Immunophenotyping
Possible to § Detect both membrane and cytoplasmic or nuclear antigens by flow cytometry § Simultaneous double, triple or quadruple immunostaining (more than 10 colours used simultaneously in research studies but this can be technically difficult) § NB this is a significant advantage over immunohistochemistry in which only one antibody is used per slide (but no morphological information is obtained in flow cytometry) § Analysis of whole blood or bone marrow without separation of mononuclear cells § Quantifies antigen levels at a single cell level and can analyse millions of cells rapidly to look for rare populations. § Analsis or quantify selected cell populations such as the estimation of the CD34 stem cells by applying gating strategies using CD45 labelled cells
Sample collection § Heparin for cytogenetic analysis § EDTA preserves cell morphology § <24h and should be kept at room temperature § Burkitts and spinal fluid samples need rapid transport § Tryptan blue survival test recommended if the sample is >24h old to check viability (>80%)
No consensus on the cut off point for a sample to be positive >20% of leukaemic cells in acute leukaemias >30% of leukaemic cells in chronic leukaemias
General recommendations § Need for positive and negative controls § Optimal dilutions of McAB § Training of laboratory staff § External quality assurance program
Whole blood § Hypotonic erythrocyte lysing solution (NH4Cl based) § Prolonged exposure changes light scatter properties § Inadequate exposure leaves intact red cells and debris making results inaccurate
Detection of intracellular antigens § Double immunostaining (extrecellular done first then intracellular) § Phycoerythrin better than fluorescein for the detection of weakly expressed antigens § Permeabilization solutions (eg Fix and Perm / Permeafix) § TdT, cytCD3, cytIg, MPO § Intracellular antigens expressed earlier and are more specific for myeloid / lymphoid lineages
CD45 gating § Allows leukocytes to be identified § Useful for estimating CD34 + cells
First line § Non lineage restricted: TdT(nuc) § B lymphoid: CD79a(cyt) CD22(cyt) CD19 CD10 § T lymphoid: CD2 CD3(cyt) § Myeloid: MPO(cyt) CD117 CD13
Second line § If B cell phenotype and TdT is negative: SmIg (k/l), § If lineage not established: CD7 (T cell) and CD33, CD41, CD42, CD61, glycophorin A (myeloid) § If all markers negative CD45 (non lineage), CytIg, CD138 (B cell) and non-haemopoietic markers
AML Immunophenotyping First line MPO(cyt), CD117, CD13 Second line CD33, CD41, CD42, CD61, glycophorin A
B ALL First line CD79a(cyt), CD22(cyt), CD19, CD10 Second line SmIg (k/l), CytIg, CD138
T ALL First line CD2, CD3(cyt) Second line CD7
Biphenotypic leukaemia
Biphenotypic leukaemia defined when the scores of myeloid and one of the lymphoid lineages are >2 points
BSH guidelines
B lymphoid: First line CD19, CD22, CD23, FMC7, SmIG (k/l), CD79b
T lymphoid: First line CD2
B and T Cell First line CD5
Second line
B Cell neoplasms
CLL
CLL scoring system – needs to be 3-5 CD23 and cyclin D1 are useful to distinguish between CLL and MCL Ki67/MIB1 also useful (rarely + in CLL but may be 30-50% MCL)
Lymphoma
T PLL CD 2, 3, 5, 7 + CD 8, 25 – Cytogenetics t(14;14)(q11;q32) – TCR alpha 14q11 with IgH 14q32 in 75%
T LGL CD 2, 3, 5, 8+ CD 4, 7, 56 – (rare cases are CD56+) TCR gene rearrangements detectable in most
NK leukaemia CD 2, 16, 56 + (CD 8 +/-) CD 3, 4, TCR a/b and g/d neg Abnormalities common but not consistent EBER (Epstein-barr early RNA in most cases No easy way to prove clonality
Mycosis fungoides / Sezary CD 2, 3, 4, 5 + CD 8-, CD25- TCR gene rearrangements common
Angio-immunoblastic lymphoma CD 2, 3, 4, 5, 7 + CD 8 – TCR gene rearrangements detectable
ATLL CD 2, 3, 4, 5, 25+ CD 7, 8 - CD1 § Expressed on common thymocytes (not prothymocytes, immature thymocytes, mature thymocytes or mature T cells)
CD1a § Langerhans cells (cyt)
CD2 § Pro and Pre T cells, T cells, thymocytes and NK cells § First line in both acute and chronic T cell malignancies
CD3 § T cell marker § Cytoplasm of immature T cells and surface of more mature cells § Negative in NK cells § First line in acute T cell malignancies § Second line in chronic T cell disorders (proportion are negative for CD3)
CD4 § T-helper cells, monocytes, dendritic cells, activated eosinophils and thymocytes § Second line in chronic T cell lymphoproliferative disorders (+ in ATLL, Sezary, and some T-PLL)
CD5 § B and T cell marker § B cell malignancies - CLL, Mantle cell lymphoma § T cell - T-ALL
CD7 § Pro and Pre T cells, T cells, thymocytes and NK cells § Also positive on some myeloblasts § Second line T cell marker in acute leukaemias
