IMMUNOPHENOTYPING Brigadier Tahir Aziz Ahmed MBBS, MCPS, FCPS, FRC Path

Armed Forces Institute of Pathology (AFIP)

INTRODUCTION Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of characterizing the presence and proportions of the various populations of interest

CPSP 4th March 2010

METHODS IMMUNOPHENOTYPING can be performed by:

Immunoperoxidase on fixed paraffin-embedded tissue

Immunoperoxidase on fresh frozen tissue (frozen-section immunoperoxidase [FSIP])

Immunofluorescence on fresh frozen tissue

Immuno-peroxidase or immunofluorescence on cytospin preparations

Flow cytometry (FCM) on cell suspensions

FLOWCYTOMETRY- gold standard Flow cytometry has become the preferred method for the lineage

assignment and maturational analysis of hematological malignancies

FAST STATISTICAL POWER SENSTIVITY (90% versus 30% when compared with frozen-section immunoperoxidase) AUTOMATION LIGHT CHAIN DEMONSTRATION (significantly higher negative predictive value (100 versus 63)

K W Biesemier Clin Diagn Lab Immunol. 1994 May; 299–303.

Flowcytometry Lab-AFIP

SCOPE OF FLOWCYTOMETRY

The scope of flow cytometric immunophenotyping is to provide an objective and reproducible method for the diagnosis and monitoring of therapy for hematological malignancies

This scope can be achieved in five different steps:

Assignment of cellular lineage of the malignant cell

Analysis of clonality

Analysis of cellular maturation Aberrant features of the malignant cell populations

Detection of minimal residual disease(MRD)

Consensus in 2 colour immunophenotyping in Leukemia

Primary panel

CD45 FITC CD3

MPO CD79a

Cy CD3 CD33

CD7 CD19

HLA-DR CD13

IgM CD10

Ig kappa CD19

Ig lambda CD19

Secondary Panel in AML

CD45 Glyco phorin A

CD14 CD15

CD34 CD117

CD2 CD13

CD41 CD42b

Prim. Panel at AFIP 1. Isotype control 2. CD5FITC/ CD10PE/

CD19PerCP 3. CD7FITC/ CD13PE 4. CD20FITC/ CD33PE 5. CD3FITC/ CD34PE 6. CD45FITC/ CD14PE 7. CD34FITC/ CD117PE

• Myeloid CD13, CD33, CD117 Mono. CD14 • Lymphoid B. CD10, CD19,CD20 T. CD5, CD7, CD3 • Maturity CD34, HLADR, CD10

Second panel in B-ALL

CD34 CD22

CD24 CD5

Secondary panel in T-ALL

CD4 CD8

CD2 CD1a

CD34 CD5

European Working Group on Clinical Cell Analysis (EWGCCA)

Consensus in 2 colour immunophenotyping in Leukemia (cont’d)

Prim. Panel at AFIP

• Myeloid CD13, CD33, CD117 Mono. CD14 • Lymphoid B. CD10, CD19,

CD20 T. CD5, CD7, CD3 • Maturity CD34, HLADR, CD10 Secondary Panel Myeloid. MPO, Glycophorin, CD41 Lymphoid. Tdt, sIgM, IgG, Kappa, Lambda, CD22, CD23, CD38 CD2,CD4, CD8, TCRa/b, TCRg/d, cCD3 Individual antibody combinations

A diagnostic laboratory performing immunophenotyping for Acute Leukemia should be able to recognize: 1. Biphenotypic Acute Leukemias: BAL

2. Acute Lymphoblastic Leukemias: ALL

a) B lineage subtypes

b) T lineage subtypes

3. Acute Myeloblastic Leukemias: AML (Mo-M7)

In addition, diagnosis of lymphoma, non haematological neoplasms and reactive cytopenias should be excluded.

Scoring System for Biphenotypic Leukemia

SCORE B-Lymphoid T-Lymphoid Myeloid

2 CD79a CD3 MPO

cCD22 TCR αβ

cIgM TCR γδ

1 CD19 CD2 CD117

CD20 CD5 CD13

CD10 CD8 CD33

CD10 CD65

0.5 Tdt Tdt CD14

CD34 CD7 CD15

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

B-Cell Development `

Stem cell

Pro Pre Immature PC

HLA-DR

TdT, CD34

cyIg SIg

CD19

CD10

Mature

CD5 FMC7 CD38

CD79

CD22 cCD22

T- Cell Development `

Stem cell

Pro-T Pre-T Immature Activated

TdT

cyCD3

sCD3

CD2, CD7

CD5

Mature

CD4/8 CD4 or CD8

CD1a

HLA-DR

Immunophenotypes of B-ALL Subtypes

B-ALL Subtypes Common Phenotypes

B- precursor ALL DR, CD19, CD20, CD24, CD10, CD34, TdT

Pre-B ALL DR, CD19, CD20, CD24, CD9, CD10, CD34(-), cIgM, TdT+/-

B-ALL DR, CD19, CD20, CD22, CD24, CD10, CD34(-), TdT(-), sIg

C. Darrell Jennings et al. The Journal of American Society of Hematology

T-ALL subtypes T-ALLSubtypes Phenotypes

Pro-T cCD3, CD7, CD2(-), CD1a(-),CD34+/-, CD4(-), CD8(-)

