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LABORATORY TESTING IN THE RHEUMATIC DISEASES: PART 2

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  1. LABORATORY TESTING IN THE RHEUMATIC DISEASES: PART 2 Judith A. James, MD, PhDUniversity of Oklahoma Health Sciences CenterOklahoma Medical Research Foundation

  2. LEARNING OBJECTIVES • To understand the strengths and weakness of anti-nuclear antibody testing in systemic rheumatic disease. • To be able to define and understand the role of select, clinically-relevant autoantibodies in the diagnosis, monitoring and prognosis of Systemic Lupus Erythematosus (SLE). • To understand historical and new autoantibody testing in Rheumatoid Arthritis (RA) and the most common cause of false positive results. • To understand the major common autoantibodies and their clinical associations with vasculitis, scleroderma, Sjogren’s syndrome (SS) and inflammatory myositis.

  3. MODULE OUTLINE • General Concepts • History • Methods of Detection • Indirect immunofluorescence ANA test (ANA) • Immunodiffusion • Counterimmunoelectrophoresis (CIE) • Immunoprecipitation • Immunoblotting • Enzyme Linked Immunoabsorbent Assays (ELISA) • Enzyme Inhibition Assays • Anti-Nuclear Antibodies (ANA) • Anti-Deoxyribonucleic Acid (dsDNA) antibodies • Anti- Extractable Nuclear Antigens (ENAs) • Anti-Smith (Sm) • Anti-Ribonucleoprotein (RNP) • Anti-Ro/SSA • Anti-La/SSB Antibodies • Antiphospolipid Antibodies • Anticardiolipin antibodies (aCL) • Lupus Anticoagulant • Antineutrophilcytoplasmic antibodies (ANCAs) • Anti-Centromere • Anti-Scl-70 • Anti-Histone Antibodies • Antiribosomal P (Ribo P) Antibodies • Autoantibodies in Inflammatory Muscle Diseases • Myositis-specific autoantibodies (MSAs) • Overlap antibodies • Rheumatoid Factor (RF) • AnticyclicCitrullinated Peptide Antibodies (Anti-CCP) • Clinical Vignettes/Test Questions

  4. Autoantibodies • Immunoglobulins (immune proteins) that mistakenly target and sometimes damage specific tissues or organs of the body; directed against autologous (self) antigens • Intracellular, cell surface or extracellular antigens • Autoantibodies are usually Immunoglobulin G (IgG) class and somatically mutated, reflecting T cell help in the germinal center • Used for diagnostic purposes and may be used to predict future clinical manifestations in: • Inflammatory rheumatic diseases • Autoimmune conditions not often thought of as rheumatic diseases • Primary biliary cirrhosis, autoimmune hepatitis, some forms of glomerulonephritis • Can also be detected in healthy individuals usually in low titers and with low affinities

  5. Autoantibodies • Produced by B cells • May be pathogenic (eg. immune complexes in lupus nephritis) • Markers of certain diseases • Not diagnostic • Apart from rheumatic disorders, they may be found in normal population & with other conditions • Antinuclear antibodies (ANA) are a group of autoantibodies that react with a variety of nuclear antigens • hallmarks of systemic autoimmune diseases • Therefore only test when clinically indicated.

  6. Autoantibodies • ANA • An autoantibody against a part of the nucleus • Screening test for SLE: so if negative it makes SLE highly unlikely • High sensitivity for SLE • If negative, usually Ro/SSA autoantibodies are detected • Strong positive titers have a larger dilution (higher denominator) • Ex. 1:1280 is a strong positive • Titers in rheumatic disease patient often times exceed 1:1000 • Can exceed 1:10,000 in Mixed Connective Tissue Disease (MCTD) • Highest titer at which fluorescence can still be clearly seen is reported • Titers do not correlate with disease activity • Pattern can change depending on the dilution • Ex. 1:80 speckled and homogenous and 1:640 homogenous

  7. Lab Med 124:71, 2000.

  8. Autoantibodies • ANA Patterns • Observation of the staining pattern • Reflect reaction to specific antigens • Most not unique to identify specific antibody • Most common: • Nulcear homogeneous • Nuclear speckled • Nucleolar

