Diagnosis of autoimmune diseases

autoimmune diseases and their diagnosis

1. Diagnosis of autoimmune diseases Presented by, S.Nagaraj , M.Sc. 3 rd year.

2. Autoimmunity Introduction the response of the immune system against self-components is termed autoimmunity. Earliest example of autoimmunity. Metalnikoff , 1900. Guinea pigs injected with their own spermatozoa produced antibodies against sperms.

3. The markers are one of the evidence for the autoimmune diseases. they are: Positive family history for the same disease, or other diseases known to be autoimmune. Presence in the same patient of other new autoimmune diseases. Presence of infiltrating mononuclear cells in the affected organ or tissue. Preferential usage of certain MHC class 2 allele. High serum levels of IgG autoantibodies. Deposition of antigen-antibody complexes in the affected organ or tissue. Improvement of symptom with the use of immunosuppressive drugs.

4. Mechanisms of autoimmunity Antigenic alteration Cells or tissues may undergo antigenic alteration to form neoantigens which induces an immune response. Neoantigens can arise by physical agents. such as irradiation, photosensitivity and cold allergy. Infectious microorganisms, particularly viruses and other intracellular pathogens, may induce alteration of cell antigens. Bacterial enzymes also induce alteration of cell antigens. Neuraminidases formed by myxoviruses and many bacteria act on erythrocytes releasing the T antigen.

5. 2. Sequestered antigens Certain self-antigens are present in a closed system and are not accessible to the immune apparatus. these are known as sequestered antigens. Examples are lens antigen, sperm antigen. 3. Cross-reacting foreign antigens the similarity between some foreign and self-antigens is the basis of cross-reacting antigen theory of autoimmunity. Organ specific antigens are present in many species. injection of these heterologous antigens may induce an immune response, damaging the particular organ or tissue in the host. For example, anti- rabic immunisation with the neural vaccine of infected sheep brain tissue. Streptococcal M protein and the heart muscle Nephritogenic strains of streptococci with antigens in the renal glomeruli.

6. 4. Molecular mimicry The antigenic determinants that are identical or similar to normal host-cell components. Such molecular mimicry appears to occur wide variety of organisms. Examples are : arthritogenic shigella flexneri and HLA-B27, mycobacterium tuberculosis and joint membrane.

7. 5. Polyclonal B cell activation In this hypothesis, antigen generally activates only its corresponding B cell, certain stimuli non-specifically turn on multiple B cell clones. Such stimuli includes chemicals (2-marcaptoethanol), bacterial products (PPD, lipopolysaccharide), enzymes (trypsin), antibiotics (nystatin), and infections with some bacteria (mycoplasma), viruses (EB virus) and parasite (malaria). Multiple non-specific antibodies are form during some infectious diseases, such as anti-human erythrocyte cold antibodies in mycoplasma pneumonia and anti-sheep erythrocyte antibodies in infectious mononucleosis. Theses polyclonal antibodies are IgM in nature.

8. Altered T and B cell function Enhanced helper T cell and decreased suppressor T cell functions have been suggested as cause of autoimmunity. Defects in the thymus ,in stem cell development and macrophage function have also been postulated as cause. Defects in the idiotype-anti-idiotype network also lead to autoimmunity.

9. Classification of autoimmune diseases Organ specific autoimmune diseases Disease Self-antigen Immune response Addison’s disease Adrenal cells Autoantibodies Autoimmune haemolytic anaemia RBC membrane proteins Autoantibodies Goodpasture’s syndrome Renal and lung basement membrane Autoantibodies Grave’s disease TSH receptor Autoantibody (stimulating) Idiopathic thrombocytopenic purpura Platelet membrane proteins Autoantibodies Hashimoto’s thyroiditis Thyroid proteins and cells T DTH cells, autoantibodies Myasthenia gravis Acetylcholine receptors Autoantibody (blocking) Myocardial infraction Heart Autoantibodies Pernicious anaemia Gastric parietal cell, intrinsic factor Autoantibody Poststreptococcal glomerulonephritis Kidney Antigen-antibody complex Spontaneous infertility Sperm autoantibodies

