Gujral FCM 2

Flow Cytometry – Principles and Applications

Second Basic Hematopathology CourseTMHJune 11-12, 2011

Dr Sumeet Gujral, MDAssociate ProfessorDepartment of PathologyTata Memorial Hospital, Mumbais_gujral@hotmail.com

Role of a Hematopathologist

• Establishing Diagnosis

• Prognostication (sub-classification)

• Prediction

• Evaluation of treatment effectiveness (MRD)

Leukemia/Lymphoma - Diagnosis

• Clinical: History and Examination

• Routine investigations

• Morphology (smear, biopsy)

• Ancillary techniques

Ancillary techniques

• Immunohistochemistry, • Flow Cytometry, • Cytogenetics, • Molecular Diagnostics

• Diagnostic• Prognostic• Predictive

100bpladder

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RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) detects diagnostic translocation markers like BCR/ABL in CML - BCR/ABL: b3a2 437bp

Flow Cytometry

Localization of antigens or proteins in cells using labeled antibodies through antigen-antibody interactions,

Reaction visualized by a marker (fluorescent dye, enzyme, colloidal gold etc)

Immunophenotyping

Complementary

FCMmulticolor immunophenotypingfluids

Immunohistochemistry mostly single colorbiopsy

Our practice

Flow cytometry – Liquid and Solid

Immunohistochemistry – Solid and Liquid

flow + cyto + metry

• 1953 - The first impedance-based flow cytometry device, using the coulter principle (Wallace A Coulter)

• 1968 - The first fluorescence-based flow cytometry device (ICP 11) by Wolfgang Göhde, University of Munster

It is the measurement of cellular properties as cells move in a fluid stream (flow), past a stationary set of detectors (thousand events per second)

Technique of quantitative single cell analysis

Principle

It analyses - physical, and - chemical properties (immunofluorescence) of cell

Components of a Flow Cytometer

• Fluidics: a flow cell with sheath fluid (hydrodynamic focussing)

• Optics: LASERS, single wavelength,

coherent light (however incoherent light is of random phase varying with time and position)

• a detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer

• an amplification system – linear or

logarithmic

• a computer for analysis of the signals- Single or multiple Lasers- Sorter

Types

- Single Laser or Multiple Lasers(1 laser three color, 4 lasers 18 fluorescence detectors)

- Sorter (so as to purify populations of interest )

- Laser scanning cytometers

Advantages of a FCM

• Study of cells, chromosomes and particles (analysis, counting and sorting)

• Thousand of particles per second

• Multiparametric analysis at a single cell level

• Pattern studies

• Sorting

Research Applications• Autofluorescent Proteins • Antigen or Ligand Density • Apoptosis • Enzyme activity • DNA, RNA content and changes in the cell cycle • Membrane Potential • Cytokine receptors and it's synthesis • Drug uptake and efflux • Phagocytosis • Viability • Changes in Intracellular pH • Changes in Intracellular calcium • Changes in Intracellular glutathione • Changes in Oxidative Burst

Diagnostic Applications

1. Monitoring AIDS patients 2. Immunophenotyping: Diagnosis, subtyping and

prognostication of hematolymphoid malignancies3. Monitoring Minimal Residual Disease 4. Determining CD34 counts5. Reticulocyte Counts 6. Diagnosis of PNH 7. DNA analysis of S-phase fraction8. Platelet counts

It requires a suspension of single cells No information on tissue architecture

Expensive, maintenance

Shortcomings of a FCM

ImmunophenotypingSampleProcessAcquireAnalysisFinal report

Flow cycle

Referring physician Sample collection/transportation

Preparation of cells of interest Add antibodies tagged with fluorochromes

Acquire (flow) the cells LASER

Amplified signals Digitized

Analyze Report Referring physician

Antigen expression:RBCs, WBCs, Platelets, Others

CD3

Cyto CD3

Tdt

Lysis of red cells

Add reagents

Process

Intracellular staining

For Intracellular staining, cells are first fixed in suspension and then permeabilised before adding the fluorochrome

This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact

Commercial kits/In-house

Cells are fixed – permeabilised –add antibody (FL)