  9. Autoantibody patterns detected on HEp-2 cells Nuclear homogeneous Nuclear coarse speckled Centromere Nucleolar

  10. Autoantibodies • Anti-DNA antibodies • First characterized antigen targeted by anti-nuclear antibodies • Anti-ss-DNA antibodies • Not specific for given rheumatic disease and no longer generally tested. • Anti-ds-DNA antibodies • Anti-ds-DNA antibodies appear in approximately 73% of SLE patients, whereas low titers appear much less often in patients with Sjogren’s, RA, other disorders, or in normal individuals • Strongly correlate with nephritis and disease activity Crithidialuciliaeshowing staining of dsDNA in the kinetoplast and more faintly, in the nucleus

  11. Autoantibodies • Anti-DNA antibodies • Contribute to disease pathology through: • High avidity to DNA • Immune-complex formation • Complement fixation • Renal antigen cross-reactivity • Direct cellular toxicity upon intracellular penetration • Combination of all above mentioned pathways • Pathogenic anti-DNA are thought to be high avidity IgG reacting with double stranded DNA

  12. Autoantibodies • Anti- Extractable Nuclear Antigens (ENAs) • Antibodies to extractable nuclear antigens • Defined with the relatively insensitive double immunodiffusion and most not confirmed with the new ELISAs or bead-based assays • Many antigens within the nucleus, but only a small number have currently known clinical utility: • Anti-Smith (Sm) • Anti-Ribonucleoprotein (RNP) • Anti-Ro/SSA (in the cytoplasm) • Anti-La/SSB Antibodies • Anti- Topoisomerase 1 (Scl-70) • Anti Jo-1

  13. Autoantibodies • Anti-Smith (Sm) • Directed against spliceosomal proteins, • Associated with multiple U RNAs • Detected by immunodiffusion, ELISAs or bead based assays • Immunologic criterion for SLE classification • Nearly only found (especially by immunodiffusion) in SLE • Within SLE may be associated with increased mortality, renal and Central Nervous System involvement • More common in African-American SLE patients (but only 10-15% of SLE patients) • Usually patients with anti-Sm also have anti-nRNP responses • Usually nuclear speckled ANA (as are anti-nRNP) • Anti-Ribonucleoprotein (RNP) • Directed against spliceosomal proteins, associated with U1 RNA • Detected by immunodiffusion, ELISAs or bead based assays • Found in about 20 to 40% of patients with SLE (also enriched in AA patients) • Defining feature of MCTD (Mixed Connective Tissue Disease) • In SLE, usually absence of renal involvement or milder form if anti-nRNP alone • Testing should be limited to patients with a + ANA test • Clinical features associated with anti-nRNP responses: Raynaud’s phenomenon, myositis, arthritis and arthralgias, esophageal dysmotility, sclerodactly

  14. Autoantibodies • Anti-Ro/SSA and Anti-La/SSB Antibodies • Detected by immunodiffusion, ELISAs or solid phase assays. • Two antibody specificities that target distinct ribonucleoprotein particles • Prevalence: • High frequency in patients with Sjögren’s syndrome (SS) and in SLE. • 40% of SLE patients (particularly those who are ANA negative) • 75% of patients with primary SS • Low prevalence in Lupus nephritis and high prevalence in RF positivity. • Present in 0.1% to 0.5% of healthy adults • Anti-La is rarely seen without anti-Ro antibodies

  15. Autoantibodies • Anti-Ro/SSA Antibodies • Associated with: • Photosensitive skin rash • SubacuteCutaneous Lupus Erythematosus • Pulmonary disease • Interstitial lung disease • Lymphopenia and Thrombocytopenia within SLE • Neonatal lupus syndrome • Cardiac fibroelastosis in Congential Heart Block • Primary biliary cirrhosis • High titers in Sjogren’s syndrome have greater incidence of extra glandular features, especially purpura and vasculitis • Hypergammaglobulinemia • C2 and C4 deficiency

  16. Autoantibodies • Anti-La/SSB Antibodies • Present in 87% of SS (Sjogren’s syndrome) patients • Present in 10-15% of SLE patients • Participate in the termination of transcription of RNA polymerase III • Associated with: • Neurologic involvement in SS and SLE • Vasculitis in SS • Glandular dysfunction in SS • Palpable purpura in SS • Leukopenia in SLE • Lymphopenia in SLE • Hypergammaglobulinemia in SS • Rheumatoid factor positivity in SLE and SS

