Flow Cytometry – Principles and applicationsBasic Hematopathology CourseJune 12-13, 2010TMH

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

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

• Flow cytometry• Immunohistochemistry• Immunofluorescence

Monoclonal antibodies

• Sensitize mouse to antigen• Harvest spleen B cells• Fuse with myeloma cells• Select hybridoma clones for antibody production• Label antibody with fluorochrome dye / color

FCM and IHC: complementary

FCMmulticolor immunophenotypingfluids

Immunohistochemistry mostly single colorbiopsy

flow + cyto + metry

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

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

Present

- 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

It requires a suspension of single cells or other particles, with minimum clumps and debris.

To analyze solid tissues, a single-cell suspension must first be prepared

No information on tissue architecture

Shortcomings of a FCM

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

PMT

PMT

PMT

LASER Sample in a hydrodynamically focused stream Detectors

Amplification and computer

Properties of FCM

Physical properties

Hydrodynamic focussing

Forward scatter Side scatter

Size Granularity

SS detector - Granularity

SS detecter Granularity

FSsize

FSsize

Laser

Laser

Size and granularity

Data shown either as a

- single parameter histograms, or - two parameter correlated plots

Data may be shown as

• Linear scale The scale on which the output is directly proportional to the input.

• Logarithmic scale The scale on which the values increase logarithmically

Data

Single parameter (Histogram), with dye (PI), tissue showing DNA content, linear scale

DNA

Count

256

384256128

128

384

G0G1 G2M

S phase

Two Parameter (Dot Plots), without dye,lysed peripheral blood, logarithmic scale

Dot plot Density plot Contour plot

Contours as a percentage of the maximum event number

Contours as a percentage of the total number of events

Scatter pattern lysed peripheral blood

Forward

Side

Properties of FCM

Chemical properties

Flow cytometry measures fluorescence per cell or particle

Spectrophotometry measures the percent absorption and transmission of specific wavelengths of light for a bulk volume of sample

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 Applications1. 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

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

Certain issues with FCM

Garbage in garbage out

Background, non specific staining

Antigen expression:RBCs, WBCs, Platelets, Others

CD3

Cyto CD3

Tdt

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

Leukemia/lymphoma immunophenotyping

Lysis of red cells

Add reagents

Forward scatter – Side scatter

CD45 – Side scatter

CD19 – Side scatter

Gating (cells of interest)

FSC vs SSC: Dot plot / Scatter plot

Forward

Side

Cells of interest

Forward

Side

Normal peripheral blood

Leukemic peripheral blood

Scatter pattern showing a single dense cluster

Scatter pattern – peripheral blood

Normal peripheral blood

Peripheral blood full of tumor cells

Different patterns on FS versus SS

Problem when the tumor cells are scanty

1. FSC vs SSC

Peripheral blood - 92% tumor cells

2. CD45 gating

Helps differentiate blasts from lymphocytes

Problem when the tumor cells are scanty

Cells of interest are few

CD45 gating

CD45 strongest in lymphocytes

CD45 weakest in blasts

CD45 gating means CD45 in each tubeTracking marker

CD45

Myeloblasts

2. CD45 vs SSC

Peripheral blood - 12% blasts

Guess

1. FSC vs SSC

3. CD19 vs SSC2. CD3 vs SSC

T and B cells

3. CD19 vs SSC

4. Reverse gating

ISHAGE

5. Sequential gating

CD19+, CD5- B cell lymphoma

6. Multiple gates (6 color IPT)

CD3 - FITCCD19 - PE CD13 – PerCPCD45 – TR

Multicolor immunophenotyping

Brent Wood et al

Multicolor FCM - issues

Expression of myeloid antigens

CD117APC

CD11bPECy7

CD15FITC

CD45APC Cy7

CD13PECD16PECy5

CD3 FITC

Diagnostic Hematopathology

• Morphology H&E stain - BiopsyGiemsa stain – Aspirate

• Cytochemistry (MPO, NSE)

• Immunohistochemistry, Flow cytometry

• Cytogenetics, FISH

• Molecular methods

Centers doing management of hematolymphoid malignancies

IHC/FCM - Must

All lymphoid cells CD45+ (LCA)

