Understanding Clinical Immunology Laboratory Tests

1. Clinical Immunology Overview and use of the Laboratory

2. This lecture will cover • What the immunology lab measures • How they are measured • Why they are measured • (You will have other lectures expanding on this)

3. Immune System Cells Complement Antibodies T-cells B cells Prevent infection Macrophages Neutrophils

4. Components of the immune system are measured for a variety of reasons: • Their amounts can vary with infections • Abnormal components can be present with certain diseases • Immunological components can be deficient Questions • Are the components of the immune system present in normal concentrations? • Do these components function normally? • Are there known abnormal immunological components present? e.g. autoantibodies, paraproteins

5. Immune System Cells Complement Antibodies T-cells B cells Prevent infection Macrophages Neutrophils

6. Immunoglobulin concentrations in serum Normal ranges vary with age • IgG 6-16 g/L • IgA 0.8-2.8 g/L • IgM 0.5-1.9 g/L • IgD 0.1 g/L • IgE 0. 0001 g/L

7. Antibodies Allergen specific IgE Total levels of Ig G, A, M Specific anti-microbial antibodies autoantibodies

8. Measurement of IgG, A and M concentrations - Nephelometry • A fixed amount of antibody specific to the immunoglobulin of interest is mixed with the patient sample (serum) • Light is directed onto the reaction chamber • Light is scattered by the presence of antibody-antigen complexes • The amount of light scatter is detected

9. Small amount of protein. No large complexes: little light scattering

10. Large amount of protein. Large complexes: light scattering

11. Very large amount of protein. Small complexes: Little light scattering

12. LightScattering Protein concentration

13. Abnormalimmunoglobulins • Monoclonal / paraproteins • Immunoglobulin components – light chains in serum or urine (BJP) • Protein electrophoresis • Separating serum proteins by charge to look for abnormalities

14. Autoantibodies • Many different autoantibodies have been found. • Each binds to a specific self antigen • May be found at low levels in healthy people. • Not always associated with disease • Most important are of IgG class • But IgA and IgM in some cases

15. Autoantibodies • May be pathological • i.e. the antibody causes the disease • More often found in association with disease • Eg cellular attack on an organ releases neoantigens to which antibodies develop • Indirect measure of disease state or progression

16. Methods for measuring autoantibodies • Indirect Immunoflourescence • ELISA • Line blot

17. + Antibodies bind to proteins on tissue section Antibodies in Patient Sample Tissue section on microscope slide Fluorescently labelled antibody to human immunoglobulin Fluorescence Microscope Indirect Immunoflourescence

18. Section of stomach tissue Gastric parietal cells stained due to presence of autoantibody

19. ELISA

21. Line blot Recombinant Antigens fixed on a cellulose strip

23. Measurement of allergen specific IgE • Commonly known as RAST -RadioAllergoSorbentTest • (Misnomer as radioactivity not used)

24. IgE levels in serum arevery very low • IgG 8.0 g/L • IgA 2.0 g/L • IgM 1.0 g/L • IgD 0.1 g/L • IgE 0.0001 g/L

25. To detect one allergen specific IgE requires a very sensitive method

26. Immune System Cells Complement Antibodies T-cells B cells Prevent infection Macrophages Neutrophils

27. Classical pathway (CP) Antibody mediated C5 convertase (CP) C5 convertase Terminal pathway Lysis of bacteria ALTERNATIVE pathway (AP) C5 convertase (AP)

28. Complement measurement • Complement components C3 & C4 • Complement control proteins C1 esterase inhibitor • Nephelometry as for immunoglobins • Complement functionality • Haemolytic assay – if all the components of the pathways are present lysis of red blood cells occurs

29. Immune System Cells Complement Antibodies T-cells B cells Prevent infection Macrophages Neutrophils

30. Lymphocytes

31. Distinguished by their cell surface markers (cluster of differentiation markers or CD) Detected by fluorescently labelled monoclonal antibodies to these CD markers

32. Lymphocytes • T cells CD3+ • CD4 lymphocytes are CD3+ and CD4+ “T helper” • CD8 lymphocytes are CD3+ and CD8+ “cytotoxic T cells” • B cells CD19+

33. Flow Cytometry • Cells can be differentiated by there size and granularity • Whole blood (or fractions thereof) can be incubated with fluorescently labelled monoclonal antibody to cell surface markers • Different cell types are detected by their different surface (CD) markers

34. Flow Cytometry Principle

35. Granulocytes Granularity Monocytes Lymphocytes Size CD8 CD4 CD4 CD3 CD3 CD8

36. Functional tests • Lymphocyte activation • In response to mitogens • In response to antigens • Neutrophil activation

37. When are Immunology tests useful?

38. Are Immunology tests ever urgent? NO Not in the sense of say potassium (which can kill you) BUT Occasionally rapid testing is required to make an early diagnosis so that treatment can be instigated.

39. Circumstances when rapid testing can be helpful • Autoimmune renal disease • Rapidly progressive renal disease • When Goodpasture’s syndrome (anti GBM disease) or vasculitis is suspected • Anti GBM and ANCA should be measured • Suspected primary immune deficiency • Severe combined immune deficiency • Lymphocytes subpopulations should be measured

40. Measurement of Total IgG, IgA and IgM concentrations

41. Non specifically in RA, SLE autoimmune liver disease etc Increased production Infection Myeloma (monoclonal) Primary Decreased production Immunodeficiency Secondary

42. Allergy

43. Specific IgE testing • May contribute to the diagnosis of allergy • But only in conjunction with a careful history • A positive sIgE does not always mean clinical sensitivity to an allergen • A negative sIgE does not exclude allergy • Allergy is a clinical diagnosis

44. Clinical significance of Autoantibodies : • Presence or absence may not rule a disease in or out • Key is understanding the clinical significance of antibodies for diseases – • We use clinical sensitivity and specificity Clinical sensitivity = % of patients with given disease who have positive antibody Clinical specificity = % of healthy people who don’t have the antibody

45. Anti-Nuclear Antibodies (ANA) • Homogeneous ANA pattern consistent with anti-double stranded DNA Ab in SLE • Speckled ANA pattern to Anti Ro (SS-A)/ Anti La (SSB) in Sjogren’s Syndrome • ANA pattern consistent with Anti Scl 70 in Systemic Scleroderma • Centromere ANA pattern in Limited Cutaneous Scleroderma (formally CREST)

46. More autoantibodies • IgM Rheumatoid Factor present in about 80% of patients with rheumatoid arthritis and about 10% of patients without • Anti-CCP is highly specific for RA in patients with clinical features of disease (not to be used as screen) • Anti tissue transglutaminase (TTG) is highly sensitive and specific for coeliac disease • Anti intrinsic factor antibodies with anti gastric parietal cell ab specific for pernicious anaemia

47. Anti Neutrophil Cytoplasmic Antibody (ANCA) in renal disease • P-ANCA • Anti MPO (ELISA) • Microscopic polyangiitis> Churg Strauss > polyarteritisnodosa C-ANCA • Anti PR3 (ELISA) • Wegener’s Granulmoatosis

48. Anti GBM antibodies in Goodpasture’s • Anti Mitochondrial Antibodies in Primary Biliary Cirrhosis (kidney section) Antigen is PDH (pyruvate dehydrogenase) • Anti Smooth muscle Antibody in Autoimmune Hepatitis Type 1

49. Use of flow cytometry • Monitoring CD4 counts in HIV • Looking for lymphocyte defects in primary immunodeficiency

50. Immune System Cells Complement Antibodies T-cells ANA ANCA Anti TTG RF etc etc B cells Igs Autoantibodies Allergen specific IgE RAST