10. Systemic autoimmune diseases Disease Self-antigen Immune response Ankylosing spondylitis Vertebrae Immune complexes Multiple sclerosis Brain or white matter T DTH and Tc cells, autoantibodies Rheumatoid arthritis Connective tissue, IgG Autoantibodies, immune complexes Scleroderma Nuclei, heat, lungs, GIT, kidney Autoantibodies Sjogren’s syndrome Salivary gland, liver, kidney, thyroid Autoantibodies Systemic Lupus Erythematosus (SLE) DNA, nuclear protein, RBC and platelet membrane Autoantibodies, immune complexes

11. Diagnostic methods of autoimmune diseases The diagnostic methods of autoimmune diseases includes, Initial laboratory evolution Immunological studies Inflammatory markers Flowcytometry Cytokine studies Major histocompatibility complex

12. 1. Initial laboratory evaluation Inflammatory diseases will cause abnormalities in routine laboratory studies. which include a normochromic, normocytic anaemia indicating the chronicity or severity of disease. Common hematologic parameters also include an elevated or decreased platelet count and/or white blood cell count. Leukopenia and thrombocytopenia are common in patients with systemic lupus erythematosus (SLE). Testing will find aberrations in serum levels of specific organ enzymes or abnormalities in metabolic processes that are reflected in the comprehensive metabolic panel. For example, autoimmune hepatitis can be manifested by elevations of transaminases, bilirubin, and serum proteins. these abnormalities can also be associated with drug toxicity.

13. Coagulation studies such as a prolongation of the activated partial thromboplastin time ( aPTT ) and/or the prothrombin time (PT) ,suggests an inhibitor of the clotting process is present as seen in the antiphospholipid syndrome. Hypercalcemia can be observed in approximately 30% of patients with sarcoidosis. An increase in muscle enzymes, [creatinine kinase, alanine transaminase (ALT), and aspartate aminotransferase (AST)] can be seen in autoimmune inflammatory myopathies. Serum protein levels are helpful to screen for abnormal elevations of immunoglobulin. The urinalysis is commonly used to assess renal injury (glomerulonephritis, interstitial nephritis) and will show proteinuria, haematuria or active sediment . Many other illnesses such as diabetic nephropathy, poorly controlled hypertension, or infections will test similarly but when autoimmune disease is suspect, the common laboratory evaluation will serve as an initial detection to pursue further testing.

14. 2. Autoantibodies and Immunologic Studies The presence of an autoantibody in a patient does not assure a diagnosis of an autoimmune disease. Rather, a positive serologic test in the company of appropriate signs and symptoms helps to support a diagnosis. Serologic testing is flawed by the presence of autoantibodies in healthy individuals and other patients with non-autoimmune diseases and imperfect testing systems. Historically, many different methods were used to test for the presence an autoantibody. Today, testing is principally done with enzyme immunosorbent assays (EIA) because of cost saving measures with mechanization .

15. Enzyme Linked Immunosorbent Assay Enzyme-linked immunosorbent assay (ELISA) ELISA is an immunometric method for detecting and measuring specific antibodies. The basic components of this laboratory method include a substrate where an antigen is fixed (typically a 96 well micro-well plate), patient's sera, washing solutions and a detection method where an enzyme is linked to an antibody that detects the antigen. In a typical double-antibody sandwich ELISA, an antibody that is attached to the bottom of a well provides both antigen capture and immune specificity, while another antibody linked to an enzyme provides detection and acts as an amplification factor. This allows for accurate and sensitive detection of the antigen of interest. The performance is largely dependent on antibody quantity, kit manufacturer, operator skill and experience. ELISA permits measurement of only one antigen at a time for a given aliquot of sample and it has a limited dynamic range .

