This document provides an overview of flow cytometry. It begins by defining flow cytometry as a technique for quantitative single cell analysis that counts, examines, and sorts cells based on optical properties like light scattering and fluorescence. It then describes the basic principles, components, and working of a flow cytometer. Key components include lasers, optical filters, and detectors. Cells in suspension pass through the laser one by one, with signals detected by photodiodes and photomultiplier tubes. Applications discussed include cell sorting, apoptosis analysis using markers like Annexin V and PI, and cell cycle analysis using DNA binding dyes or BrdU incorporation. Clinical uses involve hematologic malignancy diagnosis, residual disease detection, and monitoring treatments.

1. Flow Cytometry
Pradeep Singh
M.Sc. Med. Biochemistry
HIMSR, JAMIA HAMDARD

2. Contents
1. Introduction
2. Basic Principles of Flow Cytometry
3. Working of Flow Cytometer
4. Applications of Flow Cytometry

3. 1. Introduction
• Flow cytometry is a technique of quantitative single cell analysis.
• This technique was first described by Wallace Coulter in the 1950s.
• The flow cytometer was developed in the 1970’s and applied to
automated cell counting.
• Flow cytometer count, examine and sort cells based on their optical
properties (Scattering and fluorescence).
• The present “state-of-the-art” flow cytometers are capable of
analyzing upto 13 parameters (forward scatter, side scatter, 11 colors
of immunofluorescence).

4. Components of flow cytometer:
1. Lasers
2. Dichroic mirrors
3. Filters
4. Detectors

5. 2. Basic Principle of Flow Cytometry
• Prepared single cell or particle
suspension is necessary for flow
cytometric analysis.
• The suspension of cells or particles
is aspirated into a channel
surrounded by a narrow fluid
system.
• They pass one at a time through a
focused laser beam.
• The light is either scattered or
absorbed when it strikes a cell.

6. • Light scattering is dependent on
the internal structure of the cell
and its size and shape.
• Absorbed light of the appropriate
wavelength may be re-emitted as
fluorescence. (The cell may have a
naturally fluorescent substance or
one or more fluorochrome-labelled
antibodies are attached to surface
or internal cell structures).

7. • Light and/or fluorescence scatter signals are detected by a series of
photodiodes and amplified.
• Optical filters are essential to block unwanted light and permit light of
the desired wavelength to reach the photodetector.
• Fluorescein isothiocynate (FITS), Texas red and phycoerythrin (PE) are
the most common fluorescent dyes used in the biomedical sciences.
• Large number of cells are analysed in a short period of time
(>1,000/sec).

8. Sample
Loading
Flow
Chamber
Light
source
Scattering/
Absorption
Light
Detectors
Computer

9. 3. Working of Flow Cytometer
• A flow cytometer is composed of three main systems:
1. Fluidics – Transport cells in a stream to the laser beam for interrogation.
2. Optics – Consist of lasers to illuminate the cells in the sample stream and
optical filters to direct the resulting light signals to the appropriate
detectors.
3. Electronics – Converts the detected light signals into electrical signals that
can be processed by computer.

10. 3.1 Fluidics System
• Flow cytometers use the
principle of hydrodynamic
focusing for presenting cells one
at a time to a light source.
• The fluidics system consist of a
central channel through which
the sample is injected, enclosed
by an outer sheath that contains
faster flowing fluid.

11. • As the sheath fluid moves, it creates a massive drag effect on the
narrowing central chamber. This alters the velocity of the central fluid.
• The velocity of the central fluid becomes parabolic i.e., greatest
velocity at the center and zero velocity at the wall..
• This effect creates a single stream of particles or cells and is called
hydrodynamic focusing.
• Under laminar flow conditions, the fluid in the central chamber will
not mix with sheath fluid.

12. 3.2 Optics
• Light scattering or fluorescence
emission provides information
about the cell’s properties.
• Light that is scattered by an object
is detected by different detectors.
• One detector is placed in line with
the beam to measure the forward
scatter (FSC) from the objects.
Detectors perpendicular to the
beam measure side scatter (SSC)
and fluorescence.

13. • Forward scatter is based on two
properties: size and refractive
index
• The FSC intensity roughly
equates to the particle’s size and
can also be used to distinguish
between cellular debris and
living cells.
• Dead cells have lower FSC and
higher SSC than living cells.

14. • Side scatter is based on the
granularity or internal
complexity.
• The more granular the cell, the
more side scatter light is
generated.

