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; 299303.

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 Analysis of clonality Analysis of cellular

lineage of the malignant cell

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 MPO Cy CD3 CD7 HLA-DR IgM Ig kappa Ig lambda Secondary Panel in AML CD45 CD14 CD34 CD2 CD41

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

CD3 CD79a CD33 CD19 CD13 CD10 CD19

Myeloid CD19 CD13, CD33, CD117 Mono. CD14 Lymphoid Glyco phorin A B. CD10, CD19,CD20 CD15 T. CD5, CD7, CD3 Maturity CD117 CD34, HLADR, CD10

CD13 CD42b

Consensus in 2 colour immunophenotyping in Leukemia (contd)

Second panel in B-ALL CD34 CD24

Prim. Panel at AFIP

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

CD22 CD5

Secondary panel in T-ALL CD4 CD2 CD34

CD8 CD1a CD5

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 European Working Group on Clinical Cell Analysis (EWGCCA) 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 2 B-Lymphoid CD79a cCD22 cIgM 1 CD19 CD20 CD10 T-Lymphoid CD3 TCR TCR CD2 CD5 CD8 CD10 0.5 Tdt CD34 Tdt CD7 CD117 CD13 Myeloid MPO

CD33
CD65 CD14 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

Mature

PC

HLA-DR TdT, CD34 cyIg CD19 CD10 CD5 CD79 cCD22 CD22 FMC7 CD38 SIg

T- Cell Development
Stem cell Pro-T Pre-T
Immature

Mature

Activated

TdT cyCD3 sCD3 CD2, CD7 CD4/8 CD5 CD1a CD4 or CD8

HLA-DR

Immunophenotypes of B-ALL Subtypes

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

Pre-B ALL

DR, CD19, CD20, CD24, CD9, CD10, CD34(-), cIgM, TdT+/DR, CD19, CD20, CD22, CD24, CD10, CD34(-), TdT(-), sIg

B-ALL

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

T-ALL subtypes
T-ALLSubtypes Phenotypes Pro-T Pre-T Cortical Medullary cCD3, CD7, CD2(-), CD1a(-),CD34+/-, CD4(-), CD8(-) cCD3+, CD7, CD2, CD1a(-), CD34+/-, CD4(-), CD8(-) CD4, CD8(dual positive), CD1a CD3, CD1a(-), CD4+ or CD8+, TCR

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

AML subtypes
AML Subtypes Phenotypes Mo M1 M2 M3 M4, M5 M6 M7 DR, CD13, CD33, CD34, CD7(+/-) Similar to Mo except CD15(+/-) DR, CD13, CD33, more CD15 and less CD34 than M1 DR(-), CD13, CD15, CD33, CD34+/DR, CD15, CD14+/-, CD33>CD13, CD34+/-, CD4 weak DR, CD13+/-, CD33+/-, CD34, CD45weak, Glycophorin 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 CLL PLL Mantle cell Follicular Marginal Zone Hairy cell Common Phenotypes
DR, CD19, CD20, CD5, CD22(-), CD23, CD10(-), CD11c+/,CD25+/-, CD43, weak SIgM and SIgD DR, CD19, CD20, CD5(-), CD22, CD23(-), CD10(-), bright SIg DR, CD19, CD20, CD22, CD5, CD23(-), CD10(-), CD43 DR, CD19, CD20, CD22, CD5(-), CD23+/-, CD10, CD11c(-), CD43(-), bright SIg DR, CD19, CD20, CD22, CD5(-), CD23(-), CD10(-), CD11c, CD25(-),CD103(-) 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 (23ml 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
Incubation with fluorochrome-conjugated mAbs for 30 min in dark. 2. Lysis of erythrocytes
1.
a) b) c)

Ammonium chloride buffer Hypotonic sodium chloride solutions Commercial reagents

Washing of cells 500xg for 5 min. 4. Fixation using buffered solutions of formaldehyde or paraformaldehyde 1-3% (if not analyzing immediately)
3.

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

15 Side Scatter 10 Y-axis 5 5 10 15 X-axis Forward Scatter

Forward and Side Scatter

Laser Beam

FSC Detector

Collection Lens

SSC Detector
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

Granulocytes Lymphocytes
SSC

Monocytes

RBCs, Debris, Dead Cells

FSC

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

PMT

530nm band pass FL1 560nm short pass dichroic mirror

585nm band pass FL2

PMT

488nm band pass SSC

PMT

510nm long pass dichroic mirror

488nm band pass FSC 488nm laser beam

flow cell PD

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`

SSC

Patient

Healthy

FSC

FORWARD and SIDE SCATTER

Isotype control

CD19 + CD10 (B lymphoid markers-56%)

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?

Mature T cells

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

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 (contd)

Markers of immaturity
CD10 CD34

SSC

FSC Healthy

Forward and Side Scatter

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
LineageProbably 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