16. Anti-nuclear antibody (ANA) Autoantibodies to nuclear antigens are a diverse group of antibodies that react against nuclear, nucleolar or perinuclear antigens. These antigens represents cellular components such as nucleic acid, histone, chromatin, nuclear and ribonuclear proteins. The ANA hallmarks the serologic diagnosis of SLE, but finding an ANA is common to most other autoimmune diseases. Immunofluorescence is one of the method for testing of the patient's serum, at various dilutions, using a cell substrate. Typically, screening patient's serum for the detection of an ANA with ELISA provides high sensitivity but lacks specificity. The HEp2 cells (a human laryngeal epithelioma cancer cell line) have been used as the cell substrate because the result offers the advantage of detecting a nuclear fluorescent pattern. The fluorescent patterns suggest clinical associations with certain autoimmune diseases. Due to time and expense for testing with HEp2 cells, the assay procedures are largely done by ELISA methods.

17. Principle The assay is for detection of antibodies is a solid phase immunosorbent assay in which the analyte is indicated by a colour reaction of enzyme and substrate. The ELISA wells are coated with purified antigen On adding diluted serum to the wells the antibodies present bind to the antigen After incubating at room temperature and washing away unbound material, HRP conjugate anti-IgG monoclonal antibody is added, which binds to the immobilised antibody. For further incubation and washing, TMB is added to the each well. The presence of antigen-antibody-conjugate complex turns the substrate to a dark blue colour. Addition of stop solution turns the colour to yellow. The colour intensity is directly proportional to the amount of autoantibodies present in the original serum sample.

18. ANA Ds DNA Histone Nucleoprotein Riboprotein (Sm) SLE  Sensitivity >95% 70% ∼50% 60% 25%  Specificity 60 95 50 Medium 99 RA  Sensitivity 45 1 Low 25 1  Specificity 60 Low Scleroderma  Sensitivity 60 <1 <1 <1 <1  Specificity 50 PM/DM  Sensitivity 60 <1 <1 <1 <1  Specificity 60 Sjögren's  Sensitivity 50 <5 Low Medium <5  Specificity 50 Low Medium

19. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody RF is an autoantibody that reacts to the Fc portion of polyclonal IgG. But, they can be any class of immunoglobulin. RF is helpful when evaluating patients who may have rheumatoid arthritis as the sensitivity is ∼70% with a specificity of ∼70%. Rheumatoid factor is absent in ∼15% of patients with rheumatoid arthritis. However, ∼15% of the healthy population may have a low titre RF. Rheumatoid factor positive patients are more likely to have progressive, erosive arthritis with loss of joint mobility and also have extraarticular manifestations including rheumatoid nodules, vasculitis, Felty's syndrome and secondary Sjögren's syndrome. In addition, the presence of RF is seen in other autoimmune disorders including Sjogren's syndrome, SLE, cryoglobulinemia, in pulmonary diseases such as interstitial fibrosis and silicosis and various infectious diseases.

20. The mechanism includes, Inflammation activates the enzyme peptidyl-arginine deiminase which incorporates citrulline into certain proteins. -- In RA, autoantibodies are formed against the citrullinated protein (anti-CCP). The presence of serum anti-CCP antibodies are ∼95% specific for the diagnosis of RA, with sensitivity similar to rheumatoid factor. Testing for both anti-CCP and RF is beneficial when excluding the diagnosis of RA rather than testing for either antibody alone. In early undifferentiated disease, anti-CCP positive patients tends to more severe, erosive and aggressive disease. Anti-CCP can also be present in other disease states such as some children with JIA, psoriatic arthritis, lupus, Sjögren's syndrome, inflammatory myopathies and active tuberculosis.

21. Anti-cyclic citrullinated peptide (IgG) Anti CP IgG is an indirect solid phase enzyme immunometric assay, designed for the quantitative measurement of IgG class antibodies directed against citrullinated peptides (CP) in human serum or plasma. Significance Rheumatoid arthritis is one of the most common autoimmune disease (1-2% of European population) The most significant clinical symptom is an inflammation of the synovial membrane, which causes a painful swelling of an articulations and the ankylosis. This diagnostic process plays an important role in the determination of rheumatoid factor (RF) antibodies of class IgM, detectable in 60-80% of patients with RA. So ,RF antibodies are sensitive but not very specific marker. Anti-citrullinated peptides are hallmark of RA and are used in diagnostic assays, through the use of citrullinated sequences as antigens.