15. • Fluorescence measurements taken at different wavelength can
provide quantitative and qualitative data about fluorochrome-labelled
cell surface receptors or intracellular molecules such as DNA and
cytokines.
• When a fluorescent dye is conjugated to a monoclonal antibody, it
can be used to identify a particular cell type based on the individual
antigenic surface markers of the cell.
• The stating pattern of each subpopulation, combined with FSC and
SSC data, can be used to identify which cells are present in a sample
and to count their relative percentages.

16. Optical detectors
• Scattered and emitted light from cells are converted to electrical pulses by
optical detectors.
• Once a cell or particle passes through the laser light, the scattered and
fluorescence signals are diverted to the detectors.
• Detectors are either silicon photodiodes or photomultiplier tubes (PMTs).
• The photodiode is less sensitive to light signals than the PMTs and thus is
used to detect the stronger FSC.
• PMTs are used to detect the weaker signals generated by SSC and
fluorescence.

17. Optical
detectors
Photodiodes
Used to detect
FSC
Photomultiplier
tubes
Used to detect
SSC and
fluorescence

18. Optical
Detectors
Scattered
light detector
FSC and SSC
Fluorescence
light detector
FL-1, FL-2, FL-
3, FL-4

19. Filters
• All the signals are routed to their
detectors via a system of filters and
dichroic mirrors.
• Each PMT fluorescence detector is
placed behind a series of dichroic
mirrors and filters, so that it only
receives and detect light within a
particular range of wavelength.
• A particular color of light is split off
from the incoming mixture and
directed to the detectors by dichroic
mirrors.

20. Types of filters:
• There are three major type of
filters:
1. Longpass Filter: Transmits
wavelength of light equal to or
greater than the spectral band
of the filter.
2. Shortpass Filter: Transmits
wavelength of light equal to or
shorter than the spectral band
of the filter.
3. Bandpass Filter: Transmits
wavelength of light within a
specific range of wavelengths.
LP 500 SP 500
Longpass
480 500 520
Shortpass
480 500 520
Bandpass
480 520
460 500 540
BP 500

21. 3.3 Electronics (Signal Processing)
• Scattered and emitted light data can
be converted to electrical pulses by
optical detectors.
• Flow cytometry data may be
represented as histograms or dot
plots.

22. Histogram: A histogram quantifies the
intensity of a single parameter, be it
fluorescence or scattering (SSC or FSC).
Dot plot: A dot plot is a two parameter
representation of a sample’s properties.

23. Gating
• Gating – It is a procedure to selectively visualize the cells of interest
while eliminating results from unwanted cells and debris.
• For example, if one has a heterogeneous population of cells which
contain lymphocytes, monocytes and granulocytes and is only
interested in evaluating the fluorescence of the lymphocytes
subpopulation.
• In this situation one could define an analysis gate around the
lymphocyte population. The resulting display would reflect the
fluorescence properties of only lymphocytes.

24. • For example, if we want to analyse a sample of peripheral blood that had been stained with
fluorochromes to identify the CD4 and CD14 surface markers but we are only interested in
knowing the percentage of monocytes that contain CD4 and CD14 surface markers. We will
place a gate around the monocyte population of the FSC versus SSC scatter plot.

25. Applications of Flow Cytometry

26. 4.1 Cell Sorting (FCAS)
• A major application of flow
cytometry is the physical
separation of sub-population of
cells of interest from a
heterogeneous population.
• This process is known as cell
sorting of Fluorescence
Activated Cell Sorting (FACS)
New drop
Positive Charge
Negative Charge
No Charge

27. • Most commonly used cell sorting method is electrostatic deflection of droplets.
• In this method, the stream is focused in a vibrating nozzle and exits in a jet which
is broken into regularly spaced droplets.
• The cells of interest are charged electrically (positively or negatively). The
electrical charging actually occur at a precise moment called the ‘break-off point’.
• The cell of interest gets charged at the break-off point.
• When a charged droplet passes through a high voltage electrostatic field,
between the deflection plates, it is deflected and collected into the
corresponding collection tube.
• The deflection of the droplets is towards the oppositely charged plate, so that
this droplet is separated from uncharged and oppositely charged droplets.

28. 4.2 Apoptosis

29. The following features of the apoptotic cascade
can be observed using flow cytometry:
• Altered phospholipid composition in the plasma membrane
• Activation of caspases
• Chromation condensation
• DNA fragmentation
• Expression of proteins involved in apoptosis
• Changes in mitochondrial membrane potential
• Decrease cytosolic pH
• Altered membrane permeability

30. Detection of apoptotic cells based on changes
in forward scattering
• During apoptosis there is an initial increase in SSC (probably due to
the chromatin condensation) with a reduction in FSC (due to cell
shrinkage).
• Drawback: In many cases, the forward light scattering histograms of
apoptotic and live cells overlap and make it difficult to discriminate
apoptotic cells based solely on this parameter.

