Final Fc Ppt

7/28/2019 Final Fc Ppt

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Flow cytometry,

App l icat ions in TM

Rashmi Tondon


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Introduction

Flow cytometry

flow

cells move in single file

cytometry

measurement of numerous cell properties


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Definition

Measurement of physical and /or chemical

characteristics of cells or,by extension, of

other biological properties.

-Howard Shapiro

It is a process in which such measurements are made

while the cells or particles pass, preferably in single file,

through the measuring apparatus in a fluid stream.


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Definition

Flow cytometry The study of cells in

suspension

Three components:

1. Fluidics

2. Optics

3. Electronics


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History

Dates back to nineteenth century

German Paul Ehrlich described the fundamental

extrinsic properties of leucocytes

Conjugation of fluorescein to antibodies byCoons & Kaplan at Harvard in 1940s

Caspersson and colleagues worked out the

fundamental aspects of modern cytology Mack Fulwyler built one of the first sorting FC


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Modern Era

Multiparameter analysis by the use of

highly specific fluorochrome-labeled monoclonal

antibodies

fluorescent dyes for measurement of total DNAcontent


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Types of flow cytometer

two types

sorters

separateone particular

cell type

research purposes

analysers

cell analysis

clinical use


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Principle

3 main compartments

Sample handling Flow cell

fluidics Light sensing

Light source

Optics

detectors

Signal processing-electronics Data collection &analysis


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Injector

Tip

Fluorescence signals

Focused laser beam

Sheath

fluid

Flow cell fluidics


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Direction of flow

The Bernoulli Effect

Velocity Gradient

Viscous drag along walls.

Hydrodynamic Focusing

Sheath fluid

Lower pressure

Particles move to low pressure are

Laminar Coaxial Flow


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Cells/particles must be individually suspended,

hence individually counted Cells are made to move (or focused) in single file

using liquid pressure through a small (50-300 m)orifice = hydrodynamic focusing

Fluidics

Injector

Tip

The flow cell

Cells in a single file

Sheath

fluid


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Light source

Stationary laser light Sources

Argon laser

Krypton ion

Helium/neon

Diameter of beam-650m 2 beam focusing lenses

Horizontal horizontal axis - resolution

Horizontal axis - sensitivity

Vertical


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Light sensing

Observation/interrogation region

Spot where moving cell intercepts the

stationary laser light

Two events take place

Light scattering

Emission of fluorescent light


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Direct beam stop.Laser

Light

High angle scatter :

Reflection & refraction.

Cell structure.

Low angle scatter :

Diffraction. Cell size.

Fluorescence at longer

wavelengths.

Intrinsic

(autofluorescence)

and extrinsic.


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Scattered light

Occurs when light is deflected off the cells

Related to Intrinsic property of the cell

Detected in two different directionsAlong the axis of the beam - FS

At right angles - SS/90scatter


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Forward scatter

Along the axis of beam

Light scattered b/w .5-1 &10-20 from axis ofbeam

Proportional to the size of the cell Blocker/obscuration bar

To stop beam at 0

To assure only FS is collected

Other factors affecting Refractive index of cell

Absorptive properties


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FALS Detector

Laser

Forward scatter


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Side scatter

90 scatter perpendicular to the axis

Light reflected from internal structures

Correlates with granularity of the cell 3 major leukocyte populations in 2

parameter histogram

Lymphocytes,monocytes,granulocytes


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FALS Detector

90LS Detector

Laser

Side (90o) scatter


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Electronic gating

Gate -Electronically framed region/window

drawn around the desired cell cluster

Shape of gated area varies-

Rectangle

Polymorphous

unrestricted


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Sample : peripheral blood after red cell lysis.Data collected on 10,000 cells.