22. Anti-double stranded DNA (anti-dsDNA) antibodies reacting with native double stranded (ds) DNA as regarded as being specific for SLE and have been observed in approximately 50-80% of the patients. Antibodies against ds-DNA are found during active phase of SLE, the amount of the serum concentration is positively correlate with the severity of the disease.so, detection of these antibodies is important for the diagnosis and clinical monitoring of SLE. Most patients with SLE display IgG class antibodies against ds DNA. These auto-antibodies are associated with lupus nephritis. The more common current tests are an immunofluorescence assay (IFA) or ELISA. The IFA utilizes a target antigen  Crithidia luciliae , a flagellated protozoa containing a dsDNA-containing small organelle called a kinetoplast . The antibodies to dsDNA are detected semi quantitatively by demonstrating IgG bound to the kinetoplast . In contrast, with ELISA testing, the dsDNA is bound to the solid phase of the micro well plate. The serum is incubated and then the bound IgG is detected.

23. Specificity and sensitivity The microplates are coated with recombinant human ds-DNA. No cross reactivities to other autoantigens have been found. It shows sensitivity of 85% for SLE, thus allowing differentiation from other inflammatory rheumatic diseases.

24. Lupus Anticoagulant (LAC)/anti-cardiolipin ( aCL )/antiphospholipid autoantibodies ( aPL ) The antibodies are directed against the phospholipid diphosphatidyl -glycerol (cardiolipin) which is the complex of cardiolipin and plasma protein B2-glyceroprotein-1. Anti-phospholipid syndrome is an autoimmune disease characterised by thrombophilia. haematological signs are mainly venous or arterial thrombosis, hemocytopenia, pregnancy complications, neurological failures, pulmonary or cutaneous tissue damage. Other APS-induced organ manifestations can include Addison’s disease caused by thrombosis of suprarenal vessels, intestinal necrosis caused by occlusion of the intestinal vessels, hepatic venous thrombosis and liver and spleen infraction. According to “international consensus statement on an update of classification criteria for definite anti-phospholipid syndrome (APS) 2006” the presence of anti-cardiolipin antibodies is a serological criterion in APS diagnostics.

25. The specificity of ACA is slightly limited since ACA can also be detected in infections. They occur temporarily in syphilis, borreliosis or malaria without the cofactor B2-GP1, which means that they are not associated with APS. A positive ACA result should always be assessed after 12 weeks to conform APS diagnosis.

26. Line Immune Assays ANA profile IgG Clinical significance : antibodies against anti-nuclear antigens are directed against various cell nuclear components. These encompass nucleic acids, cell nucleus proteins and ribonucleoproteins. They are characteristic finding in many diseases, in particular rheumatic diseases. The frequency of anti-nuclear antibodies in inflammatory rheumatic diseases between 20% and 100%, the lowest occurring in RA at between 20% and 40% So, differential ANA diagnosis is indispensable in the identification of individual rheumatic diseases as well as useful in the diagnosis of further Autoimmune diseases. Indications of the test are MCTD, SLE, sjogren’s syndrome (SS), progressive systemic sclerosis, poly/ dermatomyositis, overlap syndrome, CREST syndrome, primary biliary liver cirrhosis.

27. Principles of the test It provides a qualitative in vitro assay for human autoantibodies of the IgG class to 14 different antigens : nRNP , Sm, SS-A(SS-A native and Ro-52), SS-B, Scl-70,PM-Scl, Jo-1, CENP B, PCNA, ds DNA, nucleosomes, histones, ribosomal P-protein and AMA M2 in serum or plasma. Strips are coated with parallel lines of highly purified antigens. In the first reaction step, diluted patient samples are incubated with the immunoblot strips. In the case of positive samples, the specific IgG antibodies will bind to the corresponding antigenic site. To detect the bound antibodies, a second incubation is carried out using an enzyme labelled anti-human IgG catalysing a colour reaction

28. Neuronal antigens(IgG) profile2 Indications : paraneoplastic neurological syndrome (PNS). Principles of the test It provides a qualitative in vitro assay for human autoantibodies of the IgG class to 6 different antigens : amphiphysin , CV2, PNMA2 (Ma2/Ta), Ri, Yo and Hu in serum or plasma. The strips coated with parallel lines of highly purified antigens and antigen fragments. In the first reaction step, diluted patient samples are incubated with the immunoblot strips. In the case of positive samples, the specific IgG antibodies will bind to the corresponding antigenic site . To detect the bound antibodies, a second incubation is carried out using an enzyme labelled anti-human IgG catalysing a colour reaction.