  17. Autoantibodies • Autoantibodies in vasculitic syndromes • Antineutrophilcytoplasmic antibodies (ANCAs) • Autoantibodies to the cytoplasmic constituents of granulocytes • Detected by indirect immunofluorescence on ethanol-fixed neutrophils and produce a characteristic cytoplasmic fluorescence (c-ANCA) or perinuclear fluorescence (p-ANCA) • Most common antigens: • Proteinase 3 (of c-ANCA) • Myeloperoxidase (of p-ANCA)

  18. Antineutrophilcytoplasmic antibodies (ANCAs) c-ANCA p-ANCA Directed against different cytoplasmic constituents of neutrophils including myeloperoxidase, lactoferrin, elastase and other unspecified antigens Not useful unless it is confirmed by testing for antimyeloperoxidase antibodies, which may occur in several related diseases: Churg–Strauss syndrome, crescentic glomerulonephritis and microscopic polyarteritis • Antibodies to proteinase-3 • Occur in > 90% of patients with systemic Wegener’s granulomatosis (with renal or pulmonary involvement, or both) • 75% of patients with limited Wegener’s granulomatosis (without renal involvement) • 50% of patients with microscopic polyarteritis • 98% specific for Wegener’s granulomatosis, microscopic polyarteritis or idiopathic crescentic glomerulonephritis • Often precede disease activity and may guide treatment

  19. Autoantibodies • Systemic sclerosis autoantibodies • Anti-Nucleolar, Anti-Centromere and Anti-Scl-70 • Anti-Nucleolar • Common autoantibody response in systemic sclerosis (SSc) • Usually in systemic disease, but can be found in limited scleroderma • Anti-Centromere • Found almost exclusively in patients with limited cutaneous systemic sclerosis (lcSSc) • calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactily and telengiectaesia (CREST) syndrome • Associated with an increased risk of malignancy

  20. Autoantibodies • Anti-Topoisomerase-1 (Scl-70) • Target the catalytic carboxyl-terminal region of DNA topoisomerase I • 22 to 40% of scleroderma patients and generally predicts: • Diffuse cutaneous disease and proximal skin involvement • Longer disease duration • Association with cancer • Pulmonary fibrosis • Digital pitting scars • Cardiac involvement • The usual method for detection is by ELISA or immunodiffusion for confirmation

  21. Autoantibodies • Anti-Histone Antibodies • Target the protein components of nucleosomes, the DNA-protein complexes that form the substructure of transcriptionally inactive chromatin • Sensitive but nonspecific for drug-induced lupus • May be present in SLE • Useful in patients with a positive ANA test and history of exposure to medications associated with drug-induced lupus: • Chlorpromazine • Hydralazine • Isoniazid • Methyldopa • Penicillamine • Procainamide • Quinidine • Sulfasalazine

  22. Autoantibodies • Antiribosomal P (Ribo P) Antibodies • Antibodies to ribosomal components, cytoplasmic and nucleolar constituents of cells • 10% to 20% of patients with SLE in the United States and 40% to 50% of Asian patients with SLE • Rare in other rheumatic diseases • Has been variably associated with neuropsychiatric lupus, including psychosis • Associated with hepatitis and nephritis in SLE

  23. Autoantibodies • Autoimmune Hepatitis • Smooth Muscle Antibody (SMA) • Positive Titer >1:80 • Chronic active hepatitis • Titer <1:80 • Primary biliary Cirrhosis • Epstein Barr Virus (EBV) • Cytomegalovirus (CMV)

  24. AUTOANTIBODIES TO INFLAMMATORY MUSCLE DISEASES • Comprise a diverse group of illnesses often characterized by autoantibody responses against cytoplasmic antigens (90%) • Between 40% and 80% of PM/DM patients have positive ANA • Categorized by: • Myositis-specific autoantibodies (MSAs) : • Anti-synthetase antibodies (Anti-Jo-1) • Anti-signal recognition particle (SRP) • Anti-Mi-2 • Overlap antibodies : • Anti-nRNP • Anti-PM-Scl

