Flow cytometry,
Applications in TM

Rashmi Tondon

Introduction

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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.

Definition

Flow cytometry

The study of cells in suspension

Three components:

Fluidics OpticsElectronics

History

Dates back to nineteenth centuryGerman Paul Ehrlich described the fundamental extrinsic properties of leucocytesConjugation of fluorescein to antibodies by Coons & Kaplan at Harvard in 1940sCaspersson and colleagues worked out the fundamental aspects of modern cytologyMack Fulwyler built one of the first sorting FC

Modern Era

Multiparameter analysis by the use of

highly specific fluorochrome-labeled monoclonal antibodies fluorescent dyes for measurement of total DNA content

Types of flow cytometer

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Principle

3 main compartmentsSample handlingFlow cellfluidicsLight sensingLight sourceOpticsdetectorsSignal processing-electronics Data collection &analysis

Flow cell fluidics

Injector

Tip

Fluorescence signals

Focused laser beam

Sheath

fluid

Direction of flow

The Bernoulli Effect

Hydrodynamic Focusing

Sheath fluid

Particles move to low pressure area

Laminar Coaxial Flow

Velocity Gradient

Viscous drag along walls.

Lower pressure

Fluidics

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

Injector

Tip

The flow cell

Cells in a single file

Sheath

fluid

Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.

Light source

Stationary laser lightSourcesArgon laserKrypton ionHelium/neonDiameter of beam-650m2 beam focusing lensesHorizontalhorizontal axis - resolutionHorizontal axis - sensitivityVertical

Light sensing

Observation/interrogation regionSpot where moving cell intercepts the stationary laser lightTwo events take placeLight scatteringEmission of fluorescent light

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.

Scattered light

Occurs when light is deflected off the cellsRelated to Intrinsic property of the cellDetected in two different directionsAlong the axis of the beam - FSAt right angles - SS/90scatter

Forward scatter

Along the axis of beamLight scattered b/w .5-1 &10-20 from axis of beamProportional to the size of the cellBlocker/obscuration barTo stop beam at 0To assure only FS is collectedOther factors affectingRefractive index of cellAbsorptive properties

Forward scatter

FALS Detector

Laser

Side scatter

90 scatter perpendicular to the axisLight reflected from internal structuresCorrelates with granularity of the cell3 major leukocyte populations in 2 parameter histogramLymphocytes,monocytes,granulocytes

Side (90o) scatter

FALS Detector

90LS Detector

Laser

Electronic gating

Gate -Electronically framed region/window drawn around the desired cell clusterShape of gated area varies-RectanglePolymorphousunrestricted

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)

CD14 PE (log)

Forward Scatter (linear)

Side Scatter (linear)

Fluorescent light

FluorescenceCertain dyes absorb laser light & emit light at longer wavelengthargon absorbs at 350nm & emits at 488nmPick up lenses/spatial filter assembly Fluorescent light collected at 90 angles to laser beam

Filters

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PMT

PMT

PMT

PMT

Dichroic

Filters

Bandpass

Filters

Laser

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2

3

4

Flow cell

Flow cytometer optics

Fluorochromes

PrerequisitesLight absorption spectrum should match the wavelength of emitted light(488nm)High extinction coefficientHigh quantum yield

Fluorochromes

3 group of dyesLMW organic dyesFluorescein isothiocyanate(FITC)Biological pigmentsPhycoerythrinPeridinin chlorophyll protein(PerCP)Tandem dye systemsCyChrome

Signal processing

Sensors convert photons to electrical impulsesImpulses photons fluorochrome mol.Processing in 2 waysPeak-sense-hold (process brightest signal)Integrated signal (process all signals)

Fluorescence detectors

Laser

Fluorescence

FALS Detector

Fluorescence detector

(PMT3, PMT4 etc.)

Frequency

Data presentation

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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

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

Fluorescent activated cell sorting

SortingPhysically separate the cells based on differences of any measurable parameterComponentsDroplet generatorDroplet charging & deflection systemCollection componentElectronic circuit

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 cells

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or by applying an acoustic pulse to the stream to divert the cell into the tube.

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Hydrodynamic focusing

takes place within a flow cell.