29. sensitivity Antibodies N sensitivity Anti- amphyphysin 2 100% Anti-CV2 4 100% Anti-PNMA2 9 89% Anti-Ri 8 100% Anti- Yo 20 100% Anti-Hu 27 100 % Specificity is 100%

30. Anti-MPO, -PR3 and –GBM (IgG) Indications : It provides a qualitative in vitro assay for human antibodies of the immunoglobulin class IgG against the three different antigens myeloperoxidase (MPO), proteinase3 (PR3) and glomerular basement membrane (GBM antigen) in serum or plasma for the diagnosis of granulomatosis with polyangiitis (GPA), microscopic polyarteritis with Goodpasture syndrome. Principle Strips are coated with parallel lines of highly purified antigens. In the first reaction step, diluted patient samples are incubated with the immunoblot strips. In the case of positive samples, the specific IgG antibodies will bind to the corresponding antigenic site. To detect the bound antibodies, a second incubation is carried out using an enzyme-labelled anti-human IgG catalysing a colour reaction.

31. Clinical significance Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies against antigens localised predominantly in the cytoplasmic granules of neutrophil and monocytes. ANCA showing a granular florescence in IIFT that is evenly spread over the entire cytoplasm of the granulocytes, excepting the nuclei, are called cANCA . It produce a predominantly smooth, partly fine granular florescence wrapped ribbon like around the cell nuclei (perinuclear) of the granulocyte are known as pANCA . ANCA associated antigens include MPO (myeloperoxidase) mostly the pANCA -associated enzyme. PR3 (proteinase3, myeloblastin ), mostly the cANCA -associated enzyme. ANCA associated autoimmune diseases Granulomatosis with polyangiitis (GPA) Microscopic polyangiitis (MPA) Eosinophilic granulomatosis with polyangiitis (EPGA) Sensitivity is 96% and specificity is 93%

32. Immunoglobulins Immunoglobulins Measuring total quantitative immunoglobulin levels are a key component to any immunological evolution. Ig levels reflects B cell function. serum Ig levels aid in disease detection. The Quantitative measurements of serum immunoglobulins are measured by nephelometry. Simple qualitative measurements of serum immunoglobulins reflect an individual's ability to mount a humoral immune response. Titres to tetanus, Haemophilus influenza type B (HiB), and pneumococcus can easily be tested to evaluate the quality of the immune response. These levels assess the function of B cells and also detect defects that may indicate immunodeficiency.

33. To assess antibody production, responses to protein and polysaccharide antigens should be evaluated. B-cell testing is done primarily by  in vivo (vaccination) studies. Protein vaccinations, like tetanus toxoid, measure T-cell dependent responses. Polysaccharide vaccines, like Pneumovax, measure T-cell independent responses Testing of specific antibody titres (such as to influenza immunization) are reported relative to protective values. These values are based on epidemiologic data regarding protection in larger populations.

34. For randomly acquired antibody levels, an initial comparison to protective values can be used to decide if a proper immune response was achieved. A four-fold increase in titres to protein vaccination indicates a normal response. A two-fold increase in titres to a polysaccharide antigen indicates a normal response. Failure to mount an appropriate response to antigen is a clue to the physician to pursue T and B cell function further.

