Presented By

Dr.Mohammed AttiaDirector of flow cytometry Facility

Tanta University

Introduction To Flow Cytometry ApplicationsIntroduction To Flow

Cytometry Applications

•FCM is a technique for counting, examining and sorting microscopic particles suspended in a fluid

• It allows simultanous analysis of the physical and /or chemical charactaristics of the examined particles

Definition Definition

•FCM involves the analysis of flourescence and light scatter properties of single particle ( e.g. Cell, nuclei, chromosome, bacteria, bead ect) during its passage within a liquid stream

Basics Basics

Light scatter

Light scatter Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Light scatter

To Distinguish Between 2 Cell typesA. Size Has To Be Different ORB. Internal Complexity

If These Two Parameters Are The Same, Then No Distinction Can Be Made

Limitations of Light Scatter

Immuno Cytology

membranous /intracellular

structures

dye-coupled monoclonal

antibody (mab)

Y

Multiparametric AnalysisMultiparametric Analysis

Simultaneous Detection of up to 20 Parameters

Cell size Cell structure Dyes up to 18 colors

Y

Data display

Data display

Data display

Advantages Analysis of high cell numbers (107 cells)

Short time of analysis (sec)

Gating allows detection of cell sub-populations

Measurement of rare events

Objective measurement of fluorescence intensity

Simultaneous detection of many parameters per cell

Advantages Analysis of high cell numbers (107 cells)

Short time of analysis (sec)

Gating allows detection of cell sub-populations

Measurement of rare events

Objective measurement of fluorescence intensity

Simultaneous detection of many parameters per cell

Flow cytometry

Disadvantages

Relatively expensive technology

Test cell in suspension

Tissue architecture is lost

Disadvantages

Relatively expensive technology

Test cell in suspension

Tissue architecture is lost

Flow cytometry

FCM SamplesFCM Samples Blood Bone

marrow BAL C S F Effusions Cell culture Sperms Tissue cells

e.g. tumor

Prerequisite

Single Cell Suspension

Prerequisite

Single Cell Suspension

FCM APPLICATION

Research Laboratories

Clinical Laboratories

FCM APPLICATION

Research Laboratories Immune function studies Hematopoietic stem cell research and

regenerative medicine Apoptosis Cell cycle and cell Kinetics Intracellular cytokine production Intracellular signaling Sperms and IVF Flow and FISH Microbiology and virology

FCM APPLICATION

Clinical Laboratories Haematology Haemato-oncology Clinical immunology Organ transplantation and Stem

cell therapy Cancer Rare event applications Application in clinical microbiology

Haematology

RBCs

Haematology -RBCs

Autoantibodies in H A Fetal RBCs in fetomaternal hge Blood groups Membrane proteinsFragility of RBCs ( OFT)PNH

PNH

• Principle of FCM: Absent or markedly diminished expression of glycosyl phosphatidylinositol-anchored protein (GPI-AP) on red cells and/or white cells in the appropriate clinical setting.

• GPI-AP - CD59 (MIRL) & CD55 (DAF)• Use two GPI-AP for confirmation

Haematology

Blood transfusionQuality control of blood products

*Enumerate number of residual WBC contaminating

1- Leucodepleted RBCs unit 2- Platelets unit

*Enumerate number of residual WBC, RBC & platelets in FFP units

PLATELETS

Haematology

Platelets

GPIIb-IIIa CD41/CD61 GPIb-IX CD42b/CD42aGPIa-IIa CD49b/CD29GPIc-IIa CD49e/CD29GPIc-IIa CD49f/CD29 GPIIa CD31GPIV CD36GP53 CD63Vitronectinreceptor a CD51Vitronectinreceptor b CD61ThrombinreceptorThrombospondin

Thrombinreceptor

CD62p

Haemato-oncology

Haemato-oncology

# Acute leukemia

# LPD

Acute leukemia's

ALL AMLM0M1M2M3M4M5M6M7

T-ALL B-ALLpre-T-ALL

Common-T-ALL

early T-ALL

mature T-ALL

pro-B-ALL

C-ALL

pre-B-ALL

B-ALL

BAL

Haemato-oncology

# CLL

# NHL

( FCL, MCL, SLVL , LPL etc)

# Hairy cell leukaemia

Chronic Lymphoproliferative Disorders

FCM in Haemato-oncologyDiagnosis and classification of L&L

Detect malignant cells

Define lineage (Myliod , T or B)

Sub-typing and classification

Prognosis (DNA Ploidy, CD38 & ZAP 70)

Follow up and therapy monitoring (MRD)

Acute Leukaemia Panel

LPD Panel

Case study

Seven (7) year old, male patient, referred with Clinical history of Mediastinal Mass.

P B Examination showed high Leukocyte Count with 26% Blasts.

Looking at this case display you can tell that there is an abnormal population (green) that is v.bright CD45 “brighter than normal lymphocytes” and has a high side scatter. There is almost no normal lymphocyte in this bone marrow specimen.

This is a very informative slide and probably gave you the diagnosis already. Looking at the CD3/CD4/CD8 patterns we can see that the majority of the gated cells are dual positive for CD4/CD8 and negative for surface CD3. This pattern is very uncommon in bone marrow and indicate the immature T-cell nature of the blasts. Among the abnormal cells you can recognize the normal lymphocytes pattern (see red arrow).

Looking at this slide we learn more about the nature of these cells. We find that they are negative for CD10,CD19,CD34,CD33,CD13 and positive for CD7. The later antibody “CD7” confirm the T cell lineage of the abnormal cells.

The abnormal cells are positive for CD2 and CD11b and negative for HLA-DR. Usually in most cases of the T-cells they tend to be negative for HLA-DR. The positivity of CD11b is also atypical of this kind of malignancy.

