Flow cytometry allows for rapid analysis of physical and chemical characteristics of single cells. It measures properties like cell size, granularity, and surface antigens by passing single cells through a laser beam and detecting light scattering. This provides quantitative results on multiple cell parameters simultaneously. Cells are stained with fluorescent antibodies targeting specific antigens. When excited by lasers, the antibodies emit light of distinct wavelengths, allowing identification of cell types. Flow cytometry is useful for applications like phenotyping, cell cycle analysis, and measuring intracellular proteins. It requires cells in single suspension, fluorescent reagents, and a flow cytometer instrument.

1. Flow Cytometry
Dr. Ankita R Kapoor
Radiation Oncology

2. Flow Cyto Metry
Flow Cells Measurements

3. Introduction
• Measuring multiple physical and chemical characteristics of single cell
or particles by optical means
• nuclei
• microorganisms
• chromosome preparations
• latex beads
• Physical properties: size (forward angle light scatter)
• internal complexity (right-angle scatter)

4. Why use Flow Cytometry?
• Rapid analysis
• Individual event analysis
• Quantifiable results
• Multiple parameter analysis
• Statistical relevance

5. Concept of Scattering
• FSC collects light at 180°, correlated with cell size
• SSC collects right-angle light at 90°, correlated with cytoplasmic
granularity and nuclear configuration

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8. SSC
• High SSC: PMN (including precursors), granular blasts
• Intermediate SSC: Monocytes, blasts, Hairy cell leukemia cells
• Low SSC: Lymphocytes, blasts

9. FSC
• High FSC: monoblasts and monocytic cells
• Intermediate FSC: Myeloblasts, Large lymphoma cells (DLBCL, ALCL)
• Low FSC: Lymphoblasts, lymphocytes

10. Results
• Histogram
• Dot plot graph

12. Applications
• Cell size
• Cytoplasmic granularity
• Cell surface antigens (phenotyping)
• Apoptosis
• Intracellular cytokine production
• Intracellular signalling
• Cell cycle, DNA content, composition, synthesis
• Bound and free calcium

13. Requirements
• Cells in single suspension
• Antibody
• Flurochromes/Fluorescent probes
• Cytometer

14. Single Cell Suspension
• Cells isolated in suspension buffer
• To maintain cells at optimal density
• To avoid cell clumping & clogging of chamber
10/25/17

15. Antibodies
• Based on cluster of differentiation (CD)- protocol used for
identification and distinction of cell surface antigens
• Using CD system, cells identified by presence or absence of
particular surface markers for e.g. CD3+ or CD20- etc

16. Fluorochromes
• Fluorescent dyes intercalate with different cellular components: DNA
or RNA
• Antibodies conjugated to fluorescent dyes bind specific proteins on
cell membranes or inside cells
• When labeled cells are passed by a light source, the fluorescent
molecules are excited to a higher energy state

17. • Returning to resting states: the fluorochromes emit light energy at
higher wavelengths
• Use of multiple fluorochromes with similar excitation wavelengths
and different emission wavelengths (or colors), allows several cell
properties to be measured simultaneously

18. FLUOROCHROMES CONJUGATED TO
ANTIBODIES
• Fluorescein isothiocyanate (FITC)
• Phycoerythrin (PE)
• PE-Texas Red
• PE-Cy5
• Peridinin chlorophyl protein(PerCP)
• Allophycocyanin (APC)
• APC Cy7

19. SPECIMENS SUITABLE FOR FLOW
CYTOMETRY
• Any specimen from which a single cell suspension can be
generated
• Limitation:
• lack of distinct antigens or markers in the cells of interest or tissues

20. Common specimens suitable for flow
cytometry analysis
• Peripheral blood
• Bone marrow
• Body fluids
• Cerebrospinal fluid
• Urine
• Lymph node
• Any fine-needle aspirates

21. Sample collection Protocol
1. Transported to lab ASAP at Room Temperature
2. Never be frozen
3. Stored at Room Temperature for up to 24hrs
4. Refrigerate if to be tested next day

22. Tissue or FNA
• Sample in a conical tube or capped sterile specimen container
• RPMI or Hank's solution
• May be placed in saline or saline soaked gauze (if to be transported to the Flow lab within 1 hour)
Bone Marrow or Peripheral Blood:
• One 3ml sodium heparin green top tube or heparinized syringe
• 1ml whole blood or bone marrow minimum required
Body Fluids:
• Sample in a conical tube or capped sterile specimen container
• As much fluid as possible

23. Components of Flow Cytometer
1. Fluidics
2. Lasers (2-3): each produces a single wavelength of light
3. Dichroic and bandpass filters : split light (spatial separation), and
block or pass on light of selected wavelengths
4. Light Detectors: PMT tubes and silicon photodiodes: detect light,
produce electrical pulses
5. Electronics: filtering and amplification of signals
6. Software: data presentation

24. COMPUTER
SYSTEM
ELECTRONIC
SYSTEM
OPTICAL
SYSTEM
FLOW SYSTEM
For cell analysis, the basic components of a flow
cytometer include:-

26. Brown et al. Clinical Chemistry. 2000:46:8(B) 1221–1229.

27. Disadvantages
• Need for liquid cell suspension; lack of correlation with
histomorphologic features ( tissue architecture)
• Requires viable, fresh (unfixed) material

28. Conclusion
• Rapid analysis of multiple characteristics of single cells
• Information both qualitative and quantitative
• Used for immunophenotyping of variety of specimens (whole blood, bone marrow, serous cavity
fluids, cerebrospinal fluid, urine, and solid tissues)
• Applications in hematology include
• DNA content analysis
• leukemia and lymphoma phenotyping
• immunologic monitoring of HIV-infected individuals
• assessment of structural and functional properties of erythrocytes, leukocytes, and platelets

29. THANK YOU