Immunophenotyping - An introduction by Brig Tahir Aziz Ahmed
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Transcript of Immunophenotyping - An introduction by Brig Tahir Aziz Ahmed
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IMMUNOPHENOTYPING Brigadier Tahir Aziz Ahmed MBBS, MCPS, FCPS, FRC Path
Armed Forces Institute of Pathology (AFIP)
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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
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CPSP 4th March 2010
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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
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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.
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Flowcytometry Lab-AFIP
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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)
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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
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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
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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.
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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
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B-Cell Development `
Stem cell
Pro Pre Immature PC
HLA-DR
TdT, CD34
cyIg SIg
CD19
CD10
Mature
CD5 FMC7 CD38
CD79
CD22 cCD22
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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
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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
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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
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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
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Immunophenotyping for lymphoproliferative diseases aim to identify
CLL
HCL
Burkitt Follicular lymphoma Mantle cell lymphoma Marginal zone lymphoma
LGL NK proliferative disorders
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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
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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
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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.
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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.
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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.
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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
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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)
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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
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Measurements in Flow Cytometry
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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
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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
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Forward and Side Scatter
FSC Detector
Collection Lens
SSC Detector
Laser Beam
Original from Purdue University Cytometry Laboratories
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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
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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.
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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
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CASE SERIES
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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`
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FORWARD and SIDE SCATTER
FSC
SS
C
Patient Healthy
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CD19 + CD10 (B lymphoid markers-56%) Isotype control
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HLA-DR (B lymphoid+ Act T-82%)
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`
CD13 (Myeloid-40%) CD7 (T lymphoid-28%) CD13 Not present on T cells so must be present on B cells
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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?
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Weak CD45 (61%) Q2: Which cells express weak CD45?
Mature T cells
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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
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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?
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Final Diagnosis
Precursor B-ALL
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Patient-2 Boy, 9 years of age
Fever Hepatomegaly Left sided pleural effusion
CBC 65000x 109/L 72% Lymphocytes
HEMATOLOGICAL OPINION: 55% blasts
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Panel selected B-lineage markers
CD19 CD20
T-lineage markers CD3 CD5 CD7
Myeloid markers CD13 CD33 CD117
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Panel selected (cont’d) Markers of immaturity
CD10 CD34
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Forward and Side Scatter
FSC
SS
C
Healthy
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CD10 + CD5(52%)
Isotype control
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HLA-DR(41%)
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CD7(54%) Q5: What is the percentage positivity for CD13?
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CD20(7%) Q6: Which cells are CD20 positive?
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CD3(36%) Q7: Which cells express CD3?
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WEAK CD45(49%)
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Report: CD10 + CD5(51%)
CD7(53%)
HLA-DR(42%)
Weak CD45(50%)
Q8: What is the diagnosis?
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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
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Secondary Panel To check maturity To confirm lineage
Tdt
CD4
CD8
CD2
Q9: Which cells express CD4 and CD3 antigens?
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CD7 + Tdt(59%) Tdt is nuclear stain. Performed after permeabilisation of the cell membrane. Combination of staining possible as monoclonal antibodies acquired individually
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CD5(73%) CD5+CD2(14%) Loss of CD2 on malignant T cells Q10: What are these cells?
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CD2 ( 0%) Tdt (49%)
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CD4+CD8+(33%) Q11: What are the normal counterpart of these cells?
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Report Lineage:
Lymphoid T-cell (Tdt is expressed on CD7 cells)
Maturation Stage: Tdt and CD7(58%) CD4+CD8+(33%) Cortical-T-ALL
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Final Diagnosis
Cortical T-ALL
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What if flowcytometer is not available?
Immunophenotype is still possible!
Remember Immunoperoxidase PAP APAAP
Only an ordinary light microscope is required
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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.
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Spring in my home 28 March 2010