  25. AUTOANTIBODIES TO INFLAMMATORY MUSCLE DISEASES • Anti-Jo-1 • Target class II histidyltRNAsynthetase • Most common (20-30 %) of PM/DM populations • Correlate with the "antisynthetase syndrome“ • interstitial lung disease, arthritis, Raynaud's phenomenon, mechanic's hands, hyperkeratotic lines, sclerodactyly, facial telangiectasia, calcinosis, and sicca • Anti-SRP (signal recognition particle) • 5% Polymyositis • Associate with acute onset, severe disease, resistance to therapy, cardiac involvement • Poorest overall prognosis • lower frequency of interstitial lung disease and arthritis

  26. AUTOANTIBODIES TO INFLAMMATORY MUSCLE DISEASES • Anti-Mi-2 • 15 to 20% of DM patients • More than 95 percent of patients with anti-Mi-2 have DM rather than PM • Generally associated with greater dermatologic involvement (“shawl” and “V “signs) • Anti-PM-Scl • Presence associates with myositis-scleroderma overlap without SLE features • Associated with: • Arthritis • DM skin lesions • Calcinosis • Mechanic's hands • Eczema

  27. Antiphospholipid Antibodies • Antiphospolipid Syndrome • arterial and venous thromboses • Recurrent miscarriages • Thrombocytopenia • Could be primary or secondary • Present in 30% of SLE patients • Antibodies: • Anti-Cardiolipins • Lupus Anticoagulant • Beta 2 Glycoprotein 1 (B2GP1)

  28. ANA Negative Positive Peripheral (rim) Centromere Nucleolar Diffuse (homogeneous) No disease Lab error Treatment Remission Nephroticsyndrome Anti-dsDNA Histone Raynaud’s phenomenon Scleroderma Anti-nucleoprotein SLE SLE RA Drug Induced SLE SLE RA Drug Induced SLE CREST Sdleroderma Speckled RNP Sm RO (SS-A) Scl-70 PM/Jo/SRP/Mi La (SS-B) No specificity Undifferentiated Connective Tissue Disease (UCTD) SLE RA Liver disease Any chronic inflammatory disease SLE SLE Scleroderma PM/DM SLE SLE MCTD RA Scleroderma UCTD Sjogren’ssyndrome Sjogren’ssyndrome Immunofluorescence autoantibody binding patterns and their disease associations

  29. Rheumatoid Factor (RF) • Autoantibodies directed against the Fc (constant) portion of immunoglobulin G • IgM (most common) and IgG, IgA antibodies • Measurement detected with a variety of techniques • Agglutination of sheep red blood cells • Latex particles coated with human immunoglobulin G • Enzyme linked immunosorbent assay or nephelometry • Present in most people at very low levels, but higher levels are present in 5%–10% of the population, and this percentage rises with age • Low positive predictive value • Not specific for Rheumatoid Arthritis and not Sensitive to establish diagnosis

  30. Rheumatoid Factor (RF) • Rheumatoid factor is present in ≈80% of patients with rheumatoid arthritis (RA) • 20 % of RA patients are seronegative (negative RF) • 40% seronegative in early RA • Can be found in other diseases like Sjogren’s (75-95%), scleroderma (30-40%), polymyositis (30-40%), vasculitis (30-40%), SLE (15-35%) and cryoglobulinemia (40-100%) • Can be found in non-rheumatic diseases like TB, chronic infections, other pulmonary conditions (like sarcoidosis), aging, primary biliary cirrhosis, etc. • Almost always present and in higher titer in RA when the following are present: • Subcutaneous nodules • Interstitial lung disease or any extra-articular feature of RA • Vasculitis • Also present in chronic infections (subacute bacterial endocarditis), abcesses, certain chronic liver diseases

  31. Anti-CCP Antibodies • Targets a post-translationally modified arginine residue, citrulline • Highly specific (98%) and moderately sensitive (68%) for RA • May be detected many years before the diagnosis of RA • Can also be predictive of the development of RA in patients with undifferentiated arthritis • Useful in predicting progression to RA in patients with palin- dromic rheumatism

