pleuralfluidwere shown tobe strongly expressingCD30 (not shown).
Histopathology
An H&E-stained core biopsy of a cervical node is shownin Figure 1.11. The node is replaced by an infiltrate of
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Figure 1.9 CD2/CD19.
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Figure 1.10 CD7/CD16.
undifferentiated pleomorphic large cells with prominentnucleoli.
Immunohistochemistry showed the large cells to expressCD45, epithelial membrane antigen (EMA), CD2 focally(Figure 1.12), CD7, granzyme B and CD30 (Figure 1.13).In addition, there was strong nuclear and cytoplasmicstaining for anaplastic lymphoma kinase (ALK) protein(Figure 1.14).
The CD30 staining was particularly useful in demon-strating lymphatic invasion within the capsule of the node(Figure 1.15).
4 Practical Flow Cytometry in Haematology
Figure 1.11 H&E, ×400.
Figure 1.12 CD2, ×400.
FISH studies
A t(2;5)(p23;q35) translocation, rearranging the ALK andNPM1 (nucleophosmin) genes, was shown by FISH studieson paraffin-embedded lymph node tissue. The presence ofthis specific translocation is highly associated with bothnuclear and cytoplasmic positivity for ALK.
Figure 1.13 CD30, ×400.
Figure 1.14 ALK, ×400.
Discussion
Anaplastic large cell lymphoma (ALCL) is an aggressivemature T-cell neoplasm with pleomorphic, often large cell,morphology. It frequently fails to show surface expressionof T-lineage-specific markers and to potentially furthermislead may express aberrant myeloid antigens. This is an
Case 1 5
Figure 1.15 CD30, ×100.
important condition to recognise; it frequently shows rapidprogression with extranodal tissue involvement and it canrarely appear in the blood. Treatment of ALK+ ALCL isusually rewarding, particularly in paediatric patients, withprompt response to chemotherapy and frequent durableremissions.
Final diagnosis
Anaplastic large cell lymphoma (ALK+)
2 Case 2
A 72-year-old woman presented with a few months’ historyof fatigue and the more recent onset of breathlessnessand night sweats. On clinical examination she had alarge right-sided pleural effusion but no palpable lymph-adenopathy.
Laboratory results
FBC: Hb 158 g/L, WBC 16.6 × 109/L (neutrophilia andmonocytosis) and platelets 502 × 109/L.
U&Es: normal. LFTs were mildly deranged (ALT 52U/L,alkaline phosphatase 173U/L). Albumin was low at 29 g/Land serum LDH was raised at 584U/L.
Imaging
A CT scan demonstrated a large right-sided pleural effusionwith collapse of the right middle and lower lobes and partialcollapse of the upper lobe (Figure 2.1). In addition, therewere large volume, confluent, necrotic nodal masses inthe right hilar, mediastinal, retrocrural, paracardiac andpara-aortic areas (not shown) as well as pleural deposits(arrow, Figure 2.1).
Pleural fluid biochemistryand cytology
The pleural fluid LDH was markedly elevated at 2171U/Lwith relatively low glucose at 7.2mmol/L (patient diabetic)and protein of 43 g/L.
Practical Flow Cytometry in Haematology: 100 Worked Examples, First Edition. Mike Leach,Mark Drummond, Allyson Doig, PamMcKay, Bob Jackson and Barbara J. Bain.© 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.
Figure 2.1 CT.
Microscopy of the pleural fluid showed lymphoid cellsadmixed with neutrophils, histiocytes and mesothelial cells.Most of the lymphoid cells were small but an admixedpopulation of medium-sized cells with slightly irregularnuclei was also present. Onmorphology alone, the lymphoidcells were thought likely to be reactive but the reportingpathologist suggested that a fresh pleural fluid specimenshould 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 prepara-tion showed a cellular specimen with notable macrophages,neutrophils and small lymphocytes. In addition, some largeblastoid lymphoid cells were seen (Figures 2.2–2.5).
6
Case 2 7
Figure 2.2 MGG, ×500.
Figure 2.3 MGG, ×500.
Flow cytometry (pleural fluid)
The FSC/SSC plot shows the orientation of the different pop-ulations of cells described above. The small lymphoid popu-lation (Figure 2.6) comprised mainly reactive T cells (blackevents with mixture of CD4+ and CD8+ cells) and normalB cells (blue events). Seventeen per cent of all leucocytes wereCD19+ B cells (Figure 2.7) showing a mature pan-B pheno-typewithCD20 positivity and a hint ofmonoclonality (kappa
Figure 2.4 MGG, ×500.
Figure 2.5 MGG, ×500.
73%, lambda 18%). The SSC analysis in Figure 2.7, however,defines 2 B-cell populations with different scatter character-istics, populations P1 and P2.
By gating on the higher SSC profile B cells (P2 redevents, Figure 2.7, which are larger indicated by highFSC in Figure 2.6), a clear clonal population was demon-strated showing strong CD20 positivity, expression ofCD10/HLA-DR (Figure 2.8), CD38, FMC7, CD79b andCD22 with kappa light chain restriction (Figure 2.9).
8 Practical Flow Cytometry in Haematology
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Figure 2.6 FSC/SSC.
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Figure 2.7 CD19/SSC.
Note the phenotype of the blue events, population P1,representing residual small polyclonal reactive B cells.
Lymph node biopsy
A CT-guided core biopsy of a paravertebral node showedlymphoid infiltration by predominantly small centrocyticcells with occasional larger centroblasts. The cells werepositive for CD20, CD10, BCL6 and BCL2 and negativefor CD3, CD5, cyclin D1, CD23, CD43 and CD21. Theproliferation fraction was low (∼10%). The histological andimmunohistochemical appearances were in keeping with fol-licular lymphoma, grade 2, with no evidence of high-grade
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Figure 2.8 CD10/HLA-DR.
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Figure 2.9 Kappa/lambda.
transformation. FISH for the t(14;18) translocation waspositive.
Bone marrow aspirate and trephinebiopsyThese were both normal.
Discussion
The clinical presentation, imaging, pleural fluid morphologyand flow cytometry were most in keeping with a diagnosis ofan aggressive, mature CD10+ B-cell neoplasm. Diffuse large