B-cells CD19, CD10, cCD22

T-cells CD3, CD5, cCD3

Myeloid cells CD13, CD33, CD117, anti MPO

Megakaryocytic CD41, CD61

Blasts CD34, Tdt, CD99

Other: HLA-DR, CD23, FMC-7, CD43, CD11c, CD25, CD103, CD38, CD138, CD20, CD79a, Kappa and Lambda light chains, TCR alpha beta, TCR gamma delta, CD4, CD64, CD55, CD59

Common CD markers – Leukemia lab

CDs: Large number of antibodies are available

Most of the leukocyte surface antigens are lineage associated, and not specific to a single lineage or stage of cellular maturation

Lineage specific markers

Blasts, Maturation patterns

Clonality

Minimal panels – Guidelines

10 antibodies plus controls for AL

9 antibodies plus controls for CLPD

IJPM, 2008

B-cell maturation (Brent wood et al)

B-cell maturation (Brent wood et al)

Case

56 year old male with lymphocytosis

• Provisional diagnosis: Lymphocytosis

• Advise: ?

• Provisional diagnosis: Lymphocytosis

• Advise: FCM immunophenotyping – Lymphoma panelLymph node biopsy

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

Impression: B-cell lymphoma, CLL

Another case

Bone marrow from a 3-year-old boywith megakaryocytic thrombocytopenia.

Hematogones bear close resemblanceto the neoplastic lymphoblasts

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

Hematogones B-cell ALL

Patterns of antigen expression - Granulocytic maturation

CD45 vs SSC

Normal MDSCD13 CD16CD11c CD16

MDS and Hematogones

• Pattern analysis

LymphocytosisCD10+, CD19+, HLADR+

CALLA, BL

FL, DLBCL Hematogones

Lymph node FNAC and FCM

Lymph node Biopsy and FCM

Primary folliclesIgM+ IgD+

Secondary follicles, IgD-

Mantle zoneIgM+ IgD+

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

Single Platform Immunoplatelet method

Single Platform Bead Assay

Absolute platelet count =

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

Sehgal K, Cytomerty B, Clinical Cytometry, 2010

Lymphocyte subset analysis

CD4/CD8 Counts

Lymphocyte subset analysis

Normal peripheral blood – Lymphocyte gateFor CD4 counts, add CD3

CD4 PE

CD8FITC

Normal peripheral blood – Lymphocyte gateFor CD4 counts, add CD3

PNH studies

Normal Neutrophils

Abnormal Neutrophils

Reticulocyte counts

Minimal residual disease

DNA Ploidy

CD34 stem cells enumeration

ISHAGE protocolDual 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

Minimal residual disease in ALL

(eg., MRD Lite)

Three color Immunophenotyping

5 year old boy with fever – 1 month

6 year old girl, Diagnosis

MPO negative NSE negative

Diagnosis

Immunophenotyping

3 Color FCM

FSC vs SSC

Blasts only

DiagnosisCALLA- ALL

FL, BL, DLBCL,

Hematogones

MCP 841 protocol

Day 18 BM

Morphology

MRD Lite by flow

Day 18 post induction

MRD lite

• Day 18 post induction, bone marrow is done

• No hematogones

• CD19, HLADR, CD34, syto 13

Quality control

Instrument setup, compensation, titration/validation, Isotype (background staining), FMO (spillover)

EQAS/Proficiency Testing

Cell viability

Clinical history, morphology

Panels: adequate, combinations, weak fluorochromes

Signatory

Final report (Positive/negative, intensity, CD45 gating or FSC/SSC, how many cells gated and studied)

Single/double platform, CVs

strong

dim

crossroads

do not give percentages

For Referring Oncologist/Pathologist

Discuss with cytometrist, history and choice of panels

Indications and Transportation

Stem cells, CSF, Lymph nodes

Reading the report

Indian data on hematolymphoid malignancies and guidelines for IPT

• Panel selection

• ICMR Taskforce

• EQAS / PT

Cytometry B Clin Cytometry, 2008IJPM, 2009Leukemia Lymphoma 2009Leukemia 2009IJC, 2010

Final comprehensive report

• Morphology• Cytochemistry• Flow cytometry / IHC• Cytogenetics• Molecular diagnostics