18-11-2014

Flowcytometry

Moderator - Dr. S.S.Ronad

Presenter - Dr.Vatsala Kishore
 Introduction
History
Principle
Components
Cell Sample Preparation
Fluorescein Activated Cell Sorting
Measurable Parameters
Applications
Quality control
Advantages
Drawbacks
Summary
 Introduction
Measurement of physical or chemical
characteristics of cells or, in general,
biological particles is known as
cytometry
When such measurements are
performed while the cells or other
biological material pass in a single file
through the measuring apparatus in a
fluid stream, the process is known as
flow cytometry
 Flow cytometry has allowed detailed
insight into the cellular biology of
normal, reactive and neoplastic
tissues objectively
 History
In 1947, Gucker developed Flow
Cytometer for detection of bacteria in
aerosols
The first fluorescence-based flow
cytometry device was developed in
1968 by Wolfgang Gohde
It was introduced to the clinical
laboratory in the 1980s. Original name
of fluorescence-based flow cytometry
technology- "pulse cytophotometry"
Eight years later(1968) - name "flow
cytometry"
 Principle
Analysis of cellular characteristics
as the cells pass singly in a fluid
suspension through a beam of light
Scattered light and fluorescence
intensity measured by set of
photodiodes
Data collected by these
photodiodes are transmitted to the
computer for further processing and
data output
 Components
1. Fluid System
2. Optical System
3. Signal Detector System
4. Data Management System
 Cell sample
preparation
Blood cells are prime candidates
Whole blood and buffy coat
samples are subjected to NH4Cl2
buffer for lysis of RBC
Flow cytometry used for
immunophenotyping of a variety
of specimens, includes whole
blood, bone marrow, serous cavity
fluids, cerebrospinal fluid, urine,
and solid tissues
The tissue sample broken up into single cells in
test tube

placed into the flow cytometer

liquid containing the cells drawn up from the

test tube
 Fluid System
Responsible for movement of
sample through flow chamber from
sample separation to sample waste
disposal

Follows the principle of hydro-

dynamic focusing
Suspension of cells funneled into
file stream of cells.
Cells injected into rapidly moving
column or sheath of fluid results in
alignment of cells into a single array
Typically moving
fluid sheath is at a
velocity of 10,000
times faster than the
injected cells (10
m/sec Vs. 1 mm/sec)

Single file stream of

cells in turn is
directed past a
precisely focused
light stream
 Optical
System
Single file stream of cells directed
past a precisely focused light
stream.
Presence of cells in the laser path
scatters light in all directions.
Laser illuminates cells uniformly
and intensely, but for a short
period.
 Forward Angle Light Scatter

Laser

FALS Sensor

2o to 18o relative to the laser

path
Co-related with cell size
 90 Degree Light Scatter

Laser

FALS Sensor

90LS Sensor

Correlates the internal cellular structure and cell shape

 Signal Detector
Systems
Condensed light emissions detected by
individual photo diodes and photo
multiplier tubes
Photo diodes change light photons into
electronic signals which are proportional
to the amount of light photons detected
Photo multiplier tubes are useful in
collection and amplification of weaker
side scatter light and fluorescent
emissions. PMTs direct their electronic
Use of dichrome reflection and narrow
band pass filters- light of specific
wavelength can be directed to PMT,
allowing several fluorescents tags to be
detected and distinguished.
 Data Management
Systems
Peak voltages from each analyzed cell
sent to data management system
where the electronic signals are
amplified, digitalized and displayed or
sorted by computer.
Routine FCM data can be collected and
expressed in several ways-
Histogram for single color
fluorescence data
Dot plot for dual colour immuno
Sample plot of various T cell populations
isolated from flow cytometry - the cell
population shown in the scattergram can be
broken up into 4 subpopulations: cells that are
CD45RA+ but CCR7- (green), CD45RA+CCR7+
(red), CD45RA-CCR7- (blue), and CD45RA-
 Fluorescence

