2. What is Flow Cytometry?
• ‘Flow Cytometry’ as the name suggests is a technique for cell
counting and measurement of different properties of the cell
(‘cyto’= cell; ‘metry’=count/measurement).
• It is a laser based technology that measures and analyses
different physical and chemical properties of the cells/particles
flowing in a stream of fluid through a beam of light.
4. Historical Perspective -
Evolution of Flow Cytometry
17th
Century
1934
1947 to
1949
1953 1965
1879 1968
1970s
onwards…
Development of
light microscope
by
Leeuwenhoek.
Principles of Droplet
formation by Lord
Rayleigh.
Counting of RBCs
by Moldavan by
forcing a
suspension of cells
through capillary
tube.
Development of
Coulter Principle by
Wallace Coulter and
counting of RBCs
using the first Coulter
Counter.
Optical counting of
RBCs by Crosland-
Taylor by use of
laminar flow
principles
Development of
electrostatic
inkjet droplet
deflection by
Richard Sweet
Application of
Sweet’s principle
and Coulter principle
to develop the first
cell sorter by M.
Fulwyler
Development of
fluorescence
based cell sorter
by Wolfgang
Gohde
Development of
FACS and other
advances.
5. Principles of working of
Flow Cytometer
Coulter
Principle
Principle s
of Laminar
Flow
Electro
statics
Optics
& Light
Scattering
Flow
Cytometry
6. Components of a Flow Cytometer
• A flow cytometer is made up of three main systems: fluidics, optics
and electronics.
The fluidics system transports particles in a stream to the laser
beam for interrogation.
The optics system consists of lasers to illuminate the particles in
the sample stream and optical filters to direct the resulting light
signals to the appropriate detectors.
The electronics system converts the detected light signals into
electronic signals that can be processed by the computer.
For some instruments equipped with a sorting feature, the
electronics system is also capable of initiating sorting decisions to
charge and deflect particles.
7. Working of a Flow Cytometer
• In the flow cytometer, particles are carried to the laser intercept in a
fluid stream. Any suspended particle or cell from 0.2–150
micrometers in size is suitable for analysis.
• The portion of the fluid stream where particles are located is called the
sample core. When particles pass through the laser intercept, they
scatter laser light.
• Any fluorescent molecules present on the particle fluoresce. The
scattered and fluorescent light is collected by appropriately positioned
lenses.
• A combination of beam splitters and filters steers the scattered and
fluorescent light to the appropriate detectors. The detectors produce
electronic signals proportional to the optical signals striking them.
8. Applications of Flow Cytometry
• Flow cytometry is the sine qua non (without which, nothing)of the
modern researcher’s toolbox.
• Flow cytometry measures multiple characteristics of individual
particles flowing in single file in a stream of fluid.
• Light scattering at different angles can distinguish differences in size
and internal complexity, whereas light emitted from fluorescently
labeled antibodies can identify a wide array of cell surface and
cytoplasmic antigens.
• This approach makes flow cytometry a powerful tool for detailed
analysis of complex populations in a short period of time.
9. Applications
Immunophenotyping
• Cell subsets are measured by labeling population-specific proteins
with a fluorescent tag on the cell surface.
• In clinical labs, immunophenotyping is useful in diagnosing
hematological malignancies such as lymphomas and leukemia.
Cell Sorting
• The cell sorter is a specialized flow cytometer with the ability to
physically isolate cells of interest into separate collection tubes.
• The sorter uses sophisticated electronics and fluidics to identify
and "kick" the cells of interest out of the fluidic stream into a test
tube.
10. Cell Cycle Analysis
• Flow cytometry can analyze replication states using fluorescent
dyes to measure the four distinct phases of the cell cycle.
• Along with determining cell cycle replication states, the assay can
measure cell aneuploidy associated with chromosomal
abnormalities.
Apoptosis
• The two distinct types of cell death, apoptosis and necrosis, can be
distinguished by flow cytometry on the basis of differences in
morphological, biochemical and molecular changes occurring in
the dying cells.
11. Cell Proliferation Assays
• The flow cytometer can measure proliferation by labeling resting
cells with a cell membrane fluorescent dye, carboxyfluorescein
succinimidyl ester (CFSE).
• When the cells are activated, they begin to proliferate and undergo
mitosis. As the cells divide, half of the original dye is passed on to
each daughter cell.