CD8 § Cytotoxic T cells, NK cells, thymocytes § Second line in chronic T cell lymphoproliferative disorders § + in T-LGL leukaemia and some T-PLL (< than CD4), +/- in ALCL
CD10 § B lymphoid (acute leukaemia) § Positive in common ALL – negative in Prepre / precursor B-ALL § Germinal centre cells and neutrophils § + Follicular lymphoma (60%), Large cell lymphoma (25-50%) and Burkitt § + AIL T NHL
CD11b § NK cell / LGL leukaemia § Moncytic
CD11c § Hairy cell leukaemia § Monocytes, macrophages, granulocytes
CD13 § Monocytes, neutrophils, eosinophils and basophils § First line marker for AML § Can sometimes be expressed in B-ALL
CD14 § Monocytoid marker (also positive on macrophages, subsets of granulocytes and B cells but less strongly)
CD15 § Myeloid cells and monocytes § + in Hodgkin (Reed-Sternberg cells + in classical Hodkins) / - in B NHL lymphoma
CD16 § NK cell / LGL leukaemia
CD19 § B cell marker (first line in chronic lymphoproliferative disorders) – not positive in myeloma
CD20 § Mature B cell marker § Negative in myeloma and weak in CLL
CD21 § Mature B cell marker, follicular dendritic cells also a subset of thymocytes
CD22 § B cell marker (first line in chronic lymphoproliferative disorders)
CD23 § B cell marker (first line in chronic lymphoproliferative disorders)
CD 25 § Activated T cells § HTLV-1 associated ATLL § Hairy cell leukaemia
CD30 § + Reed – Sternberg cells – classical Hodkin’s § + Anaplastic large cell lymphoma
CD33 § Second line marker for AML § + in all subtypes of AML except M6 (M7 is +/-)
CD34 § Uncommitted haemopoietic progenitors (CD34+ / CD38-) § + in AML M0 / M1 § +/- in AML M2,4, 7 and – in AML M3, M5, M6
CD38 § Expression is a marker of initial lineage commitment § Plasma cell marker
CD40 § B cell
CD41 § Megakaryocitic marker – glycoprotein IIb § Second line marker for AML (+ in AML M7)
CD42 § Promegakaryocytic / megakaryocytic / platelet – glycoprotein Ib § Second line marker for AML
CD45 § Second line marker for non-lineage restricted haemopoietic cells § Negative in Hodgkins
CD45 RO § T cell marker
CD52 § Target for campath / alemtuzumab
CD56 § Negative in PNH § NK cells, myeloma cells, some B cells
CD57 § NK cells (particularly CD3+, CD8+)
CD61 § Megakaryocitic (glycoprotein IIb/IIIa) § Second line marker for AML
CD64 § Monocytic
CD68 § Monocytic
CD71 § Transferrin receptor § Erythroid marker
CD72 (DBA.44) § Hairy cell (+)
CD79a § Intracellular epitope of the alpha chain of the B-cell receptor § Highly sensitive and specific for B-lineage § Present from the earliest stages of B cell malignancy through to plasma cells (though a proportion of clonal plasma cells are negative)
CD 79b § Specific for B lineage § Reduced expression in CLL and hairy cell
CD117 § First line marker for AML § c-kit receptor (receptor for stem cell growth factor) § Postive in 2/3 cases of AML and less than 5% of ALL (most of which also expressed other myeloid markers eg CD13 or 33 and corresponded to early T Pro ALL
CD138 § Second line marker for acute B cell malignancies § Plasma cell marker
CD 235a (Glycophorin A) § Second line marker for AML § Erythroid marker (+ in M6 only)
CD246 (Alk) § Prognostic importance in anaplastic large cell lymphoma
BCL2 § Follicular lymphoma B cells – reactive in germinal centres § Poor prognosis in DLBCL
BCL6 § Nuclear transcription factor § Expressed by cytrocytes and centroblasts but not naïve B cells, mantle cells, memory B cells or plasmacells § Positive in Burkitt lymphoma, large-B cell lymphoma (good prognosis) and follicular lymphoma § Also undergoes mutations in somatic hypermutation as well as the IGV region
CytIg § Second line marker for acute B cell malignancies
Cyclin D1 § Mantle cell lymphoma (t11;14 translocation) or B-cell prolymphocytic leukaemia
EBV-LMP1 § EBV driven lymphomas
EMA (CD66a) § Plasma cells - +ve in anaplastic and large cell lymphoma
HLA DR § AML and ALL
Kappa / lambda § Difficult to do technically – best done by in situ hybridisation
Mast cell tryptase § Mast cells
MIB1 / (Ki67) § Proliferation marker § Can be helpful distinguishing MCL from CLL (50%+ in MCL v rarely + in CLL)
MUM1 § Plasma cells § Poor prognostic marker in DLBCL
MPO § Granulocytes
p21 § Upregulation associated with p53 overexpression
SmIg § Second line marker for acute B cell malignancies
TdT § First line non lineage restricted marker in acute leukaemia § Nuclear enzyme § T-ALL or T-lymphoblastic lymphoma § NB negative in DLBCL, T-PLL
TRAP § Hairy cell lymphoma
§ Flow cytometry is quantitative (immuno is not) § Standardised technique with little variation (cannot compare the strength of staining from samples stained at different times with immunocytochemistry). § Disadvantage – if gating set up wrongly will miss abnormal population
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