Pre-T cCD3+, CD7, CD2, CD1a(-), CD34+/-, CD4(-), CD8(-)

Cortical CD4, CD8(dual positive), CD1a

Medullary CD3, CD1a(-), CD4+ or CD8+, TCR

C. Darrell Jennings et al. The Journal of American Society of Hematology

AML subtypes AML Subtypes Phenotypes

Mo DR, CD13, CD33, CD34, CD7(+/-)

M1 Similar to Mo except CD15(+/-)

M2 DR, CD13, CD33, more CD15 and less CD34 than M1

M3 DR(-), CD13, CD15, CD33, CD34+/-

M4, M5 DR, CD15, CD14+/-, CD33>CD13, CD34+/-, CD4 weak

M6 DR, CD13+/-, CD33+/-, CD34, CD45weak, Glycophorin

M7 DR+/-, CD33+/-, CD34, CD41,CD42, CD61

Immunophenotyping for lymphoproliferative diseases aim to identify

CLL

HCL

Burkitt Follicular lymphoma Mantle cell lymphoma Marginal zone lymphoma

LGL NK proliferative disorders

B-Lymphoproliferative Disorders Disorder Common Phenotypes

CLL DR, CD19, CD20, CD5, CD22(-), CD23, CD10(-), CD11c+/- ,CD25+/-, CD43, weak SIgM and SIgD

PLL DR, CD19, CD20, CD5(-), CD22, CD23(-), CD10(-), bright SIg

Mantle cell DR, CD19, CD20, CD22, CD5, CD23(-), CD10(-), CD43

Follicular DR, CD19, CD20, CD22, CD5(-), CD23+/-, CD10, CD11c(-), CD43(-), bright SIg

Marginal Zone DR, CD19, CD20, CD22, CD5(-), CD23(-), CD10(-), CD11c, CD25(-),CD103(-)

Hairy cell DR, CD19, CD20, CD5(-), CD22, CD23(-), CD10(-), CD11c, CD25, CD103

C. Darrell Jennings et al. The Journal of American Society of Hematology

Types of specimen

Bone marrow

Peripheral blood

Malignant effusions, e.g. ascites or pleural effusions

Solid tissue, e.g. lymph nodes, after preparation of single cell suspensions

Specimen collection Sample collection only after appointment with the Lab

The date and time of specimen collection should be recorded.

Specimen should be transported to the flow cytometry laboratory as soon as possible (within hours).

Information about age, sex, presumptive diagnosis, differential blood count and status of lymph nodes and spleen should be provided.

Anticoagulant

EDTA; preferred anticoagulant (2-3ml is enough)

Cells can be analyzed by morphology and automated hematology analyzers using the same specimen.

Reduced cell aggregation

Heparin - Ficoll density gradient preparations of mononuclear cells.

Sample storage

Samples storage at room temperature (18 to 22 C) until staining & analysis is recommended.

Storage at temperatures below 10 C may lead to adsorption of immunoglobulins to cells and to a selective loss of cells or antigens.

Morphology

Morphological and cytochemical analysis should be performed on EDTA-anticoagulated specimen.

If bone marrow is collected, differential leukocyte counts should be performed simultaneously from bone marrow and peripheral blood

Opinion of experienced haematologist is invaluable

Staining Procedure

1. Incubation with fluorochrome-conjugated mAbs for 30 min in dark.

2. Lysis of erythrocytes a) Ammonium chloride buffer b) Hypotonic sodium chloride solutions c) Commercial reagents

3. Washing of cells 500xg for 5 min.

4. Fixation using buffered solutions of formaldehyde or paraformaldehyde 1-3% (if not analyzing immediately)

Flow cytometry

Flow Cytometry is the process whereby physical and chemical properties of cell are studied as they pass through a measuring apparatus (hopefully in single file) suspended in a fluid stream

Measurements in Flow Cytometry

Forward scatter Forward Angle Light Scatter 2° - 20° (FSC)

Large objects will scatter more light in the forward direction than small objects

Voltage Signals received by detecters are directly proportional to cell size

Side Scatter Side Scatter near 90° (SSC)

Cell with more granularity scatter more side light

Y-axis

X-axis 5

5

Forward Scatter

Side Scatter

10

10

15

15

Forward and Side Scatter

FSC Detector

Collection Lens

SSC Detector

Laser Beam

Original from Purdue University Cytometry Laboratories

Why Look at FSC v. SSC A correlated measurement between FSC(size) and SSC ( internal structure) can allow for differentiation of cell types in a heterogenous cell population

FSC

SS

C

Lymphocytes

Monocytes

Granulocytes

RBCs, Debris, Dead Cells

Light emission

Flowcytometer can detect light emission from single cell that binds fluorescently conjugated mAb

Can detect as many as seven fluorochrome-conjugated mAb that emit light at different wavelength.