Electrostatic Sorting : Stream-in-air

Laser interrogation and signal processing followed by sort decision : white sort right, blue sort left, green or red no sort.

Electronic delay until cell reaches break

off point. Then the stream is charged :

+ if white - if blue.

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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.

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Charged droplets deflect by electrostatic field

from plates held at high voltage (+/- 3000 volts).

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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 and free calcium, apoptosis, necrosis, pH, drug kinetics, etc., etc., etc.

Applications of Flow Cytometry.

Red cell analysis

Detection of red cell-bound Igpatients with positive DAT(quantification of IgG coating)Patients with negative DAT(detection of bound IgG)IgG subclass determinationSubpopulation of IgG sensitized red cells-sickle cell disease,red cell aging

Red cell analysis

Detecting cell bound Ig other than IgG- IgM,IgADetection & quantification of red cell antigenscommon blood group antigens-ABO, Rh, Kell, Kidduncommon blood group antigens- Kn /McC , Dr(a)cells,Cr systemRBC antigens during erythroid development-max expression at blast stage(eg.MN system)cell aging accompanied by in ABH antigen

Red cell analysis

Detection & quantitation of red cell populations

0.125%minor cell population is detectableTransfused red cellscan detect antigenically dissimilar red cells following small volume transfusions (~10 ml)determination of red cell survival after transfusiondetermination of autologous red cells in multiply transfused patient(reticulocytes separation)

Red cell analysis

ChimerismGenetically &artificial chimerismAny hematopoiesis from the recipient is considered mixed chimerismChimeras also demonstrate immune toleranceGenetically gp O person with implanted A cells does not produce antiAMixed chimeras m/b associated with a lower frequency of GVHD

Red cell analysis

Fetomaternal hemorrhageAccurately quantitate FMHUsing labeled IgG or antiHbFSensitivity equal to Kleihauer-Betke techniqueNot routinely done

Analysis of GPI-linked anchor proteins

PNHacquired clonal disorderRed cells unusually susceptible to lysis by complementSomatic mutation in PIG-A gene on X-ch essential for normal synthesis of GPI anchor proteins(CD55,DAF;CD59,MIRL)Chimeric cells ( normal moderate -extreme sensitive )

Analysis of GPI-linked proteins

CD59 inhibits the formation of the terminal complex of complementPNH III (complete deficiency)PNH I(partial deficiency)

Red cells analyzed with fluorescein- labeled antibody specific for GPI-anchor proteins-CD55,CD59,LFA-3

Presence of a population of >1 GPI-linked protein is diagnostic of PNH

Analysis of GPI-linked proteins

Mutation is identified in neutrophilsGPI-linked proteins suitable for analysis include CD16,CD24,CD55,CD59 AND CD67Analysis of neutrophils more difficultMore sensitive methodFlow cytometry has replaced Ham test as primary method for diagnosisHeavily transfused patientFollowing BMT

FLOW CYTOMETRIC DIAGNOSIS

Red cell analysis

Variant red cellsD/t mutation or recombination eventSurvivors of HiroshimaBloom syndromeAtaxia telangiectasiaCancer chemotherapyMcLeod syndrome

Platelet analysis

Technically more difficultAggregationEnsure single cell populationPlatelet fragments& microparticlesLess fluorescence b/c of small sizeIn-vitro activation

Platelet antigens

Readily used for platelet phenotypingPhenotype HPA-1a of mother, father and babyHeterogeneity of various RBC & platelet antigens on plateletsDifferentiate b/w hetero & homozygous state for HPA-1a antigen (MESF)Suitable for antenatal screening for NAIT

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 surfaceGPIV (CD36)expression of Nak platelet antigen plays a role in P. falciparum infected RBC binding to endothelial cells

Platelet antigens

Semiquantitative assay to assess the amount of bound antibody with subsequent estimation of antigens /cellUseful in Glanzmanns thrombocytopeniaBernard Soulier syndrome

Platelet function

Release, adhesion and aggregationActivated platelets exhibit alterations in expression of GPIb,GPIIb/IIIa expression of platelet activation markersCD62(P-selectin)Microparticle generationLysosomal protein CD63FibrinogenThrombospondinMultimerin

