Immunology vi lab_cell_immunol
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Immunology vi lab_cell_immunol

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Immunology vi lab_cell_immunol Immunology vi lab_cell_immunol Presentation Transcript

  • 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+
  • 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 The most important CD antigens of lymphocytes
  • 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.
  • Flow cytometer –FACS Fluorescence-Activated Cell Scanner (Sorter)
  • Flow cytometer
  • Flow cytometry http://images.google.cz/imgres?imgurl=http://www.abcam.com/ps/CMS/Images/Flow-Cytometry-Diagram2.jpg&
  • lymfocytes monocytes granulocytes granularity size
  • CD3 CD4
  • CD3 CD8
  • CD3 CD8
  • 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)
  • Delayed-type of hypersenzitivity test
  • Tuberculin test
  •  
  •  
  • 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.
  • 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)
      • Chemilu mi nescence - luminol after is oxidation spontaneously reduced and emits light
  • NBT