CD45 FITC (log)

R1= monocytes (CD14+ve)

R2=lymphocytes (CD45> monocytes)

R3=granulocytes (CD45< monocytes)

CD14P

E

(log)

Forward Scatter (linear)

SideScatter(linear)


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Fluorescent light

Fluorescence

Certain dyes absorb laser light & emit light at

longer wavelength

argon absorbs at 350nm & emits at 488nm

Pick up lenses/spatial filter assembly

Fluorescent light collected at 90 angles to

laser beam


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Filters

absorption filtersabsorbs unwanted light

5 types

bandlong, shortdichroic,notch

interference filtersreflects unwanted light

types of filters

Fl t t ti


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PMT

PMT

PMT

PMT

Dichroic

Filters

BandpassFilters

Laser

1

2

3

4

Flow cell

Flow cytometer optics


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Fluorochromes

Prerequisites

Light absorption spectrum should match the

wavelength of emitted light(488nm)

High extinction coefficient

High quantum yield


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Fluorochromes

3 group of dyes

LMW organic dyes

Fluorescein isothiocyanate(FITC)

Biological pigments

Phycoerythrin

Peridinin chlorophyll protein(PerCP)

Tandem dye systems CyChrome


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Signal processing

Sensors convert photons to electrical

impulses

Impulses photons fluorochrome mol.

Processing in 2 ways

Peak-sense-hold (process brightest signal)

Integrated signal (process all signals)


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Laser

Fluorescence

FALS Detector

Fluorescence detector

(PMT3, PMT4 etc.)

Fluorescence detectors

Freque

ncy


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Data presentation

linear formoutput proportional to inputquantification of DNA,RNA

LS of size&granularity

log formoutput proportional to log of inputTypeimmunophenotyping


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Sample : peripheral blood after red cell lysis.Data collected on 10,000 cells.

Dot

Horizontal : low angle scatter. Vertical : high angle scatter.

Density Contour


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Sample : peripheral blood after red cells lysis.Data collected on 10,000

Scatter

Low

High

Events

Events CD14 PE

CD 45 FITC

Fluorescence

Isometric Displays


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Fluorescent activated cell

sorting Sorting

Physically separate the cells based on

differences of any measurable parameter

Components

Droplet generator

Droplet charging & deflection system

Collection component

Electronic circuit


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Mechanical Sorting:

Takes place within a Flow cell

When a sort decision Green cell has been

made it is diverted into a catcher tube

either by moving the tube into the stream:

Laser beam interrogates cellsoo

o

o

o

o

o

o

o

o

o

o

o

o

oor by applying

an acousticpulse to the

stream to

divert the cell

into the tube.

ooooo

ooooo

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ooo

o

Hydrodynamic focusing

takes place within a flow cell.

o

o

o

o

o o

o

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o

o

o o

o

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o

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oo

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o o o

oo

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oo

o

o


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Electrostatic Sorting : Stream-in-air

Laser interrogation and signal processing

followed by sort decision : white sort right,

blue sort left, green orred no sort.

oooo oooo oooo

left waste right

Various collection devices can be attached :

tubes, slides, multi-well plates.

Hydrodynamic focusing in a nozzle

vibrated by a transducer produces a

stream breaking into droplets.

o o o o o

o o o o o

o o o oo o o

o

o

o

o

oo

o

o

Electronic delay until cell reaches break

off point. Then the stream is charged :

+ if white - if blue.

-+ Charged droplets deflect by electrostatic fieldfrom plates held at high voltage (+/- 3000 volts).

o-

o+

o

o+

o-

o

o

+

-

+

+


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Intrinsic : size,shape,cytoplasmic granularity,autofluorescence and pigmentation.

Extrinsic : DNA content, DNA composition, DNA

synthesis, chromatin st., RNA, protein, sulphydryl

gp,antigens(surface,cytoplasmic & nuclear), lectin

binding sites, cytoskeleton components, membrane

st.( potential, Permeability& fluidity ),enz. activity,

endocytosis,surface charge, receptors, bound andfree calcium, apoptosis, necrosis, pH, drug kinetics,

etc., etc., etc.

Applications of Flow Cytometry.


