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 flow charts1.jpg

 

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

  • + 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-)

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

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

CD52

  • Target for campath / alemtuzumab

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

CD57

  • NK cells (particularly CD3+, CD8+)

CD61

  • Glycoprotein Iib/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