CD3

Cyto CD3

Tdt

Results

Physical Properties Scatter pattern, FSC vs SSC

Forward - Size

Side - Granularity

Note: no RBCs

Chemical propertiesImmunofluoresence

How do we select cells of interest?Gating

Scatter pattern, FSC vs SSC

Forward - Size

Side - Granularity

Cells of interest: FSC vs SSC

Forward - Size

Side - Granularity

FSC vs SSC CD45 Gating CD19 Gating

Cells of interest may be scanty

CD - 23391 INDULKAR (BM).003

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FSC vs SSC

Study B-cellsStudy T-cells

T and B cells

CD - 23391 INDULKAR (BM).003

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GatingSmall cell Lymphoma

What is CD45 gating?

How is CD45 gating better than SSC/FSC gating in Acute Leukemia?

Importance of CD45 gating when blasts are few

Guess

CD45 gating means CD45 in each tube

CD45

Myeloid tube T cell tube B cell tube MDS tube Other tube

CD45 CD45 CD45CD45

Generally 3-6 tubes,How to select antibody combinations?

CD3 - FITCCD19 - PE CD13 – PerCPCD45 – TR

Four color means a cocktail of four antibodies

CD3 - FITCCD4 - PE CD8 – PerCPCD45 – TR

or of same lineage..

of different lineages..

Expression of myeloid antigens – 6 colors

CD117APC

CD11bPECy7

CD15FITC

CD45APC Cy7

CD13PECD16PECy5

CD13 FITC

Indian Guidelines, 2008

Most labs still do 3-4 colors (single Laser)

10 antibodies plus controls for Acute Leukemia

9 antibodies plus controls for CLPD

Published minimal panels may be INADEQUATE

Recommendations

IJPM, 2008

All lymphoid cells CD45+ (LCA)

B-cells CD19, CD10, CD20, CD23, FMC7, k/l LC, cCD22

T-cells CD3, CD4, CD5, CD7, CD8, cCD3

Myeloid cells CD13, CD33, CD117, anti MPO

Megakaryocytic CD41, CD61

Blasts CD34, Tdt, CD99, HLADR

Others: CD1a, CD2, CD11c, CD15, CD16, CD25, CD38, CD43, CD56,CD55, CD59 CD64, CD79a, CD103, CD138, TCR alpha/beta, TCRgamma/delta, IgM, IgD

Cytometry B Clin Cytom. 2009 May;76(3):199-205

6 color and more

CD23APC

CD5PECy7

Lambda FITC

CD19APC H7

Kappa PECD20PerCpCy5.5

Lambda FITC

Single color 6 color, all B cell lineage

Normal 6 color pattern with T and B cell markers

Multiple gates

Normal 6 color pattern with T and B cell markers

Colors unstoppable…

• Euroflow - 8 colors

• Brentwood et al - 10 colors

• Roederer et al - 15 colors

• Most labs do 3-4 color IPT

In Immunophenotyping, FCM helps in detecting following:

- presence or absence of an antigen- intensity- blasts- Clonality: LCR, V-beta repertoire- scanty cells, Maturation patterns

CD3 antibody by FCM detects the fully assembled TCR-CD3 complex, which is present on the surface of T cells only.

In contrast, the CD3 IHC stain usually detects only the epsilon component of CD3, therefore cannot distinguish between T & NK cells.

Diagnosis of Leukemia/lymphoma

• Morphology H&E stain - BiopsyGiemsa stain – Aspirate

• Cytochemistry (MPO, NSE)

• Immunohistochemistry, Flow cytometry

• Cytogenetics, FISH

• Molecular methods

Case

55 year old male

Bilateral cervical and axillary lymph nodes

Normal Complete Blood Counts

Provisional diagnosis: LymphomaAdvise: Biopsy diagnosisFNAC and flow

3 color panel

3 color panel

CD45, CD20, CD19, CD5, CD23, CD22wk, CD20, Kappa LC, CD200

Impression: B-cell lymphoma, SLL

Prognostic markers: CD38, ZAP70

3 color panel

CD19 gating

Same patient of CLL, however, CD19 gating was done

4 color panel

B-CLPD – 6 color panel

Debris

B-CLPD – 6 color panel

CD45, CD20, CD19, CD5, CD23, CD22wk, CD20, Kappa

LC, CD200

Impression: B-cell lymphoma, SLL

Subtyping of Small B cell lymphomas

Patterns recognition

• MDS• Hematogones

Normal BM Maturation patterns of myeloid / monocytic cells

NeutrophilsCD13+, CD16+

NeutrophilsCD11b+, CD117-

B-cell maturation

Cyto muheavy chain

IgM+, IgD+CD5+

IgM

IgG IgA

CD10, BCL6+

Pax5 (My+)

