FLOW CYTOMETRY

Saikat Pal
• Flow cytometry was initially conceived as a
practical methodology to count blood cells.

• In 1879 Lord Rayleigh observed that fluid

emerging from an orifice breaks into a series of
droplets. Cell sorting is based on the physics of
droplet formation.

• In 1934, A. Moldavan reported the development

of the first device that could count red blood cells
automatically while in flow.
• In 1949, Wallace Coulter filed a patent
entitled, “Means for Counting Particles
Suspended in a Fluid”. The patent was issued
in 1953. This lead to the development of the
“Model A” Coulter Counter. Today, clinical
hematology laboratory instruments used to
count blood cells employ the principles
developed by Coulter.
 Flow cytometry
• Flow cytometry is a technique used to detect
and measure physical and chemical
characteristics of a population of cells or
particles. A sample containing cells or particles
is suspended in a fluid and injected into the
flow cytometer instrument. The sample is
focused to ideally flow one cell at a time
through a laser beam and the light scattered is
characteristic to the cells and their
components.
• Cells are often labeled with fluorescent
markers so that light is first absorbed and then
emitted in a band of wavelengths. Tens of
thousands of cells can be quickly examined
and the data gathered are processed by a
compute
 Applications
• Flow cytometry is routinely used in basic research,
clinical practice, and clinical trials. Uses for flow
cytometry include:
• Cell counting
• Cell sorting
• Determining cell characteristics and function
• Detecting microorganisms
• Biomarker detection
• Protein engineering detection
• Diagnosis of health disorders such as blood disorders.
Applications in Clinical Laboratories

• Immunophenotyping (HIV)
• CD4 absolute counts
• Leukemia and lymphoma
immunophenotyping
• Cell cycle and ploidy analysis of tumors
• Reticulocyte enumeration
• Flow cross-matching (organ transplantation)
• Stem cell enumeration
• Residual white blood cell detection
• (QC platelet, red blood cells)
 Research Laboratories

• Immune function studies

• Hematopoietic stem cells
• Multi-drug resistance studies (cancer)
• Kinetics studies (cell function) Platelet
analysis (coronary disease)
• Environmental sample analysis
• Flow and FISH
• Phenotypic Analysis:
Immunophenotypic Analysis of Peripheral Blood
Lymphocytes
Detection of Cytokine Receptors
Enumeration of CD34+ Hematopoietic Stem and
Progenitor Cells
Measurement of CD40 Ligand Expression on Resting
and In Vitro-Activated T Cells
• Nucleic Acid Analysis:
Analysis of DNA Content and DNA Strand Breaks for
Detection of Apoptotic Cells
DNA Content Measurement for DNA Ploidy and Cell
Cycle Analysis
Analysis of DNA Content and BrdU Incorporation
• Cell Function:
Oxidative Metabolism of Neutrophils
Measurement of Intracellular pH
Analysis of Mitochondrial Membrane Potential
Reporters of Gene Expression
Measurement of Intracellular Calcium Ions
Intracellular Cytokines

• Microbiological Applications:
Antibiotic Susceptibility
Cell Cycle Analysis of Yeasts
DNA/RNA Analysis of Phytoplankton
• Cancer therapy monitoring:

– DNA content of tumor cells is determined to assess the prognosis of

cancer patients.

– DNA specific probes bind directly to the DNA to enable evaluation of

normal and abnormal cells.

– The measurement of DNA content in cells was one of the earliest

applications of flow cytometry.

– DNA specific dyes stain cells stoichiometricly, this means that the
amount of stain is directly proportional to the amount of DNA.
• Cell function analysis:

– Neutrophils (polymorphonuclear leukocytes) are a major

contributor to the early inflammatory response and are a
primary source of toxic oxygen metabolism.

– The function of these cells is important in combating

bacterial infections.

– Flow cytometry has been used to study many disorders of

the neutrophil.
• Intracellular organelles can be stained with
fluorescent labeled organelle-specific dyes
and assessed for function as well.
 Chromosome Karotyping
• Flow cytogenetics is the classification and
purification of chromosomes.

• Human as well as other animal chromosomes

have been isolated and genetic libraries
constructed.

• All chromosomes can be identified and sorted

by using two fluorescent probes, Hoechst
33258 and Chromomycin A3.
 Fetal Cell Detection
• Rare Cell Detection is relatively simple for an
instrument that is capable of analyzing
hundreds of thousands of cells in minutes.

• Flow cytometry is useful in detecting bacteria

in whole blood, identifying HIV-infected
lymphocytes, revealing minimal residual
disease in a malignant neoplasm and
distinguishing fetal cells in maternal blood.
• During the first trimester fetal cells cross the
placenta and can be isolated from maternal
blood. Fluorescence In-Situ Hybridization
(FISH) uses chromosome-specific DNA probes
that can identify any chromosomal
abnormalities that may be present in the
fetus. This procedure may replace
amniocentesis as a noninvasive method of
determining fetal status.
• Platelets circulate in the peripheral blood in a
quiescent state but initiate a cascade of
events resulting in the formation of a fibrin
clot when activated.

