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HUMORAL IMMUNITY (SEROLOGY)

HUMORAL IMMUNITY (SEROLOGY). GOOD LABORATORY PRACTICE (GLP). rules and recommendation for operating of clinical laboratories GLP includes: preanalytical phase analytical phase interpretations and reefering of results internal system of quality control

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HUMORAL IMMUNITY (SEROLOGY)

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  1. HUMORAL IMMUNITY (SEROLOGY)

  2. GOOD LABORATORY PRACTICE (GLP) rules and recommendation for operating of clinical laboratories GLP includes: preanalytical phase analytical phase interpretations and reefering of results internal system of quality control external system of quality control Accreditation of clinical lab by State Health Authority

  3. SEROLOGY determination of immunological parameters in body fluids (serum, plasma) optimal collection and transport of sample (time) optimal processing of sample: - centrifugation - blood clot removal - stabilisation of sample optimal storage of sample: - refrigeration: short-term - freezing: - several month stability at –20°C - long-term stability at – 70°C or in liquidnitrogen repeated freezing and thawing should be avoided

  4. IMMUNOCHEMISTRY Immunochemical reaction (IR): specific interaction between antibody(Ab) and antigen(Ag) Antigen: any molecule which is immunogenic Antibody: is raised in animal after immunization (antiserum) Characteristics of IR: optimal ratio of Ag and Ab (zone of equivalence) optimal pH, ionic strength, temperature, duration Immunochemical reaction: - agglutination - precipitation

  5. Y Y Y Y PRECIPITATION CURVE Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Amount of precipitate ZONE OF EQUIVALENCE Excess Ab Excess Ag Amount of antigen

  6. Y Y Y Y IgM IgG Y Y BACTERIA AGGLUTINATE FORMATION AGGLUTINATION Ag is corpuscular (bacteria, erythrocytes-hemagglutination, inert particles-latex)) agglutinate is formed agglutinate isvisible by naked eyes application limited to infection serology (specific antibodies determination) application limited to determination of autoantibodies (rheumatoid factors)

  7. Y Y Y PRECIPITATE FORMATION PRECIPITATION: Ag is soluble Precipitation in: semi-solid medium (gel) visual determination in fluid: instrumental determination direct determinationof precipitates: turbidimetry, nephelometry indirect determinationof precipitates by secondary antibody labeled by: - enzyme: EIA (enzyme immunoassays) - isotope: RIA (radio immunoassays) - luminofore: LIA (luminiscence immunoassays)

  8. 2. PRECIPITATION 1. AGGLUTINATION Ag CORPUSCULAR Ag SOLUBLE Y Y Y Y Y Y Y IgM IgG Y Y BACTERIA AGGLUTINATE FORMATION PRECIPITATE FORMATION

  9. DETERMINATION: - qualitative (presence) - quantitative (how much) Quantitative determination: standard sample (reference) dilution of standard calibration curve unknown sample Titer: relative quantification to express relative amount of specific antibodies reversed value of the highest dilution of examined serum in which reaction is positive ; (example: dilution 1:340  titer 340)

  10. 50l diluted serum (Ab) IInd step 1:64 1:4 1:8 1:16 1:32 Ag Ag Ag Ag Ag 4 50l suspension of Ag 3 1:4 1:8 1:16 1:32 1:2 TITER OF SPECIFIC ANTIBODIES Ist step 50l 50l 50l 50l 50l 2 50l serum (Ab) 1 50l dilution fluid

  11. PRECIPITATION IN GELS gel: hydrated polysacharides (agarose) Ag (Ab) or both diffuse through gel precipitation lineages or rings are formed in zone of equivalence simple and unexpensive technique substantial delay in obtaining of results (days)

  12. SINGLE (RADIAL) IMMUNODIFFUSION (MANCINI) antiserum is dispersed in gel precipitation rings are formed area of ring is equivalent to the concentration of antigen quantitative determination (dilution of standard, calibration curve)

  13. SINGLE RADIAL IMMUNODIFUSION (SRID) SERUM SAMPLE difusion Ag difusion PRECIPITATION RING (ZONE OF EQUIVALENCE) GEL + INCORPORATED ANTIBODY

  14. DOUBLE IMMUNODIFFUSION (OUCHTERLONY) qualitative determination both Ag and Ab diffuse through the gel precipitation arc (lineage) is formed

  15. PRECIPITATION IN FLUID most common approach now precipitates are evaluated by optical system: turbidimetry nefelometry most effective and rapid automatisation, robotisation

