the principles and the applications of flowcytometry

1. ‫دهؤك‬‫يا‬ ‫ثوليتةكنيكى‬ ‫انكويا‬‫ز‬
‫ـنية‬‫ـ‬‫ق‬‫الت‬ ‫ـوك‬‫ـ‬‫ه‬‫د‬ ‫ـة‬‫ع‬‫جام‬
Duhok Polytechnic University
Lab 11 : Flow cytometry (II)
4th year students
Practical Immunopathology
Ass. lecturer: Ahmed Jumaa Ahmed
M. Sc. Immunology and Allergy 06 /05/2018
Duhok Polytechnic University
Shekhan Technical College of Health
Dept. of Medical Laboratory Technology

2. Outline
• Introduction
• Components of a Flow Cytometer
• How Does Flow Cytometry Work?
• Applications
• Benefits
• Limitations

3. Applications

4. Applications
• wide area of application
• Clinical
• Research

5. Applications
FCM can be used for different puposes:-
1. Immunophenotyping
2. Cell Sorting
3. DNA Content Analysis
4. Cell cycle analysis
5. Apoptosis
6. Cell growth & death rates

6. 1. Immunophenotyping
 is a technique used to study the protein expressed by cells.
 This technique is commonly used in basic science research
and laboratory diagnostic purpose.
 An example is the detection of tumor marker, such as in
the diagnosis of leukemia.
 It involves the labelling of WBCs with antibodies directed
against surface proteins on their membrane.
 The labelled cells are processed in a flow cytometer.
 In clinical labs, immunophenotyping is useful in diagnosing
hematological malignancies.

7. 1. Immunophenotyping
• such as lymphomas and leukemia.
• (Lymphoma type B cell malignancy)
• Prepare Ab for any cell membrane Ag
• (CD20) cell surface protein
• Use anti CD20 Ab labeled by fluorescent dye

8. 2. Cell Sorting
• The cell sorter is a specialized flow cytometer.
• Ability to physically isolate cells of interest into
separate collection tubes, for further investigation.
• The sorter is designed with sophisticated electronics
and fluidics to identify
• And "kick" the cells of interest out of the fluidic
stream into a test tube.

9. Cell Sorting
• sample nozzle is vibrated
• sample stream breaks up
into regular droplets
• droplets are
electrostatically charged
prior to passing through
the laser
• droplets containing cells of
interest (as characterized
by scatter or fluorescence
properties) are deflected
into tubes by passing them
through a pair of charged
plates

10. Cell Sorting 3
Charged
Plates
Collection
Tubes

11. Difference from Coulter Counter
• FCM based on Ab-Ag reaction by using labeled
Ab.
• Coulter measure charged particle within a fluid
passes through champers
• E.g. RBC, WBC, Platelet

12. 3. 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. Example of Annexin V and PI staining
To study viability of cells after treatment
Annexin V
necrotic
apoptotic
viable
Murray et al Journal of Immunology, 172, 274 (2004)
Control cells (A) Tumour treated cells (B)

14. 4. Cell Cycle Analysis
• Flow cytometry can analyze replication states
using fluorescent dyes to measure the four
distinct phases of the cell cycle.
• Prophase, Metaphase, Anaphase, Telophase
• Using specific markers (DNA)

15. 5. 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.

16. Group thinking
• What is the difference between Apoptosis and
Necrosis

17. • Apoptosis:-
• is a process of programmed cell death that occurs in
multicellular organisms
• occurs as a normal and controlled part of an organism's
growth or development.
• Necrosis:-
• is a form of cell injury which results in the premature
death of cells in living tissue.

18. 6. Cell Proliferation Assays
• Cell proliferation can be measured by flow cytometer; by
labeling resting cells with a cell membrane fluorescent dye.
• 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,
cellular activation and proliferation, can be calculated.

20. Group Thinking
• In case we look for different cells in the same
sample
• We must used different labeling colors

21. Clinical uses of FCM
1. In different leukemia and lymphoma the expression of
marker in cytoplasma and cell nucleus.
2. In the diagnosis of primary immunodeficiency (PIDs).
3. By using self Ab, for the diagnosis of systemic lupus
erythematosus (SLE).

22. Summary
• Flow cytometry measures the size, shape,
granularity and fluorescence of individual cells.
• Many different markers can be measured on the
same cell simultaneously.
• Fluorescence measurements can be recorded,
standardised and calibrated.
• It is possible to identify markers of cell subset,
activation, proliferation, death, and function
(such as cytokine synthesis) simultaneously.

23. Flow Cytometry Tutorials
Invitrogen web site has useful tutorials on how
flow cytometry works:
http://www.invitrogen.com/site/us/en/home/support/Tutorials.h
tml