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Red cell analysis

Detection of red cell-bound Ig

patients with positive DAT(quantification of

IgG coating)

Patients with negative DAT(detection ofbound IgG)

IgG subclass determination

Subpopulation of IgG sensitized red cells-sickle cell disease,red cell aging


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Red cell analysis

Detecting cell bound Ig other than IgG- IgM,IgA

Detection & quantification of red cell antigens

common blood group antigens-ABO, Rh, Kell, Kidd

uncommon blood group antigens- Kn /McC ,Dr(a)cells,Cr system

RBC antigens during erythroid development-max

expression at blast stage(eg.MN system)

cell aging accompanied by in ABH antigen


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Red cell analysis

Detection & quantitation of red cell populations

0.125%minor cell population is detectable

Transfused red cells

can detect antigenically dissimilar red cells followingsmall volume transfusions (~10 ml)

determination of red cell survival after transfusion

determination of autologous red cells in multiply

transfused patient(reticulocytes separation)


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Red cell analysis

Chimerism

Genetically &artificial chimerism

Any hematopoiesis from the recipient is

considered mixed chimerism

Chimeras also demonstrate immune tolerance

Genetically gp O person with implanted A cells

does not produce antiA Mixed chimeras m/b associated with a lower

frequency of GVHD


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Red cell analysis

Fetomaternal hemorrhage

Accurately quantitate FMH

Using labeled IgG or antiHbF

Sensitivity equal to Kleihauer-Betke technique

Not routinely done


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Analysis of GPI-linked anchor

proteins

PNH

acquired clonal disorder

Red cells unusually susceptible to lysis by

complement

Somatic mutation in PIG-A gene on X-ch

essential for normal synthesis of GPI anchor

proteins(CD55,DAF;CD59,MIRL) Chimeric cells ( normalmoderate -extreme

sensitive )

A l i f GPI li k d


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Analysis of GPI-linked

proteins CD59 inhibits the formation of the terminal

complex of complement

PNH III (complete deficiency)

PNH I(partial deficiency)

Red cells analyzed with fluorescein- labeled

antibody specific for GPI-anchor proteins-

CD55,CD59,LFA-3Presence of a population of >1 GPI-linked

protein is diagnostic of PNH


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Analysis of GPI-linked proteins

Mutation is identified in neutrophils

GPI-linked proteins suitable for analysisinclude CD16,CD24,CD55,CD59 AND CD67

Analysis of neutrophils more difficult More sensitive method

Flow cytometry has replaced Ham test asprimary method for diagnosis

Heavily transfused patient

Following BMT

FLOW CYTOMETRIC DIAGNOSIS


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Red cell analysis

Variant red cells

D/t mutation or recombination event

Survivors of Hiroshima

Bloom syndrome

Ataxia telangiectasia

Cancer chemotherapy

McLeod syndrome


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Platelet analysis

Technically more difficult

Aggregation

Ensure single cell population

Platelet fragments& microparticles

Less fluorescence b/c of small size

In-vitro activation


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Platelet antigens

Readily used for platelet phenotyping

Phenotype HPA-1a of mother, father and

baby

Heterogeneity of various RBC & plateletantigens on platelets

Differentiate b/w hetero & homozygous state

for HPA-1a antigen (MESF) Suitable for antenatal screening for NAIT


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Platelet antigens

To study platelet physiology,function,and

interaction with WBC and endothelial cells

To study platelet activation-

eg,CD62(GMP-140)transferred from

granules to the surface

GPIV (CD36)expression of Nak platelet

antigen plays a role in P. falciparuminfected RBC binding to endothelial cells


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Platelet antigens

Semiquantitative assay to assess the

amount of bound antibody with

subsequent estimation of antigens /cell

Useful in Glanzmanns thrombocytopenia

BernardSoulier syndrome


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Platelet function

Release, adhesion and aggregation

Activated platelets exhibit alterations inexpression of GPIb,GPIIb/IIIa

expression of platelet activation markers CD62(P-selectin) Microparticle generation

Lysosomal protein CD63

Fibrinogen Thrombospondin

Multimerin


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Platelet activation

Measure activated Vs nonactivated cells

Cardiac surgery

Thrombosis

Atherosclerosis

Assessing platelet


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Assessing platelet

concentrates In various conditions of preparation & storage

CD62 with storage

CD 62 may serve as QC measure

Loss of GPIb /IX from pl surface No filtration enhanced activation

Platelet activation in normal donors undergoing

apheresis,persisting for up to 48hrs.