Imediate emission of light from a molecule

(fluorochrome) that has absorbed light energy
(excitation light)
Because only the part of absorbed energy is
reemitted, the wavelength of the fluorescence
light is greater than the excitation light
 Fluorochromes are the fluorescent dyes
that either bind to specific cellular
components or are linked to antibodies,
that in turn are directed to specific cellular
constituents like surface antigensApplication
Fluorochrome Excitation Emission
WL WL
FITC(flourescein 490 520 Immunofluoresc
isothiocyanate) ence
Phycoerythrin 488 575 Immunofluoresc
ence
Phycocyanin 568 640 Immunofluoresc
ence
Texas red 568 620 Immunofluoresc
ence
Acridine orange 488 650 DNA, RNA
content
 FACS
In multicellular organisms, all the cells
are identical in their DNA but the
proteins vary tremendously. Therefore, it
would be very useful to separate cells
that are phenotypically different from
each other and to know how many cells
expressed proteins of interest, and how
much of this protein they expressed.
Fluorescence Activated Cell Sorting
(FACS) is a method that can accomplish
all these goals.
 To separate a subpopulation of cells, cell of
interest is tagged with an antibody linked to a
fluorescent dye. The antibody is bound to a
protein that is uniquely expressed in the cells
to be separated.
The laser light excites the dye which emits a
color of light that is detected by the
photomultiplier tube, or light detector. By
collecting the information from the light (scatter
and fluorescence) a computer can determine
which cells are to be separated and collected
 The final step is sorting the cells which is
accomplished by electrical charge. The
computer determines how the cells will be
sorted before the drop forms at the end of
the stream. As the drop forms, an electrical
charge is applied to the stream and the
newly formed drop will form with a charge.
This charged drop is then deflected left or
right by charged electrodes and into waiting
sample tubes. Drops that contain no cells are
sent into the waste tube. The end result is
three tubes with pure subpopulations of
cells. The number of cells is each tube is
 Quantifying FACS Data

This graph shows the number of cells (Y-axis) and the level of
fluorescence emitted (X-axis) by the labeled cells. Many different
colors can be plotted on this graph
 Compensation most fluorochromes
have overlapping emission spectra
So determination of the fluorescence
due to a specific fluorochrome in a
single detector requires subtraction of
the portion of the signals contributed
by each of the other fluorochrome in
the sample
 Filters
The practical use of fluorescence
involves the selective separation or
elimination of incident and
fluorescence light
The light detectors in most cytometers
are not specific for any one type of
light
So optical barriers (filters) and mirrors
are used for this purpose
Most of these filters are called
secondary filters because they are
 Barrier filters block light above or below
a certain unique value and will allow the
other wavelengths to pass through
Band pass filters allow light between the
stated value to pass through and light with
longer or shorter wavelength is absorbed
Dichroic mirrors used to split the
spectrum of light, directing them in two
different directions Light is not absorbed
or blocked by this filters
 Gating data
To display data from a single parameter-
univariate histogram .
Correlation between two parameters -
bivariate histogram, or cytogram, in the form
of a dot, contour or density plot
Impossible to visualise the correlations in
multiparameter data
Adopted a different strategy; called regions
and gates.
Regions are shapes that are drawn around a
population of interest on a one- or two-
parameter plot.
When a region is used to limit the cells that
 Light Scatter Gating
Side Scatter Projection

1000
Neutrophils

Forward Scatter Projection

Scale
800
1000
Forward Scatter

200
100
600

50
40
30 Monocytes
400

20
15
8
Lymphocytes
0 200

0 200 400 600 800 1000

90 Degree Scatter

As cells pass through the laser beam the light is changed based on the size
(forward scatter) and granularity (side scatter) of each cell. In this manner
the large and very granular neutrophils (polys) can readily be differentiated
from the smaller less granular lymphocytes in the blood.
 Coulter
cton Dickinson FACS Epics

