Transcript Functional analysis of phagocytes Functional analysis of NK cells

Assessment of
the immune system cells
At the end of this seminar you will be able to answer
the following questions:
 How to determine cell count?
 How to separate and isolate different cell populations?
 Which in vitro and in vivo tests are used for the
assessment of the immune system
Cell count determination
Based on morphology and staining

chambers and blood smears
Based on size and density

automated cell counters
Based on specific cell markers

T lymphocytes: CD3, CD4, CD8....

B lymphocytes: Ig, CD19, CD20...

NK cells: CD16 and 56, CD161
Flow cytometry
Flow cytometry (FACS®)
Flow cytometry (FACS®)
Dot plot
Histogram
Distribution of different cell population in PBMCs
Applications of flow cytometry
• Immunophenotyping (research, lymphoproliferative
diseases, immunodeficiencies )
• Functional surface molecule detection
• Activation marker detection
• Intracellular protein detection
• Activation status (intracellular Ca2+ concentration,
protein phosphorylation, ROS , NOS)
• DNA content – cell cycle
• Apoptosis detection and quantification
Isolation of cells of
the immune system
Based on different density (specific gravity)

using density gradient (Ficoll®)
Based in different cell markers using
paramagnetic beads-labeled antibodies

MACS®
Based on different cell markers using
fluorescence-labeled antibodies

FACS® (flow cytometer)
PBMCs isolation using
density gradient
centrifugation
PBMCs isolation using
density gradient
Cell separation/isolation using paramagnetic beads
Functional analysis of phagocytes
• Chemotaxis
Boyden chamber assay
Functional analysis of phagocytes
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•
•
•
Chemotaxis
Ingestion
Respiratory burst
Intracellular killing
Functional analysis of NK cells
Cytotoxicity
Cytokine production
K 562
(Cytotoxicity test)
IFN-γ
(ELISA)
Functional analysis of NK cells
Cytotoxicity (51Cr release)
Functional analysis of NK cells
Cytotoxicity (51Cr release)
Functional analysis of NK cells
Cytotoxicity (51Cr release)
Testing of cells of adaptive immunity
(T and B lymphocytes)
Phenotyping and determination of cell count
 T cells
(proliferation, cytotoxicity, cytokine production)
 B cells
(proliferation, antibody production)
Functional analysis of T cells
Proliferation
Stimulation:
 polyclonal activator (mitogen:
 antigen
Con A, PHA.....)
Based on incorporation of 3H thymidine in DNA
Patient
medium
ConA
A.B.
430±31
43905±389
D.G.
330±84
1200±227
Functional analysis of T and B cells
 Cytotoxicity (T cells)
Cytoxicity test
 Cytokine production (T cells)
ELISA
 Antibody production (B cells)
ELISA, Nephelometry, RID
Assessment of immune response in vivo
Immediate hypersensitivity skin tests
 Prick test
 Intradermal test
Delayed hypersensitivity (DTH) skin tests
Assesment of humoral and cellular immune response after
immunization
Provocation tests
Prick test (HSR type I)
15 min.
Flare and wheel
1.
Desinficate the skin
and mark the spots
2. Put a drop of alergen
positive and negative control
3.
3.
Prick the skin by lancet
Remove the excess of alergen
edema is measured, not the redness
2mm › neg. control – positive result
≥ 5mm – clinical relevance
5-10 mm mild sensitivity
10-15 mm moderate sensitivity
› 15 mm pronounced sensitivity
Assessment of immune response in vivo
Immediate hypersensitivity skin tests
 Prick test
 Intradermal test
Delayed hypersensitivity (DTH) skin tests
 Patch test
Assesment of humoral and cellular immune response after
immunization
Provocation tests
Patch test (DTH)
after 48h
(again after 72h)
redness, vesicles and edema
Negative
weak positive
moderate positive
pronounced positive
no changes
redness
redness, vesicles
redness, vesicles, edema
Assessment of immune response in vivo
Immediate hypersensitivity skin tests
 Prick test
 Intradermal test
DTH skin tests
 Patch test
 Tuberculin test
Provocation tests
 Bronchial provocation test
 DBPCFC (Double-Blind Placebo-Controlled Food Challenge)
1. Cell isolation is based on
a. presence of allergen-specific IgE
2. Surface markers of T-cells are
b. presence of antigen-specific T-cells
3. Surface markers of B-cells are
c. chronic granulomatous disease
4. Surface markers of NK cells are
d. cytotoxicity test
5. Cytotoxic activity of NK cells can be
measured by
e. difference in density or surface
markers
6. Respiratory burst is decreased in
f. CD3 and CD4 or CD8
7. Immediate type hypersensitivity test
determine
g. membrane Ig, CD19, CD20
8. DTH tests determine
h. delayed type hypersensitivity test
9. Tuberculin test is
i. stimulation with T-cell mitogens
10. T-cell proloferation can be assessed by
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j. CD16 and CD56
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