immunophenotyping
intro
The drawing shows where you would expect to find different cell populations on a flow plot.
Forward Scatter - Size of cell
Side Scatter - Granularity of cell
NORMAL CELLS
Mature Monocytes
CD 36+ CD64+ combination
NK Cells
CD2+, CD3-, CD5-
Assess NK cell clonality by expression of KIR (Killer Inhibitor Receptor) receptors
Normal T Cells
Blood – there should be twice as many CD4 as CD8
Marrow – there should be twice as many CD8 as CD4
Large Granular Lymphocyes (LGLs)
Normal response to many inflammatory processes, but can also be malignant
Normal: CD8+ CD56+
Neoplastic: Very often CD57+ CD16+ (but both can be variably expressed on normal LGLs)
Lymphomas - Typical Markers
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 7-, 8-, CD25-
TCR gene rearrangements common
Angio-immunoblastic T-Cell Lymphoma
CD 2, 3, 4, 5, 8, 7, 10, PD-1 +
CD
TCR gene rearrangements detectable
Anaplastic T-Cell Lymphoma (Anaplastic means variable)
Very variable phenotype
Often CD30+, 4+/-, 8+/-
ATLL
CD 2, 3, 4, 7, 25+
CD 5, 8 -
Cell markers
Plasmacytoid Dendritic Cell (PDC) Neoplasm
Strong CD56+, CD4+
ALPS (Autoimmune Lymphoproliferative Syndrome)
CD3+
CD4-, CD8-
Hepatosplenic T-Cell Lymphoma
CD3+
CD4-, CD8-
Numerical List of Markers
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, PTCL, T-PLL, AITL)
CD5
B and T cell marker
B cell malignancies – CLL, Mantle cell lymphoma
Pan T cell marker
T-ALL
CD7
Pan T-cell marker, and NK cells
Also positive on some myeloblasts
Second line T cell marker in acute leukaemias
CD7 negative T-cells = PTCL, Sezary’s
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
CD9
Monocytes, variably expressed on precursor B cells
Strong marker for APML (not exclusive)
Aberrant CD9- can be used as an MRD marker in ALL
CD10
B lymphoid (acute leukaemia)
Positive in common ALL – negative in Pro B-ALL
Germinal centre cells and neutrophils
+ Follicular lymphoma (60%), Large cell lymphoma (25-50%) and Burkitt
+ AIL T NHL
Expression reduced by high dose steroids
CD11b
NK cell / LGL leukaemia
Monocytic
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)
(CD14- an indication of immature monocytes)
CD15
Myeloid cells and monocytes
+ in Hodgkin (Reed-Sternberg cells positive 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
Ki-1 or TNFRSF8 (tumour necrosis factor receptor superfamily member 8)
+ Reed – Sternberg cells – classical Hodkin’s
+ Anaplastic large cell lymphoma
CD31
Glycoprotein Ia
CD33
Second line marker for AML
+ in all subtypes of AML except M6 (M7 is +/-)
CD34
Uncommitted haemopoietic progenitors (CD34+ / CD38-)
CD36
Platelet Glycoprotein 4
Red cells, megakaryocytes, basophils, monocytes, among others
CD38
Plasma cell marker
CD40
B cell
CD41
Glycoprotein IIb
Megakaryocytes
Second line marker for AML (+ in AML M7)
CD42
Promegakaryocytic / megakaryocytic / platelet – glycoprotein Ib
Second line marker for AML
CD42a
Glycoprotein IX
CD42b
Glycoprotein Ib alpha
CD43
Strongly positive in CLL (CLL ‘classically’ CD5+, CD43+. If CD43- then question your diagnosis)
Positive in B-ALL
Negative in Follicular (FL ‘classically’ CD10+, CD43-)
CD45
Haemopoietic cells (non-lineage restricted)
Negative in Hodgkins
CD45 RO
T cell marker
CD49b
Glycoprotein IIa
CD52
Target for campath / alemtuzumab
CD55
Decay-accelerating factor (DAF)
Prevents formation of C3 convertases
Deficient on red cells in PNH.
CD56 (Neural Cell Adhesion Molecule – N-CAM)
NK cells, myeloma cells, some B cells
Negative in PNH
Aberrant marker on monocytes suggestive of dysplasia
Poor prognostic marker in AML
(fun fact: CD56 is an adhesion molecule, helps keep plasma cells in the marrow —> plasma cell leukaemia will typically be CD56-)
CD57
NK cells (particularly CD3+, CD8+)
CD59
MAC-inhibitory protein (MAC-IP)
Prevents C9 from forming the membrane attack complex
Deficient on red cells in PNH.
CD61
Glycoprotein IIIa
Megakaryocytes
Second line marker for AML
Note: Care when testing as false positives can result from platelet clumping or platelets stuck to other cells.
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
The extracellular immunoglobulin complex, ie mature B cells
Specific for B lineage
Reduced expression in CLL and hairy cell
CD81
Germinal centre marker
CD99
Found on many cells
Good marker in T-ALL
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 correspond 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
CD246 (Alk)
Prognostic importance in anaplastic large cell lymphoma
CD303 (BCDA-2)
Blastic Plasmacytoid Dendritic Cell Neoplasm
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 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, ALL, Activated T-cells
Kappa / lambda
Difficult to do technically – best done by in situ hybridisation
KIR (Killer Inhibitor Receptor)
NK Cells
5 subtypes, A to E, can be used to assess clonality
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
PD-1
AITL
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
Guess Who game for learning immunophenotyping
Dr Charlotte Helmich and Co. created this great online game - https://guesswhohaem.co.uk/#/ - to help learn flow cytometry profiles for haem malignancies. Well worth a look when studying with a friend.