1. 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

2. 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

3. Flow cytometry

4. Flow cytometry (FACS®)

6. Dot plot Histogram

7. Monocyte Ig - CD3 - CD14 + Distribution of different cell population in PBMCs

8. 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

9. 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)

10. centrifugation PBMCs isolation using density gradient

11. PBMCs isolation using density gradient

12. Cell separation/isolation using paramagnetic beads

13. Chemotaxis Functional analysis of phagocytes

14. Boyden chamber assay

15. Chemotaxis Ingestion Respiratory burst Intracellular killing Functional analysis of phagocytes

16. Functional analysis of NK cells Cytotoxicity K 562 (Cytotoxicity test) Cytokine production IFN-γ (ELISA)

17. Functional analysis of NK cells Cytotoxicity ( 51 Cr release)

18. Functional analysis of NK cells Cytotoxicity ( 51 Cr release)

19. Functional analysis of NK cells Cytotoxicity ( 51 Cr release)

20. 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)

21. Functional analysis of T cells Proliferation Stimulation:  polyclonal activator ( mitogen : Con A, PHA.....)  antigen Based on incorporation of 3 H thymidine in DNA

23. Patientmedium ConA A.B.430±3143905±389 D.G.330±841200±227

24.  Cytotoxicity (T cells) Cytoxicity test  Cytokine production (T cells) ELISA  Antibody production (B cells) ELISA, Nephelometry, RID Functional analysis of T and B cells

25. Immediate hypersensitivity skin tests  Prick test  Intradermal test Delayed hypersensitivity (DTH) skin tests Assesment of humoral and cellular immune response after immunization Provocation tests Assessment of immune response in vivo

27. 15 min. Prick test (HSR type I) 1.Desinficate the skin and mark the spots 2.Put a drop of alergen positive 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 sensitivity 10-15 mm moderate sensitivity › 15 mm pronounced sensitivity Flare and wheel

28. 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

29. Patch test (DTH) Negative no changes weak positive redness moderate positive redness, vesicles pronounced positive redness, vesicles, edema redness, vesicles and edema after 48h (again after 72h)

30. 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

31. 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. efgjdcabhi