35. Immunoglobulin Increased Decreased Ig G Infection, inflammation, hyperimmunization, IgG multiple myeloma, liver disease, rheumatic fever, systemic rheumatic disease A gammaglobulinemia, amyloidosis, leukaemia, myeloma, preeclampsia IgM Early HIV infection, infectious mononucleosis, lymphoma, macroglobulinemia, myeloma, rheumatoid arthritis Rarely a gammaglobulinemia, amyloidosis, leukaemia, myeloma IgA Chronic infections (especially of gastrointestinal tract), inflammatory bowel disease, myeloma, rheumatic fever Agammaglobulinemia, hereditary IgA deficiency, myeloma or protein-losing enteropathy

36. immunofluorescence Immunofluorescence The indirect immunofluorescence (IIF) technique, which uses various tissue sections or the human tumour cell line (HEp-2) as an antigenic source, major implications for the diagnosis of autoimmune diseases in a routine laboratory settings. The IIF staining pattern of a positive sample can be used to evaluate appropriate antigen specificities. The HEp-2 cells used for the detection of autoantibodies, that do not have a satisfactory ability to give positive IIF results for antibodies to SS-A/Ro-52 and Jo-1 ( histidyl-tRNA synthetase).

37. Indirect immunofluorescence (IIF) microscopy is a sensitive method ; but, it has some limitations. they are The lack of resources and adequately trained personnel. Low level of standardization. The photobleaching effect, which bleaches significantly in a few seconds. Time consuming and low throughput.

38. 3. Inflammatory markers Inflammatory markers Serum proteins that are produced in response to inflammation can be referred to as inflammatory markers. These proteins are mainly produced by the liver in response to stress and can also be called acute phase reactants. Pro-inflammatory cytokines such as IL-1, IL-6, and TNF-alpha induce synthesis of some acute phase proteins that include CRP, fibrinogen and haptoglobin. The inflammatory markers are not diagnostic of inflammation, but reflect abnormalities that are seen in autoimmune diseases, infections, malignancies and other illnesses.

39. Erythrocyte sedimentation rate (ESR) The ESR is the measure of the quantity of red blood cells (RBC) that precipitate in a tube in a defined time and is based on concentrations and RBC interactions with serum protein. Multiple factors influence the ESR. it includes, patient's age, gender, RBC morphology, haemoglobin concentration, and serum levels of immunoglobulin. It is not a diagnostic test, it can be used to monitor disease activity and treatment response and signal that inflammatory or infectious stress is present. For example, in rheumatoid arthritis, the ESR correlates well with disease activity

40. C-reactive protein (CRP) C-reactive protein (CRP/CRP-high sensitivity) was discovered and named for its reactivity to the C polysaccharide in the cell wall of  S. pneumoniae . CRP is an innate immune protein, helps to opsonize pathogens for phagocytosis and activates the complement system. CRP production is under the control of IL-1, IL-6, and TNF-alpha.. Unlike the ESR, CRP is a fairly stable serum protein whose measurement is not time-sensitive and is not affected by other serum components. The magnitude of inflammation directly relates to the concentration of CRP. Levels < 0.2 mg/dl are considered normal, while those >1.0 mg/ dL are suggestive off inflammation and infection. More recently, the use of high sensitivity CRP has been utilized. This test may better quantify lower levels of inflammation and has been important in evaluating cardiac disease and other inflammatory states.

41. Ferritin Serum ferritin is a storage protein for iron and its synthesis is regulated by intracellular iron, cytokines (TNF-alpha, IL-1, and IL-6), products of oxidative stress, and growth factors. Elevated levels can indicate acute or chronic sepsis, inflammation or malignancy. Diseases such as adult Still's disease, systemic-onset juvenile idiopathic arthritis, hemophagocytic lymph histiocytosis and iron overload diseases, including hemochromatosis or hemosiderosis , should be considered with elevated ferritin levels. Less common indicators of inflammatory states include: Ceruloplasmin the major copper containing protein in the blood that plays a role in iron metabolism and is increased in acute and chronic inflammatory states, pregnancy, lymphoma, rheumatoid arthritis and Alzheimer's disease.

42. Fibrinogen a haemostatic coagulation factor produced in response to tissue injury. Fibrinogen synthesis is controlled at the transcription level and is increased in the presence of inflammation and stress that is mediated by IL-6. Haptoglobin is produced in response to tissue injury. Increased levels of haptoglobin can be seen during inflammation, malignancy, surgery, trauma, peptic ulcer disease and ulcerative colitis. Decreased levels may indicate chronic liver disease or anaemia.

