Haemato-oncology‎ > ‎

Immunophenotyping

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

 


Acute leukaemias

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

 

 

HLA-DR

CD34

CD33

CD13

CD11c

CD14

CD41

CD235a

M0

+

+

+

+/-

+/-

-

-

-

M1

+

+

+

+

+/-

+/-

-

-

M2

+/-

+/-

+

+

+/-

+/-

-

-

M3

-

-

+

+

+/-

-

-

-

M4

+

+/-

+

+

+

+

-

-

M5

+

-

+

+

+

+

-

-

M6

+/-

-

-

-

+/-

-

-

+

M7

+/-

+/-

+/-

-

-

-

+

-

 

B ALL

First line CD79a(cyt), CD22(cyt), CD19, CD10

Second line SmIg (k/l), CytIg, CD138

 

 

TdT

CD19

CD10

CD20

CytIg

SIg

Pro-B

+

+

-

-

-

-

Common

+

+

+

-

-

-

Pre-B

+

+

+

+/-

+

-

B ALL

+

+

+

+/-

+

+

Burkit

-

+

+

+

-

+

 

T ALL

First line CD2, CD3(cyt)

Second line CD7

 

 

TdT

CD7

CD2

CyCD3

CD5

CD1

CD3

CD4/8

Prothymocyte

+

+

+

+

-

-

-

-/-

Immature thymocyte

+

+

+

+

+

-

-

-/-

Common thymocyte

+

+

+

+

+

+

+/-

+/+

Mature Thymocyte

-

+

+

+

+

-

+

4 or 8

Mature T cell

-

+

+

+

+

-

+

4 or 8

 

Biphenotypic leukaemia

 

Score

B-lymphoid

T-lymphoid

Myeloid

2

CD79a

cytCD22

cytIgM

CD3

antiTCRa/b

antiTCRg/d

MPO

1

CD19

CD20

CD10

CD2

CD5

CD8

CD10

CD117

CD13

CD33

CD65

0.5

TdT

CD24

TdT

CD7

CD1a

CD14

CD15

CD64

 

Biphenotypic leukaemia defined when the scores of myeloid and one of the lymphoid lineages are >2 points

 

 

 


Chronic lymphoproliferative disorders

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

Villous / Hairy cells

CD11cCD25CD103HC2

Lymphoplamacytic / plasma cell

Cyt Ig (k/l), CD79a, CD138

T-Cell

CD3CD7CD4, CD8CD25TdT, a-TCRg/d

LGL leukaemia

CD3CD4, CD8CD11bCD16CD56, CD57TIA-1

Mantle cell / unclassifiable B cell

cyclin D1

Natural killer cell

CD16, CD56, CD57, CD11b

 

 

B Cell neoplasms

 

CD 5

CD 10

CD 19

CD

20

CD 22

CD 23

 

CD 43

CD 79

CIg

SIg

Cyclin D1

FMC7

Other

 

CLL

+

-

+

+/-

-

+

 

+

b -

5%+

Weak

-

-

 

 

MCL

+

-

+

+

+

-

 

+

a+

-

IgM

+ 70%

50%

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PLL

+/- 30%

 

+

+

+

-

 

-

a+

 

+ IgM/D

-

+

 

 

HCL

-

-

+

+

+

-

 

-

a+

-

+

+/-

 

TRAP,

CD11c

CD25+ CD103+

FMC7+

DBA44+

 

LPL

-

-

+

+

+

-

 

-

a+

+

IgM

-

 

CD138+

t(9;14)

Splenic

MZL / SLVL

-

30%

+

+

+

-

 

+/-

a+

+/-

+

-

+

DBA44 most

CD25 30%

CD11c 50%

 

FCL

-

60%

+

+

+

-

 

-

a+

-

+

-

 

BCL2

t(14;18)

or 2/22

LCL

10%

<50%

+

+

 

+

 

+/-

 

+/-

+/-

-

 

30% BCL2, BCL6 in most

Ki67 high

Complex

t(14;18) BCL2

t(3;14) BCL6

Burkitt

-

+/-

+

+

+

-

 

-

a+

+

+

-

 

Ki67 >99%

t(8;14)

or 2/22

Myeloma

-

-

 

-

 

-

 

+

 

+

-

20%

 

CD56 CD38 CD138

 

LPL

-

-

 

+

 

-

 

+/-

 

+

 

-

 

 

 

 

 

CLL

Marker

CLL

Score

MCL

CD5

Positive

1

Pos

CD23

Positive

1

Neg

SIg

Weak

1

Pos

CD79b/ CD22

Weak

1

Pos

FMC7

Negative

1

Pos

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

 

CD45

CD30

CD15

CD20

CD3

Hodgkin

-

+

+

-

-

NLPHD

?

-

-

+

-

B lymphoma

+

+/-

-

+

-

T 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 -
Cell 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, 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

 


Advantages of immunophenotyping vs immunohistochemistry

§   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

 


Comments