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AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY

AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY . http://rimm.dote.hu. AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY a. diagnosis of i nher i ted and a c quired immunodeficiencies b. diagnosis of autoimmune and allergic di seases

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AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY

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  1. AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY http://rimm.dote.hu

  2. AIMS OF THE LABORATORY TESTS OF CLINICAL IMMUNOLOGY a. diagnosis of inherited and acquired immunodeficiencies b. diagnosis of autoimmune and allergic diseases c. verification of the immunological background of the disease I. SEROLOGICAL TESTS 1.Immunoglobulins IgG: IgG1-4 (infections, autoimmun dis.) IgA (infections, autoimmun diseases) IgM (infections, autoimmun diseases) IgD IgE(allergy) 2. Acute phase proteins: -positive acute phase proteins: C reactive protein(CRP), serum amyloid A (SAAcomplement factors, fibrinogen, haptoglobin, procalcitonin, etc. CRP:! Elevated in acute infections and autoimmune diseases (RA) -negative acute phase proteins: transferrin, albumin 3. Complement factors: C3, C4, C1INH Congenital deficiencies (predisposition for autoimmun diseases) Increased consumption: activity marker of SLE HANO: herediter angioneurotic oedema: C1INH deficiency

  3. 4. Determination of the level of circulating immune complexes 5. Measurement of “natural” autoantibodies: antibacterial antibody titer isohaemagglutinin titer (anti-A, anti-B) 6. Measurement of cold agglutinins 7. Detection ofcryoglobulins

  4. 8. AUTOANTIBODIES Their types: 1.natural („physiologic”) autoantibodies (IgM) 2.pathologic autoantibodies (IgG) The benefit of their verification: - markers for diagnosis and differential diagnosis, - formation of subgroups, subtypes - prognostic value estimation of disease activity and efficacy of therapy

  5. 9. AUTOANTIBODIES OF POLYSYSTEMIC AUTOIMMUNE DISEASES Rheumatoid factor a. Antinuclear autoantibodies (ANA)-collective name of antibodies directed against various nuclear antigens, detected by indirect immunofluorescence. b. Anti-double stranded DNA: one of the diagnostic criteria of SLE. c. Anti-histon antibody:marker of drug induced SLE. d. Anti-chromatin (nucleosoma) antibody: directed against the complex of ds-DNA+histon, marker of lupus nephritis. e. Anti-extractable nuclear antigen (ENA) antibodies directed against SS-A(Ro),SS-B(La),Sm, U1-RNP, Scl-70, Jo-1 antigens. f. Anti-neutrophil cytoplasmic antibodies (ANCA) c-ANCA (cytoplasmic fluorescence) p-ANCA (perinuclear fluorescence) atypic-ANCA (atypic c + p fluorescence) g. Antiphospholipid antibodies: anti-cardiolipin anti-beta 2 glycoprotein I lupus anticoagulans

  6. Autoantibodies in polysystemic autoimmune diseases: Type of antibodyDisease Anti-dsDNA SLE (renal involvement) -histon SLE(drug induced) -Sm SLE -nucleosome lupus nephritis -U1RNP MCTD, Raynauld synd. -SS-A(Ro) Sjögren, RA, SLE -SS-B(La) Sjögren, RA, SLE -Scl 70 PSS (renal involvement) -Centromer PSS (CREST synd.) -Nucleolar PSS -PM-1 PM, DM -Jo-1 PM, DM PM: serum CK elevation ANCA vasculitis

  7. Marker antibodies in polysystemic autoimmune diseases * autoantibodies of ANA positivity x also cytoplasmic positivity

  8. Organ specific autoantibodies • Antithyreoidea antibodies: - anti-thyreoglobulin (anti-TG) - anti-thyreoidea peroxidase (anti-TPO): Hashimoto-thyreoiditis - anti-thyreoidea stimulating hormon receptor (anti-TSH-receptor): Graves-Basedow disease • Anti-pancreatic islet cell antibody (anti-ICA): I. type diabetes mellitus • Anti-gastric parietal cell antibody: aneamia perniciosa autoimmune gastritis • Anti-adrenal-cortex (17,21-hydroxylase) antibody: Addison disease • Anti-glomerular basal membrane antibody: Goodpasture syndrome • Anti-smooth-muscle antibody: autoimmune hepatitis • Anti-liver-kidney-microsome (anti-LKM) antibody: autoimmune hepatitis

  9. Main laboratory tests of autoantibody determination: - indirect immunofluorescence by microscopy: positivity/negativity, pattern,titer, duration - enzyme linked immunosorbent assay (ELISA)(quantitative) - immunhistology

  10. BENEFITS OF THE LABORATORY MEASUREMENTS IN CLINICAL IMMUNOLOGY • 1. DATA FOR DIAGNOSIS • a.) immunodeficiencies: • - serology: IgG, IgA, IgM, IgE, C3, C4, level of “natural autoantibodies” • - test of phagocytes: phagocytosis, chemotaxis, “killing” assay, • chemiluminescence measurements (phagocytosis+killing) • - cell surface antigens on lymphocytes: CD3, CD4, CD8, CD56, CD19 • active immunization: Pneumococcus vaccination, measurement of • antibody titers.