 Measurable parameters
Volume andmorphological
complexity of cells
Cellpigmentssuch aschlorophyll
TotalDNAcontent
TotalRNAcontent
Chromosome analysisand sorting
Proteinexpression and localization
Protein modifications, phospho-
proteins
Transgenic productsin vivo
Cell surfaceantigens(CD markers)
Intracellularantigens (variouscytokines,
secondary mediators, etc.)
Nuclearantigens
Enzymatic activity
ph, intracellularionized
calcium,magnesium,membrane potential
Membrane fluidity
Apoptosis
Cellviability
Monitoring electropermeabilization of cells
Oxidative burst
Characterisingmultidrug resistance(MDR) in
cancer cells
Applications
Flow Cyto metric DNA analysis
DNA Ploidy analysis is useful for
identification of tumor cells.
Commonly used fluorochromes
e.g. Ethidium Bromide, Propidium
bromide binds nucleic acid
specifically
FCM quantitates the amount of
DNA present in each cell and
displays it as histogram
 Normal Cell Cycle
M G0
G2 DNA Analysis
G1
G0G1
s
C
o
u
n
t
s G2 M
0 200 400 600 800 1000
2N 4N
DNA content
DNA Index mode of DNA content of
the population of cells studied along
with mode of DNA content of
corresponding non-neoplastic normal
cells.
Normal cells are diploid 2N, tumor cells
tend to be aneuploidy
hypoploid DI < 1
hyperploid DI > 1 and
occasionally tetraploid DI=2
An abnormal DNA index is highly
specific for malignancy, particularly
in solid tumors.
Computer software for flow
cytometric cell cycle analysis has
been developed based on
assumption that the percentage of
cells synthesizing DNA (s-phase) is a
direct reflection of the tumor
proliferation.
 Cell Surface Markers
Multi parameter FCM , a standard
technique for the analysis of surface
antigens and proliferation markers in
lymphocytes and related cells
i)Lymphocyte immunophenotyping
Detection of CD4 in patients with
AIDS.
This clinical application led to
FCM entry into clinical
laboratory.
Currently it is used in the diagnosis,
staging and therapeutic intervention
AIDS &ARC, Transplantation
Diagnosis& monitoring-CD4/CD8 ratio &
absolute CD4 count
BM transplant-prediction of successful
graft by Thiazole orange stains
reticulocytes as early indicator of
functional marrow
Selection of CD34 rich peripheral blood
T cell depleted donor marrow-decreased in
GVHD
Renal/cardiac-graft rejection [IL2+ CD3+T
lympho]Vs CMV infection[CD4/CD8 ratio]
 CD4/CD8 Quadstats
1
2

100 45%
Log PE Fluorescence (CD4)
10
1 2%

27% 3 26% 4
.1

.1 1 10 100 1000
Log FITC Fluorescence (CD8)
ii)Leukemia / Lymphoma Immuno
phenotyping (IPT)
Lymphocytic leukemia and lymphomas
classified as B, T and pre-B cell
processes
Aids in the diagnosis of certain myeloid
leukemias in particular AML with
minimal differentiation (AML M0),
acute erythroblastic leukemia (AML
M6) and acute megakaryoblastic
leukemia (AML-M7).
In patient after bone-marrow
transplantation,can verify the lack of
malignant process or identify an early
relapse of malignancy.
 Fluorochrome tagged antibodies against IgM and CD43 along with
forward and side scatter were used here to determine the size and
distribution of early precursor B-cells in the marrow (four
parameter analysis).
Reticulocyte Enumeration
Used to evaluate the bone marrow
erythropoetic activity
Evaluation of EA is useful in
Differentiation of anemias
Monitoring the patient response to
therapy
Assessment of EA after bone
marrow transplantation
A fluorescent dye (e.g. - Thiazide orange)
is used to stain the residual RNA
Analyzes approximately 10,000 cells to
determine the reticulocyte percentage,
as well as absolute reticulocyte count
Newer instrumentation allows the
determination of reticulocyte
maturity
Reticulocytes with high
fluorescence ratio represent the
earliest cells with most RNA content
and those with low fluorescence
ratio represent the most mature
erythrocytes
 Reticulocyte Analysis

150
150

112
112

RMI = 34 RMI = 0

75
75

Count
Count

R4 R3 R2 R1 R4 R3 R2 R1

37
37

0
0

.1 1 10 100 1000 .1 1 10 100 1000

log Thiazole Orange log Thiazole Orange
Cytokerati
ns
Technology has been developed to
double label cells for DNA and keratin
which allows the DNA contents to be
analyzed in epithelial cells alone
This approach improving the
accuracy of tumor has been applied
successfully to endometrial, breast,
bladder, colonic and head & neck CA
Proliferation Markers
Multiparameter FCM assays that
cells labeled with
bromodeoxyuridine (BrdUrd) and
DNAare very sensitive and
comparable with tritiated
thymidine assay for the study of
proliferative activity
Another method of assessing cell
proliferation consists of
simultaneous FCM measurement of
proliferating cell nuclear antigen
Oncoproteins and Growth
Factors

Studied still at research level

Attempts are being made to assay
the nucleus associated product of
various oncogenes simultaneously
with DNA
Intracellular Calcium Flux
Cells interact with each other and
their environment through signal
transduction pathways
When these pathways are activated,
membrane-bound calcium ion channels
pump calcium into the cell and rapidly
increase the intracellular calcium
concentration
The higher calcium levels provide
energy to the cell to respond to the
external stimuli
The cytometer can monitor the flux of
Calcium Flux - Indo-1

1000
800
RATIO [short/long]
600
400
200

Stimulation
0

0 36 72 108 144 180

Time (Seconds)