Measuring intracellular Ca Changes in actin &myosin


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Platelet alloantibodies

Testing multitransfused alloimmunized

patients

HPA antibodies(15%)

HLA Vs HPA antibodies

HLA antibodies(85%)

HPA antibodies detection in NAIT

Platelet crossmatching


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Platelet autoantibodies

Autoimmune thrombocytopenia

To measure platelet associated Ig

PAIgG

PAIgM PAIgA

Even when thrombocytopenia is

severe(


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Reticulated platelets

Thiazole orange used for reticulated pl

Binds to nucleic acid esp. RNA

Measure of platelet overturn

Distinguish b/ w pl production &destruction

Measure early detection of pl recovery

from CT induced thrombocytopenia

Hematopoietic progenitor


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Hematopoietic progenitor

cells Quantification of CD34+ cells in peripheral

blood or bone marrow

Total CD 34+ cells=

CD34+ cells WBC X Vol. of product

Total nucleated cells

Light scattering also helps to differentiate

Low SS &FS


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Immunophenotyping in HIV

CD3+ CD4+ T cells enumeration

Baseline evaluation

Staging of disease

To monitor progression

To determine likelihood of opportunistic

infection

To make therapeutic decisions as surrogate marker in clinical trials


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Detection of viral antigens

Measurement of viral content

Apoptosis of CD8+ cells by blood born

viruses b/c of immune suppression

HIV,CMV,E-B virus,Varicella zoster,HTLV


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Leukocyte analysis

Leucoreduction in leukodepleted blood

products

Accurately measure 0.1 WBC/ L

Preferential depletion of WBC subsets

HLA class II bearing dendritic cells


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Leukocyte analysis

Leukocyte antigens

Detection of white cell antigens

HLA-B27 phenotyping

Quantitative analysis of HLA class 1 antigens

Determination of CD4+ lymphocyte levels

Measurement of CD4+subsets


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Leukocytes analysis

Leukocyte function

Neutrophils activation in SLE

Upregulation of CD11a density on

CD4+/CD8+ in IM Neutrophil respiratory burst

Measuring cellular glutathione content in AIN

Immune competence in SCA


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Leukocyte antibodies

antiHLA antibodies in FNHTR

Antineutrophil antibodies (GIFT)

Antilymphocyte antibodies (LIFT)

Transfusion related acute lung injury

Neutrophil associatedantibodies/complement

Detection of antiphospholipid antibodies Detection of ANCA


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Histocompatibilty testing

Pre-allograft transplant crossmatching

Crossmatching b/w donor lymphocytes &

recipients serum

Marked reduction in hyperacute rejection Improved graft survival

Detects low levels of anti donor antibodies

Identifies high risk patients

Considered definitive crossmatch technique


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Histocompatibility testing

Monitoring ALG/ATG therapy to prevent

allograft rejection

Detecting presence of anti CD3

CD3 Modulation on T cells

CD2+ or CD3+ T cells determination


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Apoptosis

Detection of abnormally activated cell

populations

Generic marker for viral infection


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Advantages of flow cytometry

Rapid assessment of large no. of cells Multiparameter analysis

High accuracy & reproducibility

Objective analysis Ability to analyze many samples quickly

Capable of data reduction

Permanent data storage

Ability to reanalyze data

Requires relatively small sample

COULTER EPICS XL and


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COULTER EPICS XL and

XL-MCL


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BD FACS Count


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