Platelet activation

Measure activated Vs nonactivated cellsCardiac surgeryThrombosisAtherosclerosis

Assessing platelet concentrates

In various conditions of preparation & storageCD62 with storageCD 62 may serve as QC measureLoss of GPIb /IX from pl surfaceNo filtration enhanced activationPlatelet activation in normal donors undergoing apheresis,persisting for up to 48hrs.Measuring intracellular CaChanges in actin &myosin

Platelet alloantibodies

Testing multitransfused alloimmunized patients HPA antibodies(15%)HLA Vs HPA antibodiesHLA antibodies(85%)HPA antibodies detection in NAITPlatelet crossmatching

Platelet autoantibodies

Autoimmune thrombocytopeniaTo measure platelet associated IgPAIgGPAIgMPAIgAEven when thrombocytopenia is severe(

Reticulated platelets

Thiazole orange used for reticulated plBinds to nucleic acid esp. RNAMeasure of platelet overturnDistinguish b/ w pl production &destructionMeasure early detection of pl recovery from CT induced thrombocytopenia

Hematopoietic progenitor cells

Quantification of CD34+ cells in peripheral blood or bone marrowTotal CD 34+ cells=

CD34+ cells WBC X Vol. of product

Total nucleated cells

Light scattering also helps to differentiateLow SS &FS

Immunophenotyping in HIV

CD3+ CD4+ T cells enumerationBaseline evaluationStaging of diseaseTo monitor progressionTo determine likelihood of opportunistic infectionTo make therapeutic decisions as surrogate marker in clinical trials

Detection of viral antigens

Measurement of viral contentApoptosis of CD8+ cells by blood born viruses b/c of immune suppressionHIV,CMV,E-B virus,Varicella zoster,HTLV

Leukocyte analysis

Leucoreduction in leukodepleted blood productsAccurately measure 0.1 WBC/ LPreferential depletion of WBC subsetsHLA class II bearing dendritic cells

Leukocyte analysis

Leukocyte antigensDetection of white cell antigensHLA-B27 phenotypingQuantitative analysis of HLA class 1 antigensDetermination of CD4+ lymphocyte levelsMeasurement of CD4+subsets

Leukocytes analysis

Leukocyte function Neutrophils activation in SLEUpregulation of CD11a density on CD4+/CD8+ in IMNeutrophil respiratory burstMeasuring cellular glutathione content in AINImmune competence in SCA

Leukocyte antibodies

antiHLA antibodies in FNHTRAntineutrophil antibodies (GIFT)Antilymphocyte antibodies (LIFT)Transfusion related acute lung injuryNeutrophil associated antibodies/complementDetection of antiphospholipid antibodiesDetection of ANCA

Histocompatibilty testing

Pre-allograft transplant crossmatchingCrossmatching b/w donor lymphocytes & recipients serumMarked reduction in hyperacute rejectionImproved graft survivalDetects low levels of anti donor antibodiesIdentifies high risk patientsConsidered definitive crossmatch technique

Histocompatibility testing

Monitoring ALG/ATG therapy to prevent allograft rejectionDetecting presence of anti CD3CD3 Modulation on T cellsCD2+ or CD3+ T cells determination

Apoptosis

Detection of abnormally activated cell populationsGeneric marker for viral infection

Advantages of flow cytometry

Rapid assessment of large no. of cellsMultiparameter analysisHigh accuracy & reproducibilityObjective analysisAbility to analyze many samples quicklyCapable of data reductionPermanent data storageAbility to reanalyze dataRequires relatively small sample

COULTER EPICS XL and XL-MCL

BD FACS Count

Flow cytometry

flow

cells move in single file

cytometry

measurement of numerous cell properties

two types

sorters

separateone particular

cell type

research purposes

analysers

cell analysis

clinical use

absorption filters

absorbs unwanted light

5 types

band

long, short

dichroic,notch

interference filters

reflects unwanted light

types of filters

linear form

output proportional to input

quantification of DNA,RNA

LS of size&granularity

log form

output proportional to log of inputType

immunophenotyping