Again the abnormal cells continue to confirm its T-cell lineage; they are positive for CD56, CD5, partially CD1a and negative for CD117 and CD64. Usually the expression of CD56 in leukemias is associated with worse prognosis.

The cytoplasmic staining of TdT and CD3 confirm the immature nature of the blasts.

Antigen profile: Positive for CD4, CD8, CD7, CD2, CD5, CD56, partially CD1a, cCD3 and negative for HLA-DR. Diagnosis: Acute T-cell

Leukemia

MRD# The lower level of disease (residual malignant cells) detectable in patients after therapy .

# Aberrant phenotypic pattern by FCM and Leukemic cell DNA OR RNA by PCR are the most widely used as they have the most sensitive and specific level

MRD detection by FCM

ALL

It requires the identification of specific pattern of

Ags at presentation which then sought at follow up

T-ALL (TdT and CD3) reliable up to 1 in 104

since normal BM doesnt contain it

B-ALL (19, 34, 10 &TdT, 20,38,58,45 )

MRD detection by FCM

FCM APPLICATION

Immunology

#Lymphocyte Sub-setting: T,B & NK cells (CD3/CD4/CD8/CD19/CD16+56) * HIV patient monitoring * SCID patients * Immuno compromised patients

FMC IN IMMUNOLOGY

#T cell function assay FCM can assess lymphocyte activation of

* T helper CD4 cells * T cytotoxic CD8 cells

FMC IN IMMUNOLOGY

Applying Gates for sub-population analysis

Simple gating stratagies…Whole blood light scatter

Gate on lymphocytes

(light scatter)

Assess T-cell population

(fluorescence)

…to more complex!

#Neutrophil Functional assay Number of ready to use kits are available assess Neutrophil

* Migratest (measure chemotactic & adhesion)

* Phagotest (determine phagocytic activity)

* Oxidative Burstest (Quantify oxidative burst

activity)

FMC IN IMMUNOLOGY

FCM APPLICATION

Organ transplantation

and

Stem cell therapy

FCM APPLICATION

Organ transplantation and Stem cell therapy

Stem cell enumeration -HSC Banking -HSC Transplantation HLA cross matching Pre & Post Transplantation

monitoring of immune system

• Stem Cell enumeration

FCM APPLICATION

Cancer

FCM APPLICATION

Cancer DNA Ploidy and cell cycle kinetics MDR Circulating Tumor cells ( CTCs)

FCM APPLICATION

DNA analysis:

Aneuploidy and/or elevated S-phase

fraction have been shown to be

prognostic indicators in breast,

colon, rectal, prostate, ALL and

bladder tumors.

Cell Cycle Analysis

Note the cell volume (size) and DNA concentration change as the cell progresses through the cell cycle

Cell Cycle Analysis

DNA probes

DAPI }

Hoechst } UV

Propidium iodide (PI) }

7-AAD } 488

TOPRO-3 }

DRAQ5 } 633

These dyes are stoichiometric – number of bound molecules are equivalent to the number of DNA molecules present

The cell cycle

l

Typical DNA histogram

Stoichiometric DNA probe binding

Cell Cycle Analysis

Cell Cycle Analysis

Assessing cell proliferation using FCM

APOPTOSIS AND

CELL VIABILITY

FCM APPLICATION

Apoptosis• Gene directed cell death

• An event that occurs during development and a response to trauma or disease

• Cancer cells develop a strategy to evade apoptosis

Apoptosis results in a number of cellular events that can be analysed by FACS:

• Fragmentation of DNA (subG1 assay, Hoechst dyes)

• Membrane structure and integrity Annexin-V, PI)

• Mitochondrial function (Mitotracker Red)

• Caspase activity (antibodies assay)

Sub G1 apoptosis assay

Sub-G1 peakDNA fragmentation allows apoptosis to be quickly assessed with eg. PI

Can be seen as a population of small peaks to the left of G1 in a histogram

Quick and easy way to determine if apoptosis is occurring

Apoptosis

Apoptosis detection using viability dye uptake

Changes in membrane permeability due to apoptosis allow intracellular dyes to stain unfixed cells

7-AAD (DNA)

Live cells exclude dye

Apoptotic cells stain 7-AADdim

Dead cells stain 7-AADbright

Apoptosis

Annexin-V/PI assay for apoptosis:

hPS normally on inside of cellular membrane hAnnV can bind to externalised PS highlighting cells that are apoptotic hPI will only go into cells with compromised membranes – dead (necrotic) cells

AnnV-FITC

PS

X

X

X

X

X

X

PI

Apoptosis

• Membrane potential of the organelle reduced

• Mitochondrial activity appears to change in parallel with cytoplasmic and plasma membrane events

• Dyes that accumulate in mitochondria can therefore play role in detecting apoptosis

-Mitotracker Red CMXRos

-JC-1

-DiOC2(3)

-Laser Dye Styryl-751 (LDS-751)

• Reagent combinations can provide a window on intracellular processes not available with the much used pairing of annexin V and propidium iodide

• Apoptosis – Organelle Analysis

Apoptosis

Live/Dead assay

Utilise the properties of dyes that are impermeable to intact cell membranes:

Propidium iodide

DAPI

TOPRO-3

+ve fluorescence indicates compromised cell membranes and therefore dead cells

Yeast cells + TOPRO-3

Dead cells show more granularity and reduced size

Live cells retain their morphology and appear larger in size and less granular

Apoptosis

CELL SORTING

Cell sorting

Allows rare

populations to be

isolated from

heterogenous

populations (cell

culture, blood

samples, etc)

Can isolate sub

cellular particles

(e.g. endosomes,

nucleus,

chromosomes)

Can produce purity

>95%

Cell Sorting

Thank You