  16. TURBIDIMETRY light absorbance detector source of light precipitate precipitate light scatter source of light NEPHELOMETRY detector

  17. IMMUNOGLOBULINS: are major blood proteins agammaglobulinemia hypogammaglobulinemia hypergammaglobulinemia: - inflammation - myeloma

  18. DETERMINATION OF IMMUNOGLOBULINS 1. electrophoresis: semiquantitative 2. immunochemistry:- radial immunodiffusion - turbidimetry, nephelometry (IgG, A, M) - ELISA (IgE) Normal ranges in adults: total IgG: 6-17 g/l IgA: 1-4 g/l IgG1: 3-9 g/l IgM: 0.5-3.0 g/l IgG2: 1-5 g/l IgG3: 0.1-1 g/l IgG4: 0.01-0.9 g/l IgE class immunoglobulins - present in a minute amount in serum (g/l) - detection by the sensitive immunoassay (ELISA)

  19. ACUTE PHASE PROTEINS (APP) Are plasma proteins which level is changed during inflammation. infection injury cancer IL-1 macrophage hepatocytes APP TNF

  20. ACUTE PHASE PROTEINS (APP) + reactants: - plasma levels are increased in inflammation - CRP: opsonisation - -orosomucoid - -antitrypsin - haptoglobin - C3, C4 complement - reactants: - plasma levels are decreased in inflammation - albumin - prealbumin - transferin

  21. DETERMINATION OF ACUTE PHASE RESPONSE erythrocyte-sedimentation rate (ESR) - reflects the changes in APP (fibrinogen) - simple, cheap electrophoresis - proteins have electrical charge - migration of proteins in DC electrical field - albumin (negative charge) -fraction: orosomucoid, antiproteases -fraction: transferin, C3 -fraction: immunoglobulins, CRP

  22. DETERMINATION OF ACUTE PHASE RESPONSE electrophoresis - overall information about the gross changes in the spectrum of APPs immunochemistry: - detailed information about particular APP - function of APPs - interpretation of results in the clinical context

  23. IMMUNOELECTROPHORESIS

  24. electrophoresis - proteins have electrical charge - migration of proteins in DC electrical field - albumin (negative charge) -fraction: orosomucoid, antiproteases -fraction: transferin, C3 -fraction: immunoglobulins, CRP

  25. E L E C T R O P H O R E S I S E L E C T R O P H O R E T O G R A M nosič elektroforézy - + START  globulins 2 2 1 1 albumin D E N S I T O M E T R Y

  26. DENSITOMETER

  27. ELECTROPHORESIS + -

  28. IMMUNOELECTROPHORESIS immunoelectrophoresis: combination of electrophoresis immunodifusion immunofixation modern approach more sensitive identification of plasma or serum proteins immunoelectrophoresis: essential for detection of paraproteins

  29. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y NORMAL AND ABNORMAL B CELLS DIFFERENTATION Ag INDEPENDENT Ag DEPENDENT PLASMA CELLS UNMATURE B-CELLS MATURE B-CELLS CD 38 POLY-CLONAL RESPONSE HLA DR HLA DR CD 19 Ag STIMULUS T HELP ISOTYP. SWITCHING AFFINITY MATURATION Ig GENES REARRANG. TdT BcR CD 19 MYELOMA CELLS GENETIC DAMAGE ACCUMULATION CD 56 HLA DR CD 38 CD 19 MONO- CLONAL RESPONSE (PARAPROTEIN) MALIGNANT TRANSFORMATION BcR

  30. PARAPROTEIN: abnormal monoclonal immunoglobulin produced by clone of malignantly transformed plasma cell (plasmocytoma) macroglobulinemia(Morbus Waldenström) lymphoma infiltrating lymphoid tissues paraprotein is of IgM class plasma viscosity multiple myeloma(M.M.) localisation of plasmocytoma cells in bone marrow osteolytic lesions paraproteins of IgG, IgA, IgD and IgE (in decreased frequency) light chain disease monoclonal light Ig chains (kappa, lambda) secreted in urine as Bence-Jones protein

  31. ELECTROPHORESIS + - lane 3: susp. paraprotein lane 7, 8: susp. paraprotein other lane: polyclonal immunoglobulins (normal)

  32. IMUNOFIXATION + - lane 1: monoclonal protein (electrophoretogram) lane 4, 5: immunochemical evidence of paraprotein IgM INTERPRETATION: Waldenström macroglobulinemia

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