  32. KEY POINTS • Clinical acumen continues to direct the utility of the test for ANAs • If one suspects an autoimmune condition based on the history, physical examination, and complete blood count (e.g., leucopenia, thrombocytopenia), request an ANA test. • Care should always be taken when interpreting positive results in patients

  33. CLINICAL VIGNETTES

  34. Mixed connective tissue disease • 19-year-old typist • Acute, bilateral arthralgia of her hands, wrists and knees • Two years later, she developed a mild degree of Raynaud's phenomenon, with a flare-up of the arthralgia and some proximal muscle weakness. • On investigation, she had a low Hgb(10.8mg/dl) but a normal white-cell count and differential. Her ESR was raised (63mm/h) and her serum contained ANA (titer 1:3,240; speckled pattern). • dsDNA binding was negative but antibodies to ENA were detected and found to be largely directed against nuclear ribonucleoprotein (RNP); there were no antibodies to the Sm antigen. A latex test for rheumatoid factor was negative. Complement levels (C3 and C4) were normal • X-rays of the hands and knees were normal. There was no proteinuria and her serum creatinine and blood urea were normal.

  35. Sjögren's syndrome • A 38-year-old woman was referred to an oral surgeon for investigation of dry mouth and progressive dental caries • She had a sister with arthritis • ESR (42mm/h) • Six months later, she developed a mild conjunctivitis and complained of sore eyes • On testing, rheumatoid factor was now positive • Abnormal Schirmer's test • She was treated with methylcellulose eye drops to prevent corneal ulceration over a period of many years. • Her rheumatoid factor titer steadily increased and ANA (1:640) and antibodies to the extractable nuclear antigens Ro/SSA and La/SSB became detectable. • Seven years after the development of the dry mouth and dry eyes she developed a mild, bilateral non-erosive polyarthritis of her hands, wrists and ankles.

  36. Polyarteritisnodosa • A 64-year-old man developed diplopia due to a right sixth nerve palsy, lethargy, weight loss and skin lesions on the right leg which were thought to be erythemanodosum • Six weeks later, he presented with aches and pains in his shoulders, which his doctor thought were due to polymyalgiarheumatica. He improved dramatically on steroids but unfortunately they had to be withdrawn because of hypertension • ESR of 104mm/h, a polymorphonuclear leucocytosis and some proteinuria (1.5g/24h) with occasional granular casts. Biopsy of a skin lesion showed non-specific changes. A renal biopsy was normal. No diagnosis was possible. • Four weeks later, he developed profound malaise with fever, marked muscle weakness and anemia • Lab at that time was: Hgb 7.7mg/dl, CRP of 70mg/l, a negative direct Coombs' test and a reticulocyte count of 5.4%. His blood urea, serum creatinine and creatinine clearance were normal, as was his serum creatinekinaselevel. • His ANA, antineutrophilcytoplasmic antibodies (ANCA) are negative, with normal C3 and C4 complement levels • Biopsy of an affected calf muscle showed a florid arteritis • All the medium-sized arteries showed reduction of their lumens or complete occlusion.

  37. Polymyositis • A 32-year-old woman with a past history of ulcerative colitis (quiescent for the last 7 years), presented with a dry cough • The cough became productive of clear sputum and she was admitted 2 months later with increasing dyspnea, myalgia and arthralgia. • A clinical diagnosis of fibrosingalveolitis was made and confirmed by transbronchial biopsy. • She was treated with prednisolone, which improved her arthralgia, and it became clear that she had a severe proximal myopathy. • Serum creatinekinase was found to be very high. • She had a circulating autoantibody to Jo1 antigen • Muscle biopsy done

  38. Autoimmune hepatitis • A 43-year-old woman presented with a 5-month history of weight loss (6kg), anorexia, irritability and generalized pruritus. • Sclera were Icteric, skin had numerous spider nevi, scratch marks on her trunk and extremities, and palmarerythema. She had hepatosplenomegaly. • Low Hgb (9.5mg/dl) with a normal white-cell count but an ESR of 140mm/h. PT was prolonged but urea and electrolytes, calcium and phosphate concentrations were normal. Although the serum albumin was normal (4.1 mg/dl), the total serum proteins were raised at 9.3 mg/dl with a raised serum bilirubin of 3.4, ALT 152iu/l and AST of 164iu/l. The alkaline phosphatase level was normal (83iu/l). • Antinuclear antibodies were strongly positive to a titre of 1:9,720. Her serum was positive for antibodies to smooth muscle to a titer of over 1:1000. • Hepatitis B surface antigen and hepatitis C antibody were absent and AFP was not detected.

