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

MonocyteIg -CD3 -

CD14+

Distribution of different cell population in PBMCs

• 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

Applications of flow cytometry

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)

centrifugation

PBMCs isolation using density gradient

PBMCs isolation using density gradient

Cell separation/isolation using paramagnetic beads

• Chemotaxis

Functional analysis of phagocytes

Boyden chamber assay

• Chemotaxis• Ingestion• Respiratory burst • Intracellular killing

Functional analysis of phagocytes

Functional analysis of NK cells

Cytotoxicity

K 562(Cytotoxicity test)

Cytokine production

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)

Phenotyping and determination of cell count

T cells

(proliferation, cytotoxicity, cytokine production)

B cells

(proliferation, antibody production)

Testing of cells of adaptive immunity (T and B lymphocytes)

Functional analysis of T cells

Proliferation

Stimulation:polyclonal activator (mitogen: Con A, PHA.....)

antigen

Based on incorporation of 3H thymidine in DNA

Patient medium ConA

A.B. 430±3143905±389

D.G. 330±84 1200±227

Cytotoxicity (T cells)

Cytoxicity test

Cytokine production (T cells)

ELISA

Antibody production (B cells)

ELISA, Nephelometry, RID

Functional analysis of T and B cells

Immediate hypersensitivity skin tests Prick test Intradermal test

Delayed hypersensitivity (DTH) skin tests

Assesment of humoral and cellular immune response afterimmunization

Provocation tests

Assessment of immune response in vivo

15 min.

Prick test (HSR type I)

1. Desinficate the skinand mark the spots

2. Put a drop of alergenpositive and negative control

3. Prick the skin by lancet

3. Remove the excess of alergen

edema is measured, not the redness

2mm › neg. control – positive result

≥ 5mm – clinical relevance

5-10 mm mild sensitivity10-15 mm moderate sensitivity

› 15 mm pronounced sensitivity

Flare and wheel

Assessment of immune response in vivoImmediate 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)

Negative no changesweak positive rednessmoderate positive redness, vesiclespronounced positive redness, vesicles, edema

redness, vesicles and edema

after 48h (again after 72h)

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)

Assessment of immune response in vivo

10.__9.__8.__7.__6.__5.__4.__3.__2.__1.__

CD16 and CD56j.T-cell proloferation can be assessed by10.

stimulation with T-cell mitogensi.Tuberculin test is9.

delayed type hypersensitivity testh.DTH tests determine8.

membrane Ig, CD19, CD20g.Immediate type hypersensitivity test determine

7.

CD3 and CD4 or CD8f.Respiratory burst is decreased in6.

difference in density or surface markers

e.Cytotoxic activity of NK cells can be measured by

5.

cytotoxicity testd.Surface markers of NK cells are4.

chronic granulomatous diseasec.Surface markers of B-cells are3.

presence of antigen-specific T-cellsb.Surface markers of T-cells are2.

presence of allergen-specific IgEa.Cell isolation is based on1.

e f g j d c a b h i