Flow cytometry,

Applications in TM
Rashmi Tondon

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Introduction
F lo w c y t o m e t r y
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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
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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 by
Coons & 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 DNA
content

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Types of flow cytometer

tw o ty p e s
s o rte rs
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re s e a rc h p u rp o s e s

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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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Flow cell fluidics

Injector
Tip

Fluorescence
Fluorescencesignals
signals
Focused
Focusedlaser
laserbeam
beam
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Sheath
fluid

Hydrodynamic Focusing
Sheath fluid

Laminar Coaxial Flow

The Bernoulli Effect

Direction of flow
Lower pressure
Velocity Gradient
Viscous drag along walls.
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Particles move to low pressure area

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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

Sheath
fluid

The
Theflow
flowcell
cell
Cells
file
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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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High angle scatter :

Reflection & refraction.
Cell structure.
Low angle scatter :
Diffraction. Cell size.
Laser
Light

Direct beam stop.

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 directions
Along 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 of
beam
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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Forward scatter

Laser
FALS Detector

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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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Side (90o) scatter

Laser
FALS Detector

90LS

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Detector
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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.

CD14 PE (log)

Side Scatter (linear)

Data collected on 10,000 cells.

CD45 FITC (log)

Forward Scatter (linear)

R1= monocytes (CD14+ve)

R2=lymphocytes (CD45> monocytes)
R3=granulocytes (CD45< monocytes)

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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
ty p e s o f filte r s
a b s o r p tio n filte r s
a b s o r b s u n w a n te d lig h t

in te r fe r e n c e filte r s
r e fle c ts u n w a n te d lig h t
5 ty p e s
band
lo n g , s h o r t
d ic h r o ic ,n o tc h

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Flow cytometer optics

PMT
4

PMT

Dichroic
Filters

Flow cell

PMT
2

PMT

Bandpass
Filters

Laser

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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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Fluorescence detectors
Laser

Frequency

FALS Detector

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Fluorescence detector
(PMT3, PMT4 etc.)
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Fluorescence

Data presentation

lin e a r f o r m
o u t p u t p r o p o r t io n a l t o in p u t
q u a n t if ic a t io n o f D N A , R N A
L S o f s iz e & g r a n u la r it y

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Sample : peripheral blood after red cell lysis.

Data collected on 10,000 cells.

Dot

Density

Contour

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

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Isometric Displays
Sample : peripheral blood after red cells lysis.
Data collected on 10,000
Events
High

Events

CD14 PE

Low
CD 45 FITC
Scatter
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Fluorescence
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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
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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.
Laser beam interrogates cells
When a sort decision Green cell has been
made it is diverted into a catcher tube
either by moving the tube into the stream:

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Electrostatic Sorting : Stream-in-air

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Hydrodynamic focusing in a nozzle

vibrated by a transducer produces a
stream breaking into droplets.
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
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Charged droplets deflect by electrostatic field

from plates held at high voltage (+/- 3000 volts).
Various collection devices can be attached :
tubes, slides, multi-well plates.
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Applications of Flow Cytometry.

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.
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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 of bound
IgG)
IgG subclass determination
Subpopulation of IgG sensitized red cellssickle 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 following
small 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 ( normal moderate -extreme
sensitive )
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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 proteinsCD55,CD59,LFA-3
Presence 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 analysis
include CD16,CD24,CD55,CD59 AND CD67
Analysis of neutrophils more difficult
More sensitive method

Flow cytometry has replaced Ham test as

primary method for diagnosis
Heavily transfused patient
Following BMT
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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 & platelet
antigens 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. falciparum
infected 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
Bernard Soulier syndrome

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Platelet function
Release, adhesion and aggregation
Activated platelets exhibit alterations in
expression 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

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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(<5x109)
Semiquantitate the amount of bound antibody

Drug dependent antibodies

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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

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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 associated
antibodies/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
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COULTER EPICS XL and

XL-MCL

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BD FACS Count

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