Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.

1. Flowcytometry
Assessment of Immune Functions
(Immunological Diagnosis of Immunodeficiency Diseases)

2. Intended Learning Outcomes
•Explain the principles of
Flowcytometry and interpret the
result.
•Understand the basic principles of
tests assessing immunodeficiency.

3. 1. Flowcytometry

4. FLOW CYTOMETRY

5. Cell
counting
Ab
Fluorochrome
Cell
Certain marker
e.g. CD4
Specific monoclonal Ab
Ab
Multiple cell surface markers can be detected
simultaneously because different cells can be
stained with multiple fluorescent antibodies,
each directed at one particular antigen and
having a different fluorescence wavelength.
Another
marker on
the cell
Detected by a light and fluorescence optical detector
system which demonstrates fluorescently labeled cells on
a computer.

6. Cell Counting & Sorting by Flowcytometry

7. Medical Microbiology & Immunology Dep
The flowcytometer represents
the fluorochrome staining
pattern of the cells in a graph
called a Dot Plot

8. Each dot on the graph
reflects the passage of
a cell with a certain
level and color of
fluorescence, so the
darkly dotted areas
of the graph reflect
the presence of many
cells of similar
attributes.
Double
Positive
Single Positive
for Vertical Axis
Double
Negative
Single Positive for
Horizontal Axis

9. In the above flow cytometry dot plot, cells were labeled with
antibodies against CD16 and CD 56. What percentage out of
the total population of cells is the population of NK cells?
a) 9%
b) 41%
c) 17%
d) 33%
e) 42%

10. In reference to the above dot plot obtained from a flowcytometric analysis,
what percentage of the cells are CD8-negative T cells?
a) 45%
b) 79%
c) 5%
d) 16%
e) 34%

11. 2. Assessment of Immune Functions
(Immunological Diagnosis of Immunodeficiency Diseases)

12. Immunological diagnosis of
Immunodeficiency diseases
Test for
phagocytic
cell
Number
Function
Ingestion
Intracellular
killing
Test for
complement
Number
Function
Test for B
lymphocyte
Number
Function
Test for T
lymphocyte
Number
Function

13. I- Defect in Phagocytic Cells
Test for
number
Test for
function
Test for
ingestion
Test for
intracellular
killing

14. 1- Tests for Number:
Total and differential leukocytic count

15. Number of monocytes can be assayed by
flowcytometry using fluorescein labeled
monoclonal antibodies directed against a
surface marker CD14.

16. Cell counting and sorting by flowcytometry

17. 2-Tests for Function
Tests for random motility and chemotaxis
Modified Boyden Chamber Assay

18. 3-Tests for Ingestion
Incubate leucocytes with heat killed yeast
or Staph aureus and count the ingested
particles by microscope.
Medical Microbiology & Immunology Dep

19. 4-Tests for Intracellular killing
Detects defects in oxygen-dependent
intracellular killing as in congenital NADPH
oxidase deficiency by Nitroblue Tetrazolium
Dye Test.

20. Addition of NBT dye
(colorless)
Production of H2O2 &
Reduction of dye to blue
color (Positive)
Isolation of
PMNLs
TLR
LPS
NADPH
oxidase
H2O2
bacterial
killing
NADPH oxidase
deficiency???

21. II- Defect in Complement
Measurement of quantity of individual complement
factors by RID or ELISA.

22. III- Tests for B Lymphocytes
1- Tests for Number
In peripheral blood and lymph node
biopsy by flowcytometry using
fluorescein labeled MABs directed
against B cell surface markers (anti CD19
& anti-CD 20).
Medical Microbiology & Immunology Dep

23. Medical Microbiology & Immunology Dep
2-Tests for Function
Serum levels of antibody are evaluated by
ELISA, radial immunodiffusion & Immuno-
electrophoresis.
➢ A serum sample is placed in a well in agar on a glass
slide.
➢ A current is passed through the agar, and the
proteins move in the electric field according to their
charge and size. Then a trough is cut into the agar
and filled with antibody.
➢ As the antigen and antibody diffuse toward each
other, they form a series of arcs of precipitate.

26. IV-Tests for T Lymphocytes:
1- Test for Number
•Counting mature T cells by flow-
cytometry using fluorescein labeled
MAbs directed against T cell surface
markers (anti-CD3).
•Counting T cell subsets using fluorescein
labeled anti-CD4 and anti CD8.
Medical Microbiology & Immunology Dep

27. Medical Microbiology & Immunology Dep
2- Test for Function
a) Delayed hypersensitivity skin testing using
intradermal injection of common antigen as
candidin or tuberculin. Absence of reaction
suggests impairment of cell mediated immunity
(CD4 T cells).
b) Assessment of circulating cytokine levels by ELISA.
Intradermal Skin Test: Results
are examined after 48 hours.
Erythema Induration

28. c) Cytotoxic T-cell activity is often
assessed by chromium release from
labeled target cells

29. 3- Assay for Lymphocyte Proliferation
Antigen-specific T-cell proliferation

30. Subgroup Activity
Group Discussion

31. Thank You!