  39. Antiphospholipidantibody syndrome • A 28-year-old woman with SLE was admitted with a stroke due to a cerebrovascular thrombosis. • She had had four spontaneous abortions in the past. • She was a non-smoker. • Cerebral angiography confirmed the thrombosis but showed normal vasculature otherwise. • Hgb, platelet and white-cell counts were normal as were her serum immunoglobulins, C3 and C4 levels. • ANA at 1:640 , double-stranded DNA (+ at 1:90) , and she did have high-titer antiphospholipid antibodies (Anticardiolipin IgG 80 and IgM 65). • Coagulation tests showed a prolonged kaolin-cephalin clotting time which did not correct with normal plasma (i.e. lupus anticoagulant).

  40. Rheumatoid arthritis • A 37-year-old woman gradually developed painful wrists over 3 months; she consulted her doctor only when the pain and early morning stiffness stopped her from gardening. • Both wrists and the metacarpophalangeal joints of both hands were swollen and tender but not deformed. There were no nodules or vasculitic lesions. • Raised C-reactive protein (CRP) level (27mg/l) but a normal hgb and white-cell count. A latex test for rheumatoid factor was positive 1:680 and anti-ccpresponses were positive. Antinuclear antibodies were not detected. • She was treated with ibuprofen. Despite some initial symptomatic improvement, the pain, stiffness and swelling of the hands persisted and 1 month later both knees became similarly affected. • Six months after initial presentation, she developed two subcutaneous nodules on the left elbow; these were small, painless, firm and immobile but not tender. • X-rays of the hands showed bony erosions in the metacarpal heads. She still had a raised CRP (43mg/l) but normal serum complement (C3 and C4) levels and, had she had a biopsy, pannus would have been demonstrable histologically.

  41. Systemic lupus erythematosus • A 26-year-old woman presented with painful, stiff hands of 4 weeks duration. • She had a 6-year history of Raynaud's phenomenon • She had active synovitis in several MCP and PIP joints, but all other joints were normal. She had a photosensitive skin rash, but no muscle tenderness, proteinuria or fever. • Antinuclear antibody positive at 1:1080, her anti-dsDNA response was 1:810, and her C3 and C4 levels were occasionally low. She was also found to have anti-Ro/SSA responses, but no other antibodies against extractable nuclear antigens tested. • Later, she developed a bilateral, blotchy rash on her hands and thighs, consistent with active vasculitis. • Her Raynaud's phenomenon concurrently became much worse. Following treatment with prednisone, the skin manifestations gradually disappeared and the steroids were tailed off. • C-reactive protein 8mg/l, RF Negative   C3 35mg/dl C4 05mg/dl • Biopsy of normal, sun-exposed skin: Granular deposits of IgG and complement at dermo-epidermal junction

  42. References • Primary Reading: Klippel JH ed. Primer on the Rheumatic Diseases. 13th edition. Pages 15-27. • Secondary Reading: • American College of Rheumatology Position Statement: Methodology of Testing for Antinuclear Antibodies. Committee on Rheumatologic Care. Jan 2009 • Bizzaro N, Tozzoli R, Shoenfeld Y. Are we at a stage to predict autoimmune rheumatic diseases? Arthritis Rheum. 2007 Jun;56(6):1736-44. • Bradwell AR, Hughes RG. Atlas of HEp-2 patterns and laboratory techniques. 3rd ed. 2007 • Harris E. Kelley’s Textbook of Rheumatology. 2005, 7thed • Sheldon J. Laboratory testing in autoimmune rheumatic diseases. Best Pract Res ClinRheumatol. 2004 Jun;18(3):249-69. Review. • Solomon DH, Kavanaugh AJ, Schur PH; American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: Antinuclear Antibody testing. Arthritis Rheum. 2002;47:434-444.