31. Detection of apoptotic cells based on Annexin
V binding
• During early apoptosis cell lose
symmetry, phosphatidylserine
on the outer leaflet of the
plasma membrane.
• Annexin V is a calcium-
dependent phospholipid-binding
protein that binds preferentially
to negatively-charged
phosphatidylserine.

32. • The assay involves incubating cells briefly in a solution containing
fluorochrome conjugated Annexin V (FITC – Annexin V) in a buffer
that facilitates its binding.
• Apoptotic cells can be detected easily by flow cytometry on the basis
of fluorescence due to increased binding of FITC-conjugated Annexin
V.
• Drawback: Unfortunately, it is not specific only for apoptosis because
whenever cell membrane integrity is disrupted (even non-ionic
detergents), cells may stain with Annexin V.

33. Detection of apoptotic cells based on PI
binding
• The intact plasma membrane of live cells have a tendency to exclude
cationic dyes such as propidium iodide (PI) and 7-amino-
actinomycin D (7-AMD).
• PI is a good staining method to distinguish apoptotic, necrotic and
normal live cells.
• Apoptotic cells show an uptake of PI that is much lower than that of
necrotic cells. It is therefore possible to distinguish live (PI-negative),
apoptotic (PI-dim) and necrotic (PI-bright).

34. • Thus, the combined use of cationic dyes (e.g. PI) with annexin V
allows the discrimination between:
Live cells = Annexin V negative/PI negative
Early apoptotic cells = Annexin V positive/PI negative
Late apoptotic = Annexin V positive/PI positive
Necrotic cells = Annexin V negative/PI positive

35. Detection of apoptotic cells based on DNA
Fragmentation
• The late stages of apoptosis are characterized by changes in nuclear
morphology, including DNA fragmentation, chromatin condensation,
degradation of nuclear envelope, nuclear blebbing and DNA strand
breaks.
• Cells undergoing apoptosis display an increase in nuclear chromatin
condensation. As the chromatin condenses, cell-permeable nucleic
acid stains become hyperfluorescent, thus enabling the identification
of apoptotic cells.

36. Assessment of mitochondrial membrane
potential and caspases level
• Assessment of mitochondrial membrane potential and caspases level
within the cell through flow cytometry is also used to analyse
apoptotic cells.
• Cells undergoing apoptosis often lose the electric potential that
normally exists across the inner mitochondrial membrane.
• A distinctive feature of early stages of apoptosis is the activation of
caspases enzymes. These enzymes can be labelled with fluorophore
which can easily be detected by flow cytometry.

37. 4.3 Cell Cycle Study
• One of the important application of flow cytometry is the
measurement of DNA content in cells.
• The duplication of the DNA occurs during the S-phase of cell cycle.
• There are two differnet methods to measure the DNA content:
1. The cells have to be stained with a fluorescent dye that binds DNA in a
stoichiometric manner.
2. Incorporation of thymidine analog bromodeoxyuridine (BrdU) during new
DNA synthesis.

38. Fluorescent dye that binds DNA in a
stoichiometric manner
• The cells are treated with a fluorescent dye that stains DNA
quantitatively. The fluorescence intensity of the stained cells at
certain wavelengths will therefore correlate with the amount of DNA
they contain.
• Dyes have different binding mechanism:
Intercalative binding dyes – Propidium iodide
A.T rich regions binding dyes – DAPI (4,6- Diamidion-2-phenylindole)
G.C rich regions binding dyes – Chromomycin A3

39. Incorporation of thymidine analog
bromodeoxyuridine (BrdU) during new DNA
synthesis
An accurate method for detection of
cell cycle progression also uses the
incorporation of thymidine analog
bromodeoxyuridine (BrdU) during
new DNA synthesis.
The incorporated BrdU is then stained
with specific fluorescently labelled
anti-BrdU antibodies, and the levels of
cell-associated BrdU measured using
flow cytometry.
By this method, the number of cells
that are proliferating rapidly & the
duration of S-phase can be calculated.

40. Clinical Applications of Flow Cytometry
1. Diagnosis of Hematologic
Malignancies
2. Detection of Minimal Residual
Disease
3. Lymphocyte Subset
Enumeration (HIV)
4. Efficacy of Cancer
Chemotherapy
5. Reticulocyte Enumeration
6. Cell Function Analysis
7. Application in Organ
Transplantation

41. Thank You !!!