Typical 4 parameter layout

530nm band pass FL1

488nm band pass FSC 488nm laser beam

560nm short pass dichroic mirror

585nm band pass FL2

PMT

510nm long pass dichroic mirror

488nm band pass

SSC PMT

PMT

PD flow cell

CASE SERIES

Patient - 1 Boy, 7 years of age

Fever Pneumonia Lymphadenopathy Patecheae

CBC 148 x 109/l Hb 7.6 g/dl Plt 55 x 109/l

Hematological opinion: 55% blasts`

FORWARD and SIDE SCATTER

FSC

SS

C

Patient Healthy

CD19 + CD10 (B lymphoid markers-56%) Isotype control

HLA-DR (B lymphoid+ Act T-82%)

`

CD13 (Myeloid-40%) CD7 (T lymphoid-28%) CD13 Not present on T cells so must be present on B cells

CD34 (Immature cells-58%) CD3 (T lymphocytes-24%) CD34 absent on T cells so must be present on B cells. Q1. Which haematopoeitic cells express CD34?

Weak CD45 (61%) Q2: Which cells express weak CD45?

Mature T cells

Report CD19 + CD10 (56%)

CD34 (59%)

Weak CD45(61%)

CD13(40%)

HLA-DR (82%)

Q3: What does HLA-DR positivity signifies in this patient? Lineage or Maturation

Interpretation Lineage:

Lymphoid (B) Lineage

Maturiton stage: Precursor B-ALL( CD10+ , CD34 +)

Abberant expression: CD13 (poor prognosis)

Q4: Which combination of antigens would you

recommend to detect MRD in this patient?

Final Diagnosis

Precursor B-ALL

Patient-2 Boy, 9 years of age

Fever Hepatomegaly Left sided pleural effusion

CBC 65000x 109/L 72% Lymphocytes

HEMATOLOGICAL OPINION: 55% blasts

Panel selected B-lineage markers

CD19 CD20

T-lineage markers CD3 CD5 CD7

Myeloid markers CD13 CD33 CD117

Panel selected (cont’d) Markers of immaturity

CD10 CD34

Forward and Side Scatter

FSC

SS

C

Healthy

CD10 + CD5(52%)

Isotype control

HLA-DR(41%)

CD7(54%) Q5: What is the percentage positivity for CD13?

CD20(7%) Q6: Which cells are CD20 positive?

CD3(36%) Q7: Which cells express CD3?

WEAK CD45(49%)

Report: CD10 + CD5(51%)

CD7(53%)

HLA-DR(42%)

Weak CD45(50%)

Q8: What is the diagnosis?

INTERPRETATION

Lineage- Probably Lymphoid(T cell) lineage

i) CD10 is expressed on CD5 positive cells ii) CD10 is not expressed on B-cells

Cellular Maturation- Not yet confirmed i) CD34 negative ii) CD10 is also expressed on T-lymphomas e.g

angioimmunoblastic T-cell lymphoma*

* Steve H. Swerdlow et al. WHO Classification of tumours of Haematopoietic and Lymphoid cells 176-177

Secondary Panel To check maturity To confirm lineage

Tdt

CD4

CD8

CD2

Q9: Which cells express CD4 and CD3 antigens?

CD7 + Tdt(59%) Tdt is nuclear stain. Performed after permeabilisation of the cell membrane. Combination of staining possible as monoclonal antibodies acquired individually

CD5(73%) CD5+CD2(14%) Loss of CD2 on malignant T cells Q10: What are these cells?

CD2 ( 0%) Tdt (49%)

CD4+CD8+(33%) Q11: What are the normal counterpart of these cells?

Report Lineage:

Lymphoid T-cell (Tdt is expressed on CD7 cells)

Maturation Stage: Tdt and CD7(58%) CD4+CD8+(33%) Cortical-T-ALL

Final Diagnosis

Cortical T-ALL

What if flowcytometer is not available?

Immunophenotype is still possible!

Remember Immunoperoxidase PAP APAAP

Only an ordinary light microscope is required

Summary Immunophenotyping is an essential aid to establish the diagnosis of haematological malignancies.

Immunophenotype is best carried out on a fresh and representative specimen (e.g. presence of blasts in peripheral blood or bone marrow).

Immunophenotype can only be useful if carried out with recommended type and number of antibodies.

Keep a record of Immunophenotype. The unique combination of antigens may be utilised to identify malignant cells in fluids or for MRD.

Spring in my home 28 March 2010