  11. b.) Antimicrobial, reactive inflammations: • serology: We (augmented), increases in the levels of C reactive protein, • immune complex, IgG, IgA, IgM, IgE, C3, C4 • cellular tests: increased white cell count, granulocytosis, lymphocytosis, • elevated phagocytic activity • increased expression of activation markers of T cells: HLA-DR, CD25, • CD30, CD38, CD45RO, CD69 • c.) Allergy: • - serology: increase in the serum levels of total and allergen specific IgE • - cellular test: allergen induced lymphocyte proliferation • d.) autoimmune diseases: • - serology: organ specific autoantibodies in organ specific diseases, • “marker” autoantibodies in polysystemic diseases

  12. 2. MEASUREMENTS FOR FOLLOWING UP THE THERAPY a.)following up the immunosuppression: antibody levels b.) following up the immunoreactivity: proliferation response (blast transformation) cytokine production active immunization: Pneumococcus vaccination c.)determination of the effect of desensibilisation in allergy d.)measurement of the effect of immunosubstitution: IVIG therapy

  13. 3. LABORATORY PARAMETERS AS CRITERIA FOR DIAGNOSIS a.) immunodeficiencies b.) antibodies:-organ specific autoantibodies -marker antibodies in polysystemic autoimmune diseases e.g. anti-U1RNP: MCTD, cAnCA: Wegener granulomatosis SLE: anti-dsDNA, anti-Sm c.)demonstration of atypic immunoglobulins: monoclonal components in myeloma multiplex

  14. 4.LABORATORY PARAMETES AS TOOLS FOR FORMATION OF SUBGROUPS OR VERIFICATION OF RISK FACTORS • Seropositive and seronegative RA (HLA-B-27) • Antiphospholipid syndrome, primer or associated with SLE (thrombosis, abortions). • Anti-Jo1 and the “synthetase syndrome”: anti-histidyl-tRNA synthetase positivity: pulmonary fibrosis (PM/DM) • Bronchial asthma, PM/DM sensitive or resistant to glucocorticosteroids • 5.VERIRIFICATION OF “OVERLAP” IN ASSOCIATED AUTOIMMUNE DISEASES • Simultaneous occurence of various marker antibodies: SLE-Ss, SLE-PM/DM,PSS-Ss

  15. 5. RELATIONS BETWEEN THE LABORATORY PARAMETERS AND THE ACTIVITY OF DISEASES • Definition of diagnosis by laboratory tests. • Differentiation of the active and inactive states of the diseases by laboratory tests: • -         identification of the autoantibody at least once • -         antibody titer and disease activity: anti-ds-DNA-SLE, • c-ANCA-vasculitis • antiphospholipid syndroma • Demonstration of activation markers in lymphocytes • Cytokine measurements in samples from biopsy • Molecular genetic methods, demonstration of oncogens, etc

  16. TYPES OF LABORATORY METHODS USED IN CLINICAL IMMUNOLOGY

  17. SEROLOGICAL TESTS: 1. QUANTITATIVE MEASUREMENTS OF PROTEINS IN SERUM, PLASMA, URINE AND LIQUOR - radial immunodiffusion (Mancini-test) - turbidimetry - nephelometry - electrophoresis 2. TOTAL SERUM COMPLEMENT ACTIVITY (CH50) –haemolytic test 3. PASSÍVE HAEMAGGLUTINATION - anti-bacterial antibodies - rheumatoid factor (Waaler-Rose) 4. INDIRECT IMMUNOFLUORESCENCE - antinuclear antibodies - organ specific antibodies - ANCA (anti neutrophil cytoplasmic antibody) 5. ELISA (enzyme linked immunosorbent assay) - autoantibodies - allergen specific IgE

  18. CELLULAR MEASUREMENTS Tests for phagocytes: microba induced chemiluminescence (phagocytosis+killing) monocyte-granulocyte functions: Fc, complementcarbohydrate receptor dependent methods  Tests for the functions of lymphocytes: Proliferation (blast transformation) induced by mitogen NK cell activity: 51Cr release from tumor cells  Measurement of CD antigens (flow cytofluorimetry): -  differentiation antigens on the cell surface, -  subpopulations of lymphocytes: CD3: T cells CD4: helper T cells CD8: cytotoxic T cells CD19: B cells CD56: NK cells

  19. Measurement of immunoglobulins Turbidimetry Light absorbtion Nephelometry Light scattering

  20. Age related changes in the serum amounts of immunoglobulins Immunoglobulins g/l IgG IgA IgM months age

  21. a1 globulins b1b2 globulins g globulins Albumin a2 globulins 1 2 3 4 5 6 7 8 9 10

  22. PROTEINOGRAM OCT 18 93 2/1 ECH: 1 AGE: 0 SEX: TOTAL PROTEINS: 61.0 G/L A/G: 1.33 NAME 0/0 G/L NORM:% G/L Albumin 57.1 34.9 52-65 34-42 Alpha 1 3.6 2.2 2-4.5 1-3 Alpha 2 14.6 8.9 10-15 6-9 Beta 12.5 7.6 6-13 4-9 Gamma 12.2 7.4 10-19 6-13 N Albumin Albumin a1 zone: a1 antitrypsin, orosomucoid, a1 antichymotrypsin a2 zone: haptoglobin, ceruleoplasmin, Gc globulin, a2 macroglobulin, a lipoprotein b1 zone: transferrin – hemopexin b2 zone: blipoprotein, C3 complement g zone: IgG, IgA, IgM, (IgD, IgE)

  23. with b mercaptoethanol reduction: correct typing of IgM K cryoglobulin. (To be confirmed after isolation of the cryoprecipitate). ELP G A M K L

  24. ELP G A M K L

  25. ELP GAM K L K L free free Image of biclonal gammpathy Presence of two Lambda monoclonal bands confirmed: l one with anti heavy chains (g, a or m) l the other, more anodic, with anti Lambda free light chain. Conclusion: presence of a Lambda Ig monoclonal component with more anodic Lambda free light chains.

  26. Ur Ser Albumin

  27. Ur Ser IgG Transferrin Albumin

  28. Ur Ser Albumin

  29. Hemolytic measurement of classical complement pathway activation (CH50)

  30. Passive hemagglutination (Waaler-Rose test)

  31. Indirect immunofluorescence

  32. C-ANCA

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