Flow cytometry is a technique used to analyze physical and chemical characteristics of single cells suspended in a fluid stream. It provides valuable information for diagnosis and classification of hematolymphoid malignancies by assessing cell antigens. The principle involves hydrodynamic focusing to pass single cells through a laser beam for light scattering and fluorescence detection. Samples are prepared using lysis and staining with fluorochrome-conjugated antibodies before analysis using gating strategies to identify abnormal cell populations and determine lineage, maturity, and antigenic profiles for diagnosis. Flow cytometry has many clinical applications including detection of minimal residual disease and monitoring response to therapy.

1. FLOW CYTOMETRY
Presenter: Dr G Santhi priya
Moderator: Dr Kavitha
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2. Contents
• Introduction
• Principle
• Fluorochromes
• Sample preparation and processing
• Antibody panel selection
• Gating
• Analysis of leukemia cells by flow cytometry
• Indications of flow cytometry
• References
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3. Introduction
• Flow cytometry is the study of physical and chemical
characteristics of a single cell in the fluid stream
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4. • Flow cytometry provides
 valuable and reliable information in the diagnosis and
 classification of hematolymphoid malignancies,
 assessing prognosis and
 response to therapy,
 and for detection of minimal residual disease.
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5. Compartments of Flow Cytometer
• Fluidics system
• Optics and detection
• Electronic system
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6. Principle of Flow cytometry
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7. • Monodisperse suspension
• Hydrodynamic focusing
• Only one cell or particle can
pass through the laser beam at
a given moment
• The sample pressure is always
higher than the sheath fluid
pressure
• A higher flow rate is generally
preferred for immuno
phenotypic analysis of cells,
while a lower flow rate is ideal
for DNA analysis
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Fluidics
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8. Optics & detection
• The light source used in a flow cytometer:
• Laser (more commonly)
• Arc lamp
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9. • This ensures that the light
gets focused into a
volume
• That is small enough to
excite cells maximally,
• While allowing a
simultaneous passage of
cells in a single file at an
acceptable flow rate of
around 1000 cells per
second.
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10. Signal Processing
• The electronic signals are converted to digital signals
with the help of convertors
• Digitalised data is stored as histograms or as list mode
data(LMD) files
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11. Graphic display of data
• Single histogram or cytogram
• Scatterogram or dot plot- FSC/ SSC and CD45/SSC
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12. Histogram Scatterogram
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13. Fluorochromes
• Fluorochromes are essentially dyes which accept energy
• From a laser, at any given wave length and re-emit, it at a
longer wavelength
• These two processes are called excitation and emission
respectively.
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14. Commonly used fluorochromes in leukemia
immunophenotyping
Flurochrome Emission
maximum
Fluorescein isothiocynate(FITIC) 530 nm
Phycoerythrin(PE) 576 nm
Peridin-chlorophyll alpha complex (PerCP) 680 nm
Texas Red 620 nm
ECD (PE-Texas Red tandem) 615 nm
Allophycocyanin (APC) 660 nm
PC5 (PE-cyanine 5 dye tandem) 667 nm
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15. Fluorochrome -criteria
1. Its light absorption spectrum should, match the
wavelength of light emitted by the Argon laser i.e.
488nm.
2. It should have a reasonably high extinction coefficient, a
measure of the probability of absorbing a photon of light
and thus being ‘bright'.
3. It should have a high quantum yield, a measure of the
efficiency to convert, the absorbed light to emitted light.
4. Other desirable properties are lack of interaction with
cellular or biologic components, and minimal overlap
with the spectrum of other fluorochromes to be used
concomitantly.
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16. • When a light intersects a laser beam at the so
called‘interrogation point' two events occur:
A) Light scattering.
B) Emission of light (fluorescence).
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17. 17
A. Light Scatter
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Forward scatter(FSC) Side scatter(SSC)

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19. B. Emission of fluorescent light(fluorescence)
• Cell subtypes can further be characterized using
fluorescent dyes conjugated to monoclonal antibodies.
• The fluorochrome conjugated to monoclonal antibodies
bind to the cell expressing the analogus antigen,& emit
fluorescent light, which can be measured.
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21. Sample preparation and processing
• Types of samples:- Can be performed on peripheral blood,
bone marrow aspirates, body fluids, lymphnode aspirates.
• Anticoagulants:- EDTA -24hrs,
Heparin – 48hrs
Acid citrate dextrose – 72hrs
• Sample storage:- Processed with in 48hrs
Temp – 18-22’ c
• Specimen integrity:- 1- Time elapsed between sample
collection and delivery to lab
2- Environmental conditions
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22. Sample processing
• Lysis of red cells :- With reagents like water, ammonium
chloride, hypotonic buffer.
• Removal of lysed red cells :- with an isotonic fluids like
phosphate buffered saline
• Staining with antibodies :- 1. Cell surface antigens
2. intracellular antigens
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23. • Intracytoplasmic markers :- some of the cytoplasmic
markers are used in lineage differentiation. Ex- in AML,
cMPO has the highest sensitivity & specificity.
• Membrane permeabilization :-Low concentration of non
ionic detergents like Saponin are used as permeabilizing
agents.
• Negative controls:- determines the level of non specific
binding & autofluoroscence.
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24. Antibody panel selection
• Antibodies are assembled into panels for the
immmophenotypic analysis of leukemias and lymphomas.
• One step analysis
• Two step analysis- Primary panel
- secondary panel
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25. Gating
• Process of identifing the abnormal cell population and to study
its characteristics
• Population of interest
• A gate is selected by defining a region on a cytogram
• Strategies: Conventional FSC/SSCgating
SSC/CD45 gating
CD19 gating for B cells
CD 3 gating for T cells
CD 38 gating for plasma cells
Reverse gating
Gating for exclusion of dead/ non- viable cells
Live gating
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26. Conventional FSC/ SSC gating
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27. SSC/ CD 45 gating
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28. CD 19 gating CD 3 gating
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29. CD 38 gating
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30. Analysis of leukemia cells by flow cytometry
1. Detection of abnormal cell population
2. Lineage assignment
3. Detailed analysis of antigenic profile of abnormal cells and
its comparison with normal cells.
4. Establish neoplastic nature of the abnormality
5. Determination of maturity level of neoplastic cells
6. Accurate characterization of aberrant, antigenic profile of
the neoplastic cells for future tracking of residual disease
7. Diagnosis of the disease
8. Prognostication
9. Identification of targets for directed therapy
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31. Indications of flow cytometry
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36. Solid tumors
 Tumors where flow cytometry for DNA analysis commonly
used are-
 Breast carcinoma.
 Lung carcinoma.
 Bladder carcinomas.
 Colorectal carcinoma.
 Prostatic carcinoma.
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38. References
• Tejendher S. Automation in cell counts, hemoglobin
seperation, immunophenotyping and coagulation:Atlas
and text of hematology, 3rd ed. Avichal publishing
company. p65-75
• Richard A, Mc Pherson M, Pincus R.The Flow
Cytometric Evaluation of Hematopoietic Neoplasia In.
HENRY’S clinical diagnosis and management by
labaratory Methods :22nd ed. Saunders elsevier , P230-
235
• Pranab D.Flow cytometry In. Diagnostic cytology:2nd ed;
2016. Jaypee Ltd, P260-6
• Martin GW. Flow cytometry: Principles and
Instrumentation. Curr. Issues Mol. Bol.(2001)3(2):39-45
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39. Thank you !!
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