  43. Autoantibodies • Methods of Detection • Indirect immunofluorescence ANA test (IF) • Immunodiffusion • Low sensitivity and requires longer time, but increased specificity • Counterimmunoelectrophoresis (CIE) • Modifies the immunodiffusion technique to generate somewhat greater sensitivity • Immunoprecipitation • Increases the sensitivity of detection for antibodies against minor cellular components (i.e., myositisautoantigens); high skill level to perform and not in widespread use for most commercial assays • Immunoblot • Less sensitive than ELISA and more laborious • Enzyme Linked Immunoabsorbent Assays (ELISA) • Highly sensitive and rapid; lower specificity; very confusing when adapted to clinical practice as the significance of low-titer antibodies in the general population are unknown • Enzyme Inhibition Assays • Not frequently used in commercial settings for rheumatic disease testing

  44. Autoantibodies • Indirect immunofluorescence ANA test (IF) • Most widely used method historically for ANA detection in past several decades • Rapid and highly sensitive • Results typically are reported by two parameters: • Titer • ≥1:120 being considered clinically significant (although 1:40 is considered positive in most labs) • Pattern • Homogeneous, speckled, nucleolar and centromere patterns refer to staining of different parts of the cell • Cytoplasmic staining refers to staining of antigens in the cytoplasm • These patterns may vary with serum dilution based upon the character and titer of various antibodies present

  45. Autoantibodies Serial dilutions IF Steps • To measure results of the IF test, human tissue culture cells (HEp-2 cell line) are grown in culture and allowed to adhere to a microscope slide • The slides are treated with methyl alcohol to “fix” the cells and make them permeable before being incubated with the patient’s serum • The slide is then washed to remove unbound antibodies, followed by incubation with fluorescent antibodies that detect the binding of human antibodies to the cells • Finally the slide is viewed using a fluorescence microscope, and staining intensity and the pattern of binding are scored at various dilutions (titers) Sera and 2nd antibody to slide

  46. Autoantibodies • Hep 2 cells in IF • More sensitive substrate in IF that allows identification of many patterns • Derived from human cell lines which ensures better specificity than animal tissues • Immunofluorescence patterns are related to the cell cycle and most patterns are seen with cells in interphase subdivided into three successive periods: G1, S and G2 (G =gap, S = synthesis)

  47. Autoantibodies • ANA Patterns • Speckled • Nonspecific • Most common pattern • Pattern occurs due to antibodies directed against "extractable nuclear antigens" • Diffuse/homogeneous • Nonspecific • Second most common pattern • Pattern occurs due to antibodies directed against histone • Rim/peripheral • Specific for SLE • Pattern occurs due to antibodies directed against DNA • Centromere • Specific for Limited Systemic Sclerosis • Pattern occurs due to antibodies directed against centromere (where two chromosomes attach to each other) • Nucleolar • Relatively specific for Scleroderma (Diffuse Systemic Sclerosis) • Pattern occurs due to antibodies directed against nucleoli

  48. Detection of two autoantibody binding patterns at two different concentrations Mixed pattern on HEp-2 cells showing few nuclear dots, mitochondrial, homogeneous and speckled staining. The sample has been titeredout, the image on the left is at 1:40 and on the right at 1:320.

  49. Detection of two autoantibody binding patterns at two different concentrations The image on the left is at 1:640and on the right at 1:1280. At the highest dilution only the nuclear dots pattern can be seen

  50. Antiphospholipid Antibodies • Anti-Cardiolipin Antibodies (aCL) • Medium-to-high levels are associated with thromboembolic events and fetal loss • These autoantibodies are also found in infections and in chronic diseases • False-positive results • serological test for syphillis, hepatitis C, leprosy, Lyme disease, mycoplasma infection, tuberculosis, HIV, Legionnaire’s disease, Q fever and varicella zoster infections • IgG and IgM isotype are part of the criteria for the diagnosis of anti-phospholipid syndrome • Lupus Anticoagulant • Specific immunoglobulin against phospholipids in the cell surface that prolongs the time it takes blood to clot but does not produce a bleeding disorder • Helps to determine the cause of an unexplained thrombosis and recurrent fetal loss • Beta 2 Glycoprotein 1 (B2GP1) • Part of the criteria for the diagnosis of anti-phospholipid syndrome • Cardiolipin binding of aCLIgG depends on the existence of B2GP1