• Many platelet defects responsible for bleeding

disorders are diagnosed using flow cytometry
to identify glycoprotein receptors.
 Flow Cytometric Crossmatch (FCXM)
• Flow cytometry has become a valuable tool to
assess potential solid organ allograph recipients.

• It is now recognized as the laboratory procedure

of choice. Circulating alloantibodies at levels too
low to be detected by standard methods can be
detected by a flow cytometric crossmatch
(FCXM).

• This means that transplants done based on a

negative FCXM are more successful.
• Patients with neoplastic disease require a
minimum number of 2-5 X 106/kg recipient
body weight of CD34+ cells for engraftment.
Flow cytometry and the identification of the
CD34 antigen on hematopietic progenitor cells
has made this possible.

• Patients with type 1 diabetes may have

pancreatic islets transplanted thus eliminating
the need for daily injections of insulin.
 Leukemia and Lymphoma
• Lymphomas are tumors of the immune
system, primarily in the lymph nodes, spleen,
and bone marrow.

• Flow Cytometry has been used since the late

1970's to diagnose and classify human
lymphomas. A standard panel of fluorescent
labeled monoclonal antibodies is used for this
purpose.
 Commercial Applications
• Biology and Cytometry of Sperm Sorting

• Spermatozoal Differences

– Sex pre-selection is based on identifying differences

between X- and Y-bearing sperm

– The X chromosome contains about 4% more DNA in cattle

and horses than the Y chromosome.

– This difference in DNA content can be used to distinguish

and select X from Y bearing sperm.
 Emerging Applications
• Microbiology
– Drug industry
– Molecular biology
– Food industry
– Dairy industry
– Water industry
– Defense industry
Immunophenotyping
Immunophenotyping is the analysis of
heterogeneous populations of cells for the
purpose of identifying the presence and
proportions of the various populations of
interest. Antibodies are used to identify cells
by detecting specific antigens expressed by
these cells, which are known as markers
• Cell markers are a very useful way to identify a
specific cell population. However, they will
often be expressed on more than one cell
type. Therefore, flow cytometry staining
strategies have led to methods for
immunophenotyping cells with two or more
antibodies simultaneously.
• . By evaluating the unique repertoire of cell
markers using several antibodies together,
each coupled with a different fluorochromes,
a given cell population can be identified and
quantified. Many immunological cell markers
are CD markers and these are commonly used
for detection in flow cytometry of specific
immune cell populations and subpopulations.
• Is performed by labeling the cells with red,
green, and / or orange labeled monoclonal
antibody.

• The cells are then interrogated in the Flow

Cytometry, and the number, percentage of
positive cells are recorded.
T-Cell Markers Myeloid Markers
CD 2 CD11b
CD3 CD11c
CD4 CD13
CD5 CD14
CD6 CD33
CD7
B-Cell Markers Miscellaneous Marker
CD10 sIgG HAL-DR
CD19 sIgM CD34
CD20 Kappa CD45
CD22 Lambda TdT
CD23 CIgM
CD25 CCd3
 Three Major Components
• Fluidics: Transports the cells to the laser
interrogation point

• Optics: Collects light signal generated by

scatter and fluorescence emission

• Electronics: Converts optical signals into digital

signals that can be processed by a computer
 Forward Angle Light Scatter

Laser

FALS Sensor
• The amount of light scattered at right
angle to the incident light beam depends
on the internal complexity of the
particle, this known as wide angle or Side
Scatter (SSC) , side scatter detected at 90,
to the laser beam
 90 Degree Light Scatter

Laser

FALS Sensor

90LS Sensor
❖As the cell passes through the laser beam,
light is scattered in all directions and that
scattered in the forward direction is
proportional to the square of the radius of
a sphere, and so to the size of the cell or
particle.

❖ The cells may be labeled with

fluorochrome-linked antibodies or stained
with fluorescent membrane, cytoplasmic or
nuclear dye.
What can a Flow Cytometer (FCM)
tell us about a cell?
• Its relative size (Forward Scatter—FSC)

• Its relative granularity or internal complexity

(Side Scatter—SSC)

• Its relative fluorescence intensity

 Fluidics
• A normal physiological saline liquid,
known as sheath fluid, is used to move
the cells through the flow cell, to the
sensing area.

• The sensing area is the point at which the

cells are interrogated by the laser beam.
The cells or particles to be analyzed are
suspended in a sample fluid.
• The sheath fluid has a higher flow rate than
the sample fluid. This difference serves to
constrict the sample stream to the center of
the flow cell.
• This method, known as hydrodynamic
focusing, aligns the cells in a single file in the
center of the stream and laminar flow allows
these two fluids to move in the same direction
through a flow cell without mixing. This design
insures that the cells in the sample are
contained in a central core surrounded by
sheath fluid and are illuminated optimally by
the light source
Electronics
Thanks