CD79a (T+)

IgG,A,M.D.EIgMIgM

BCR

T-cell maturation

Prothymocyte Subcapsular Th Cortical Th Medullary Th PTC

CD7

Tdt

CD1a

CD2/CD5

CD3

CD4/CD8

surface

DPT CD4+

CD8+

cytoplasmic

Myelodysplastic syndrome

MDS Scatter pattern and maturation pattern

• FSC versus SSC• Myeloid cells: CD11c, CD13, CD15, CD16• Monocytic: CD33, CD56, CD14• Erythroid: CD235, CD71• Megakaryocytic: CD41, CD61, Morphology

Normal MDS

Patterns of antigen expression - Granulocytic maturation

FSC vs SSC

Patterns of antigen expression - Granulocytic maturation

CD45 vs SSC

Normal MDSCD13 CD16CD11c CD16

Normally Maturing Granulocytes

Sickle/Concave pattern Wave pattern

Granulocytic maturation in MDS (CD13 vs CD16)

NORMAL MDS

Normal MDS

Granulocytic maturation in MDS (CD11C vs CD16)

NORMAL MDS

Normal MDS

Hematogones

Hematogones

medium sized lymphoid cells,scant cytoplasm, round to irregular nuclei, dense homogeneous chromatin no - very small nucleoli

Bone marrow from a 3-year-old boy with megakaryocytic thrombocytopenia. Hematogones bear close resemblance to the neoplastic lymphoblasts

Numerous lymphoblasts are present in this bone marrow smear from a 5-year-old girl with precursor B-ALL

Hematogones B-cell ALL

Another case

CD19+, CD10+, FMC7+, CD79b+, CD38+, LLC+ CD3-, CD5-, CD23-, KLC -

Diagnosis: High grade B cell lymphoma

Pleural fluid, lymphoid rich effusion

CD10+, CD19+, HLADR+

B cell ALL, Burkitt’s Lymphoma

CD10+, CD19+, HLADR+

FL, DLBCL

B cell ALL, Burkitt’s Lymphoma

CD10+, CD19+, HLADR+

FL, DLBCL

Hematogones(Tdt+/-)

B cell ALL, Burkitt’s Lymphoma

Lymph node FNAC and FCM

Lymph node Biopsy and FCM

Primary folliclesIgM+ IgD+

Secondary follicles, IgD-

Mantle zoneIgM+ IgD+

B cell clonalitySmall B cell lymphomas

CD20+

Brent Wood et al

?? Role of FCM

Follicular lymphoma , Grade 1 Follicular hyperplasia

FL (Grade 3) Reactive

Bcl2 might not help

flow flow flow

Light chain restriction

CD20 and lambda

Quantitation by FCM

Platelet counts

Various method

International Flow Reference Method

RBC/Platelet Ratio Method (Dual Platform Method)

Absolute Platelet Count=

RBCevents X RBC count Platelet events (Automated Cell Analyzer)

ISLH Task Force, Am J Clin Pathol 115, 460-464.(2001)

CD41/61

RBC

Platelets

Single Platform Immunoplatelet method

Single Platform Bead Assay

Absolute platelet count =

Gated Plt events X Bead count Gated bead events (fixed value)

PlateletsBead

s

Sehgal K, Cytomerty B, Clinical Cytometry, 2010

Lymphocyte subset analysis

CD4/CD8 Counts

Lymphocyte subset analysis

Normal peripheral blood

CD4 PE

CD8FITC

PNH studies (WBCs and RBCs)

It is a rare clonal stem cell disorder of hematopoietic cells that is associated with intravascular hemolysis, venous thrombosis, and cytopenias related to bone marrow aplasia.