43. Albumin a serum protein synthesized by the liver that aids body tissues in maintaining oncotic pressure necessary for proper body fluid distribution. The average amount of albumin in the plasma is approximately 300 to 400 grams, and about 15 grams is produced by the liver per day. the rate of synthesis can double in situations of rapid albumin loss as seen in glomerulonephritis or inflammatory bowel disease, serum levels will decline.

44. 4. Flow cytometry Flow cytometry is a technique where particles or tagged cells flow through laser light so that populations of particle/cells can be counted and phenotyped using cell characteristics and surface proteins. Initial applications of flow cytometry pertained to the interest in certain cell populations, for example the numbers of lymphocytes in patients infected with human immunodeficiency virus (HIV). The number of T cells that are CD4 positive is an important gauge of severity of HIV infection. By using flow cytometry, cell cycle analysis, quantification of malignant cells and activation status of lymphocyte subpopulations can be determined. When evaluating a patient with a suspected immunodeficiency, flow cytometry is crucial to determine the quantitative number of immune cells (typically T, B and NK (natural killer) cells). flow cytometry testing reveals numbers of cells and does not indicate cellular function. Testing for cellular functioning involves other laboratory methods, such as quantitative immunoglobulin levels to indicate proper B cell function.

45. 5. Cytokine studies Cytokine studies Cytokines are molecules secreted by a variety of cells that function in cellular communication. Immunologists are keenly interested in cytokines, particularly those that influence immune function and inflammation. this testing is largely done in research laboratories. Testing is laborious because of the labile nature of these small molecules. After phlebotomy, the serum needs to be quickly removed from the cellular components and frozen as quickly as possible and testing should not be delayed. Laboratory methods commonly used to assay cytokine levels include flow cytometry and ELISA.

46. Cytokines that influence inflammation include IL-1, IL-6 and TNF-alpha. these cytokines promote inflammation and become targets for therapy. Rheumatoid arthritis is the best example of an autoimmune illness where anti-TNF therapy has revolutionized the natural history of the disease. Targeting TNF with proteins (fusion produced or monoclonal antibodies) that antagonize TNF action results in dramatic improvement of disease activity. In fact, rheumatoid arthritis is the prototypic autoimmune disease where the efficacy of anti-cytokine therapy is best demonstrated. Currently, anti-TNF, anti-IL-1 and anti-IL-6 therapies are proven to be effective in treating rheumatoid arthritis.

47. 6. Major Histocompatibility Complex (MHC) (human leukocyte antigen (HLA)) Human leukocyte antigen (HLA) is synonymous with the major histocompatibility complex (MHC). MHC class I and II genes are the major genetic determinants of susceptibility to many autoimmune diseases. MHC class I molecules include HLA-A, -B, and –C. MHC class II molecules include HLA-DR, HLA-DQ, and HLA-DP. Detection of HLA type can be done routinely and can be assayed using several methods that include gel electrophoresis, polymerase chair reaction (PCR), ELISA, and newer methods employing high-throughput detection of nucleic acid. Many antigens of the MHC, especially of HLA class I and II, have been associated with rheumatic disorders. HLA-B27 is present in approximately 90% - 95% of white patients with ankylosing spondylitis and only 7% to 8% of the general population. HLA-DR1 and HLA-DR4 increase the risk of polyarticular juvenile idiopathic arthritis (JIA) in many populations. HLA-DR3 and HLA-DR2 are associated with lupus in Caucasian populations, while much of the risk attributable to MHC is associated with variation at HLA-DRB1 in patients with rheumatoid arthritis.

48. Conclusion Autoimmune laboratories use immunoassays as the basic technique for the determination of autoantibodies. the central and main procedure for the all autoantibody diagnostic assays is the capture of autoantibodies from the serum using immobilised autoantigens. there is an enormous variability in these tests that has lead to differences In results, a variable degree of confidence in their utility and even misdiagnosis of the patients disease. There is no universal solution to resolve these problem, but it is possible to improve the standardisation level for techniques and methods.

49. Thank you