Ion fluxes-Ca flux in triggering

target cells
 Oxidative Burst
DCF-DA
CGD-defect in
oxidative metab
of neutrophils,
lack of H2O2 DCF-DA
production
DCF-
DCF H 2O 2
DA[ dichloro
fluorescien
diacetate]
enters live cells
DCF
& H2O2 acts to
form insoluble
dihlorofluorescein
[fluorogenic]
Cytotoxic and Multidrug Resistance

FCM extensively used in the study of-

i) Cytokinetic disturbances in cells
incubated in vitro with cytotoxic
drugs
ii) Cell cycle phase sensitivity to
cytotoxic agents
iii) Drug resistance in vitro
iv) Drug resistant cells and their
isolation
v)Intensity of cellular fluorescence
can be easily measured by FCM as
Apoptosis
Apoptotic cells can be recognised by a
characteristic pattern of morphological,
biochemical and molecular changes
BIOCHEMICAL CHANGES
Free calcium ion rise
bcl2/BAX interaction
Cell dehydration
Loss of mitochondrial
membrane potential
Phosphatidylserine
externalisation
Laminin B proteolysis
 Methods
Based on the measurement of light scatter
Detection of changes in the plasma
membrane
Analysis of cell organelles
Sensitivity of DNA to denaturation.
In all methods for the detection of
apoptosis, a positive control is useful ie
cells in which apoptosis has been induced
by, for example, fas induction, treatment
with the topoisomerase I inhibitor etc
 Live cell measurement by
Fluorescein Diacetate
One of the more elegant uses of a
flow cytometer is to assess enzyme
activity by loading cells with a non-
fluorescent substrate which may be
acted on by an enzyme to yield a
fluorescent product.
Fluorescein diacetate will enter
live cells and be cleaved by
intracellular esterases to yield a
fluorescent product (fluorescein) - in
this way live cells will fluoresce green
 Tracking
cellsin vivo
Fluorescent dyes are a safe and convenient
alternative to radioactive labels for
following the migration of cellsin vivo,
particularly lymphocytes
The labelled cells are injected into an
animal and are then subsequently collected
(for example, by cannulation of a lymphatic
duct) and counted in the flow cytometer
If required, the harvested cells can be
additionally labelled with antibodies so that
different populations of cells may be
distinguished
 More
Applications
Thromboembolytic disorder-fresh whole
blood to assess platelet activation,S12ab
against alpha granule membrane protein
GMP-140,PAC1ab against activated platelet
membrane glycoprotein IIb-IIIa complex
Abnormal hematopoeisis- retic count,RMI
[retic maturity index]-based on qty of RNA
in the cells
Autoimmunity-ab assay-Tx induced
thrombocytopenia,autoimmune
thrombocytopenia,neonatal alloimmune
thrombocytopenia
 Biological response modifier therapy:
IL-2-activates antitumor cytolytic
abilities-NK cells Proliferate-
measured by FCM
Estrogen receptors
Chromosome karyotyping
Chromosome Analysis
(Bivariate Flow Karyotyping)
chromosome 1

chromosome 2
 Flow FISH

DNA is denatured via heat and formamide treatment, probe is allowed to

hybridize, excess probe is washed away, reading is taken in flow
cytometer
Binding and endocytosis
of ligands
The surfaces of cells are often
labelled with fluorescent tagged
antibodies.
If analysis is performed in the
presence of different concentrations
of ligand, the surface density of the
receptors and an affinity constant for
binding can be calculated
 Monitoring electropermeabilisation

Introduction of foreign DNA into cells.

The cells are permeabilised at a low
temperature by a high voltage pulse
and are then warmed to allow their
membranes to reseal
FCM establishes whether holes have
been punched in the plasma
membrane and whether they have
successfully resealed
Propridium iodide added immediately
after electropermeabilisation and
cells with ruptured membrane take
up the dye and their nuclei fluoresce
red. Then cells are incubated in
warm medium to allow their
membranes to reseal, fluorescein
diacetate is added so that the cells
with intact membranes fluoresce
green. The cells are excited at 488
 Microbead technology
A biomolecule can be attached to a bead and its
interaction with other molecules studied; for
example, the binding of an antigen to an antibody
A particularly powerful application is the use of
multiplexed arrays. Sets of beads have been
produced, each with a different fluorescent
intensity and each carrying a different antibody.
After incubation with a patient's serum, beads
with antigens bound to their surface can be
detected with fluorescein-labelled antibodies
A wide range of molecules, such as hormones,
cardiac markers, therapeutic drugs, drugs of
abuse and blood-borne viruses, can be measured
Identification of stem cells using
'side population' (SP) analysis
In human, rodent and non-human primates, stem
and early progenitor cells show high expression of
an ABC(ATP binding cassette) membrane pump.
This pump will act on a variety of dyes, including
the DNA binding dye.
If cells are incubated with the dye, higher
concentrations of dye accumulate in maturer
cells compared to the stem and early progenitor
cells
Cells are excited by UV and the blue versus red
fluorescence displayed. The stem cells are
found in a population which has lower
fluorescence than the main population
 Applications in
microbiology
Analysis of malaria infected blood
Effects of Ab on bacteria
Parasite studies
Kinetics of virus absorption
EBV/CMV studies
Herpes simplex studies
immunology
 Flow Cytometry Work in Marine Planktology