Somatic mutations of PIGA gene

Loss of GPI (CD55, CD59)

FLAER test: In vitro binding of a fluorescent-labeled modified toxin that is dependent on the presence of GPI anchors.

Normal Neutrophils

Abnormal Neutrophils

CD34 stem cells enumeration

ISHAGE protocol

Dual Platform - Lyse wash method

Performed in duplicate

Acquire at least 100 CD34+ events for an intra assay C.V of 10%

Four parameters are used– FSC– SSC – Intensity of CD34 staining– Intensity of CD45 staining

All initial ungated events must be acquired and isotype controlsare not required

Then analyzed in a sequential manner (BOOLEAN gating)as per the ISHAGE protocol

Isotype controls are not required with ISHAGE gating system

CD34 % =G4/G1 x100

Absolute CD34=CD34% X WBC 100

N=CD34+ events in R4

D=CD45+ events in R1

ISHAGE Single Platform

Subtract 7AAD + cells from R1

R7=bead events

Lyse no wash processing

Reverse pipetting is essential

Reticulocyte counts

DNA Ploidy

FCM studies cell surface antigens (phenotype), DNA content (ploidy) (DNA index) and DNA flow cytometric proliferation analysis (S-phase fraction or %S-phase).  

Minimal residual disease in ALL

(eg., MRD Lite)

5-year-old boy presented with fever - 1 month

On examination: Pallor, Hepatosplenomegaly

Complete Blood CountsHb - 6gm%TLC – 44,000/cmmPlatelets – 1.25lakh/cmm

Peripheral Blood Smear Examination

DiagnosisMPO, NSE negative

MPO negative NSE negative

Diagnosis

3 Color FCM

FSC vs SSC

Blasts only

CD19, CD10, HLADR positive blasts

CD34 negative

Diagnosis: B-cell ALL

Morphological differentials CD19, CD10, HLADR positive blasts

1. B-cell ALL Tdt, CD34

2. Burkitt’s Lymphoma Kappa, Lambda

LymphocytosisCD10+, CD19+, HLADR+, Tdt+

B cell ALL Hematogones

Diagnosis: Tdt + B-cell ALL

Prognostication: Cytogenetic studies

Chemotherapy: MCP 841 protocol

Evaluation of Treatment: Day 18 BM

- Morphology

- MRD Lite by flow

Day 18 post induction

MRD lite

• Hypothesis: Day 18 post induction, no hematogones in BM

• CD19, HLADR, CD34, syto 13

MRD Tube

FSC-AF

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MRD Tube

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MRD Tube

CD10 PE-A

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KAPPA/LAMBDA FITC-A

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CD34 PerCP-A

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CD10 PE-A

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KAPPA/LAMBDA FITC-A

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CD10 PE-W

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CD19 APC-A

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Gate 5

FCS Filename Gate # of Events % of Gated Cells

% of All Cells

1,3a,100_MRD TUBE.fcs

None 104464 100.0 44.75

1,3a,100_MRD TUBE.fcs

Gate 1 104464 100.0 44.75

1,3a,100_MRD TUBE.fcs

Gate 2 58041 55.56 24.86

1,3a,100_MRD TUBE.fcs

Gate3 1157 1.11 0.5

1,3a,100_MRD TUBE.fcs

Gate 4 1126 1.08 0.48

1,3a,100_MRD TUBE.fcs

Gate 5 6481 6.2 2.78

1,3a,100_MRD TUBE.fcs

Gate6 12102 11.58 5.18

1 in 100 tumor cell dilution – MRD Tube

MRD = 1.43%

Quality control

• Instrument quality controlExperiment setup includes:

- PMT Voltage setup

- Compensation setup and

- Titration of Antibodies

• Reagent/Antibody quality control

• Process quality control

• Tandem dyes

• Trained staff, CMEs

For Referring Oncologist/Pathologist

Indications of FCM, short cuts

Transportation/viability of sample

Multicolor analysis and reading the report

QC - Final comprehensive report

• Morphology• Flow cytometry / IHC• Cytogenetics• Molecular Diagnostics