• By measuring the reduction of the fluorescence signal, researchers
can calculate cellular activation and proliferation.
12. DNA Content Analysis
• The measurement of cellular DNA content by flow cytometry uses
fluorescent dyes, such as propidium iodide, that intercalate into the
DNA helical structure.
• The fluorescent signal is directly proportional to the amount of
DNA in the nucleus and can identify gross gains or losses in DNA.
13. Fluorescence Activated Cell Sorting (FACS)
• Consider a group of lymphocytes from a mouse that have been stained
with green fluorescent antibodies specific for CD4 (e.g., fluorescein
isothiocyanate, or FITC anti-CD4) and red fluorescent antibodies specific
for CD8 (e.g., phycoerythrin, or PE anti-CD8).
• Both the labeled cells generate SSC and FSC as they pass through the
laser beam creating voltage pulses that are recorded by the computer.
• However, each labeled cell will also emit light of specific wavelength as
a result of the fluorescent label. For instance, CD4 cells will emit green
fluorescent light of wavelength 525-530 nm while CD8 cells emit orange
light of wavelength 560 nm.
• These fluorescent signals pass through the Photomultiplier tubes and
generate voltage pulses. The software integrates all the information for
a particular cell allowing characterization of individual cells.
14.
15. Clinical Applications – DNA Content Analysis
• Investigators are currently using techniques of DNA flow cytometry
to measure ploidy status (DNA content) and proliferative potential
(S phase fraction) in a wide variety of solid tumors.
• These measurements have shown relevance for diagnosis, prognosis,
and treatment for patients with cancer.
• The measurement of cellular DNA content by flow cytometry uses
fluorescent dyes, such as propidium iodide, that intercalate into the
DNA helical structure.
• The fluorescent signal is directly proportional to the amount of DNA
in the nucleus and can identify gross gains or losses in DNA.
• Abnormal DNA content, also known as “DNA content aneuploidy”,
can be determined in a tumor cell population.
16. • DNA aneuploidy generally is associated with malignancy;
however, certain benign conditions may appear aneuploid.
Cell Cycle Analysis
• This technique is based on the premise that cells in G0 or G1
phases of the cell cycle possess a normal diploid
chromosomal, and hence DNA content (2n) whereas cells in
G2 and just prior to mitosis (M) contain exactly twice this
amount (4n).
• As DNA is synthesized during S-phase, cells are found with a
DNA content ranging between 2n and 4n.
• A histogram plot of DNA content against cell numbers gives
the classical DNA profile for a proliferating cell culture.
17. Flow Cytometry and Ecology
• Assessments of diversity, abundance, and activity of water column
microorganisms are fundamental to studies in aquatic microbiology.
• Currently, most applications of flow cytometry to environmental
samples make use of various morphological and physiological
characteristics of the cells (e.g., size and pigment content of
photosynthetic organisms).
• These criteria generally are not sufficient for identification at the genus
or species level. Staining with DNA-specific fluorochromes offers
information about numbers of bacterial cells but not about their identity.
• The combined use of dyes that bind preferentially to G- C or A. T base
pairs has been used to distinguish organisms of different G+C content
18. Flow Cytometry and Cancer Research
• The prognosis of patients with cancer is largely determined by the specific
histological diagnosis, tumor mass stage, and host performance status.
• Quantitative cytology in the form of flow cytometry has greatly advanced
the objective elucidation of tumor cell heterogeneity by using probes that
discriminate tumor and normal cells and assess differentiate as well as
proliferative tumor cell properties.
• Both DNA content analysis and FACS can be utilised in cancer research.
• Abnormal nuclear DMA content is a conclusive marker of malignancy and
is found with increasing frequency in leukemia (23% among 793 patients),
in lymphoma (53% among 360 patients), and in myeloma (76% among 177
patients), as well as in solid tumors (75% among 3611 patients), for an
overall incidence of 67% in 4941 patients.
19. • Flow cytometric immunophenotyping (FCI) aids in the differentiation
of chronic lymphocytic leukemia (CLL) from mantle cell lymphoma
(MCL); however, overlapping phenotypes may occur.
• CD11c expression has been reported in up to 90% of CLL cases but
has rarely been reported in MCL.
• Whether CD11c can be used to exclude MCL has not been directly
addressed.
• FCI reports were reviewed for 90 MCL cases (44 patients) and 355
CLL/small lymphocytic lymphoma (SLL) cases (158 patients).
