Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.

1. FLOW CYTOMETRY
ABU SUFIYAN CHHIPA
M. PHARM (PHARMACOLOGY) I SEM.

2.  Flow cytometry is a technology that is used to
analyse the physical and chemical
characteristics of particles in a fluid as it passes
through at least one laser.
 Flow cytometry is a laser- or impedance-
based, biophysical technology employed in cell
counting, cell sorting, biomarker detection
and protein engineering, by suspending cells in
a stream of fluid and passing them through an
electronic detection apparatus.

4. PRINCIPLE OF FLOW CYTOMETRY
 The basic principle of flow cytometry is the
passage of cells in single line in front of a laser
so that they can be detected, counted and
sorted.
 A flow cytometer is generally composed of 3
components:
1. The flow system(fluidics)
2. The optical system(light sensing)
3. The electronic system(signal processing)

5. THE FLOW SYSTEM
 The fluidics system includes a flow cell, where
the sample fluid is injected.
 The flow cell requires sheath fluid to carry and
align the cells or particles so that they pass
through a narrow channel and into the laser
intercept (light beam) in a single file.
 This hydrodynamic focusing allows the analysis
of one cell at a time by laser interrogation.

6. THE OPTICAL SYSTEM
 The optics system consists of various filters, light
detectors, and the light source, which is usually a
laser line producing a single wavelength of light at a
specific frequency.
 This is where the particles are passed through at
least one laser beam.
 Lasers are available at different wavelengths
ranging from ultraviolet to far red. Interrogation by
the laser beam excites any compatible fluorescent
probes that are conjugated to antibodies, causing
the probes to emit light (or fluoresce) at specified
wavelengths.

7. THE ELECTRONIC SYSTEM
 These light signals are converted by the
electronics system to data that can be
visualized and interpreted by software.

9. FLUORESCENCE ACTIVATED CELL SORTING
(FACS)
 Fluorescence-activated cell sorting (FACS) is a
specialized type of flow cytometry.
 It provides a method for sorting a heterogeneous
mixture of biological cells into two or more
containers, one cell at a time, based upon the
specific light scattering and fluorescent
characteristics of each cell.

10. PROCEDURE
The cell suspension is entrained in the center of a narrow, rapidly flowing stream of
liquid. The flow is arranged so that there is a large separation between cells relative to
their diameter. A vibrating mechanism causes the stream of cells to break into
individual droplets.
The system is adjusted so that there is a low probability of more than one cell per
droplet. Just before the stream breaks into droplets, the flow passes through a
fluorescence measuring station where the fluorescent character of interest of each cell
is measured.
A charge is placed on the ring based on the immediately prior fluorescence intensity
measurement, and the opposite charge is trapped on the droplet as it breaks from the
stream.The charged droplets then fall through an electrostatic deflection system that
diverts droplets into containers based upon their charge.

12. INTERPRETATION OF HISTOGRAM

13. FORWARD SCATTER(FSC)
 Scattering of light by the cells in forward direction.
 Intensity of forward scattering is directly
proportional to cell size.

14. SIDE SCATTER(SSC)
 Light scattered at degrees to the axis of laser
path is referred as side scatter.
 The intensity of side scatter is directly
proportional to the amount of cytosolic
structures(organelles).

15. APPLICATIONS OF FLOW CYTOMETRY

16. LEUKOCYTE ANALYSIS
 As HIV disease progresses, CD4-positive T
lymphocytes decrease in total number while
CD38 is a protein whose expression increases
upon activation in HIV infected individuals.
 The absolute CD4 count provides a powerful
laboratory measurement for predicting and
monitoring disease progression and response
to treatment in HIV infected individuals.

17. DNA ANALYSIS
 Flow Cytometry is used to differentiate malignant cells
from their normal counterparts. The distinction
between normal and leukemic bone marrow
precursors is essential for the diagnosis and treatment
monitoring of acute lymphoblastic leukemia.
 DNA aneuploidy generally is associated with
malignancy; often correlates with many types of
cancer such as multiple myeloma, and childhood
acute lymphoblastic leukemia (ALL).

18. ENZYMATIC DEFICIENCIES
 Flow cytometry is a tool for measuring Beta
glucocerebrosidase activity in Gaucher’s
disease and to study the abnormalities RBCs
shape.
 Gaucher disease is caused by a deficiency of
the enzyme glucocerebrosidase. Macrophages
transform into pathogenic Gaucher cells
following the phagocytosis of red blood cells
(RBCs) and subsequentaccumulation of
glucosylceramide.

19. DETECTION OF MINIMAL RESIDUAL DISEASE
 Minimal residual disease (MRD) is defined as
disease beyond the limit of morphological
detection using conventional microscopy.
 Patients with acute leukaemia were considered
to be in remission when bone marrow samples
contained <5% neoplastic cells.
 Flow cytometric methods can detect far lower
levels of disease, which can be important in the
clinical management of leukaemia.

20. PROCEDURE
 Mononucleated cells are separated on a
density gradient and labelled with three
antibodies associated with the phenotype of the
leukaemia - CD19/CD34/CD13.
 One sample contains an isotype control in
place of the CD13 antibody. The CD13 versus
CD34 plot is gated on light scatter and the
CD19, CD34 +ve cluster.
 110,000 events were recorded; there were 62
cells in the region set on the isotype control and
2021 cells in the same region set on the
positive sample.

22. DETECTION OF AUTOANTIBODIES
 Autoantibodies to leucocytes, platelets and
erythrocytes may be found in a variety of
autoimmune conditions and can cause
anaemia, leukopenia, or thrombocytopenia.
 Detected by immunofluorescence in either a
direct or an indirect assay.
 In direct assay, anti-human Ig antibodies are
used to detect Ig on the surface of the patient’s
cells.
 In the indirect assay, the reaction of antibodies
in the patient’s serum with cells from a normal
person is observed.

23. FOETO-MATERNAL HAEMORRHAGE
 Foeto-maternal bleeding can sensitise a Rhesus
blood group D-ve mother to D+ve blood cells from the
foetus.
 Haemolytic disease of the new born child can be
caused by the destruction of Rhesus D +ve blood
cells of the foetus by maternal anti-D antibodies.
 Prophylactic anti-D given to the Rhesus D -ve mother
shortly after delivery of a Rhesus D +ve child
significantly reduces the incidence of anti-D
sensitisation in the mother.
 Since the dose of anti-D given is related to the size of
the foeto-maternal haemorrhage, quantitation of
foetal-maternal haemorrhage is therefore important.

25.  Quantitation is achieved by labelling the
erythrocytes in a sample of maternal blood with
FITC-conjugated, non-agglutinating anti-D
antibodies.
 A population of as few as 0.1% foetal cells is
sufficient to sensitise the parent so at least
500,000 cells should be analysed to obtain a
statistically significant estimation.

26. RETICULOCYTE ANALYSIS
 Reticulocytes can be distinguished from
erythrocytes by their high content of RNA.
 There are several stains that can be used
for RNA. Ex. Thaizole orange