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 Prepre / precursor 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

§   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)

§  (CD14- an indication of immature monocytes)

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

CD36

§  Platelet Glycoprotein 4

§  Red cells, megakaryocytes, basophils, monocytes, among others

CD38

§   Expression is a marker of initial lineage commitment

§   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

§   Second line marker for non-lineage restricted haemopoietic cells

§   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 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, 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