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Lymphocytes

Lymphocytes. Methods to Obtain Non-coagulating Blood - Heparin (5-10 IU/ml): activates antithrombin III, blocks the action of thrombin - EDTA: binds Ca2+ - Citrate: binds Ca2+. The most important CD antigens of lymphocytes. Marker: Cell Subpopulation CD3 , CD4, CD8 T-lymfocytes

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Lymphocytes

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  1. Lymphocytes

  2. Methods to Obtain Non-coagulating Blood - Heparin (5-10 IU/ml): activates antithrombin III, blocks the action of thrombin - EDTA: binds Ca2+ - Citrate: binds Ca2+

  3. The most important CD antigens of lymphocytes Marker: Cell Subpopulation CD3 , CD4, CD8 T-lymfocytes CD19, CD20, CD21 B-lymfocytes CD16/CD56 NK cells CD14/DR monocytes HLA-DR; CD25; CD69 activation markers

  4. CD antigens • Antigens expressed on leukocytes • Abbreviation o Custer of Differentiation • Each antigen is designed: CD+ a number (CD1- CD340..) • The antigens were designated after they were recognized and characterized – no system exists.

  5. Flow cytometer –FACS Fluorescence-Activated Cell Scanner (Sorter)

  6. Flow cytometer

  7. Flow cytometry http://images.google.cz/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/Flow-Cytometry-Diagram2.jpg&

  8. granulocytes size monocytes lymfocytes granularity

  9. CD3 CD4

  10. CD8 CD3

  11. CD8 CD3

  12. Laboratory investigation of lymphocytes • Enumeration of lymphocyte subsets by monoclonal antibodies against typical cell-surface markers -CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (suppressor/cytotoxic T-lymphocytes), CD19 or CD20 (B-lymphocytes), CD16 (NK cells). • Evaluation of function -lymphocyte proliferation tests - determines response to various stimuli: • specific (antigen, anti CD-3) • non-specific -polyclonal mitogens: PHA (Phytohaemagglutinin), ConA (Concanavaline A), PWM (Pokeweed mitogen)

  13. Delayed-type of hypersenzitivity test

  14. Tuberculin test

  15. Cell-Mediated Immunity (CMI) tests • Uses the principle of delayed type of hypersensitivity • Common anamnestic antigens are used (tuberculin, candidin, trichofitin, staphylococcal antigens…) • In normal situation induration shout be formed by 48 hours. • If the patient does nor react to majority of antigens a deficiency of T- cell mediated immunity is suspected.

  16. Investigation of function of neutrophils • Baktericidial killing of various bacteria of fungi • Chemotaxis (e.g. under agarose) • Ingestion of inert particles • Metabolic activity - production of reactive metabolites of oxygen • NBT test (reduction of colourless nitro-blue-tetrazolium into blue formasan) • Chemiluminescence - luminol after is oxidation spontaneously reduced and emits light

  17. NBT

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