To estimate cell numbers of pico- and

nanophytoplankton, i.e Algal cells of less than
20 m in size,
Distribution and composition of pico- and
nanophytoplankton populations
On board application of flow cytometry allowed
distributional studies on prochlorophytes,
coccoid cyanobacteria (Synechococcus) and
small eukaryotic algae
FUTURE APPLICATIONS

Research investigation into use of FCM

in
Chromosome analysis
Platelet assays
Cell function assays
Immune complex quantification
 Why use flow cytometry
(FCM)?
FCM is FAST, analyzing 500 to 5000
suspended particles (usually cells) per
second.
FCM is MULTIPARAMETRIC, allowing for
the measurement of up to six structural and
functional phenomenon at the same time.
FCM is not a bulk assay; measurements are
made on INDIVIDUAL CELLS, one at a
time.
Finally, FCM allows the SORTING of
heterogeneous populations
 Quality control
Monitoring instrument setup &
performance
Optimizing sample prep & reagents
Standardising controls & data
interpretation
Participation in inter lab profeciency
testing surveys- triannual sample
provided by college of american
pathologists
 Advantages
It can provide objective measurement of
representative sample of cells from large
volume of tissue, rapidly and easily than
histology and image analysis.
Provides statistical accuracy,
reproducibility and sensitivity.
Compared to IHC, which gives useful
qualitative information but is usually
subjective, only moderately sensitive and
difficult to standardize, FCM can overcome
some of these problems and is
complementary to classical techniques.
 As FCM records parameters of each
individual cell of a sample, small sub-
population of cells can be easily
identified.
Yield of tumor cells enriched by
sorting cells for further bio-chemical
analysis.
Allows simultaneous measurement of
several constituents on a cell to cell
basis by using multi-color staining
 Drawbacks
Requires a suspension of single cells or
other particles, with minimum clumps
and debris.The tissue architecture and
any information about the spatial
relationship between different cells are
lost when single cells or nuclei are
prepared
Hardware is very expensive and not
widely available.
Paraffin wax embedded material for
FCM- only bare nuclei can be analyzed.
 Summary
FCM simultaneously measures and
analyzes multiple physical and
chemical characteristics of single cell
as it flows in a fluid stream through the
light
With this technique any suspended
particle ranging in size from 0.2 to
150m can be analyzed
It is composed of the fluid system,
optical system, and electronic system
 Analyzed parameters include size,
granularity, structure complexity, surface
receptors, cytokines, enzymes and
nucleic acid contents
Multiple structural and functional
parameters can be quantified
simultaneously on a single-particle basis,
whereas up to thousands of biological
particles per second may be examined.
FCM is increasingly used for basic,
clinical, biotechnological, and
environmental studies of biochemical
relevance.
 Smaller, less expensive instruments
and an increasing number of
clinically useful antibodies are
creating more opportunities for
routine clinical laboratories to use
flow cytometry in the diagnosis and
management of disease
 References
Richard McPherson, Matthew Pincus ,Henrys Clinical
Diagnosis and Management by Laboratory Methods,
22nd edition,2011
Michael G. Ormerod, Flow cytometry-a basic
introduction .
http://flowbook.denovosoftware.com/FlowBook
Carl A.Burtis,Edward R ashwood,David E Bruns.Tietz
Textbook of clinical chemistry and molecular
diagnostics,5th edition,2012
Alice L. Givan. Flow Cytometry: First Principles, 2 nd
Edition ,2001
N. Baumgarth, M. Roederer. A practical approach to
multicolor flow cytometry for immunophenotyping,
Journal of Immunological Methods 243 (2000) 7797
Jose-Enrique OConnor etal. Critical Review The
Relevance of Flow Cytometry for Biochemical