D.Antova, MD,M.Sc.

1. IMPORTANCE OF AUTOANTIBODIES IN THE CLINICAL EXPRESSION AND ACTIVITY OF AUTOIMMUNE CONNECTIVE TISSUE DISEASES D.Antova, MD,M.Sc. PHI University Clinic for Rheumatology, Faculty of Medicine, University "St. Cyril and Methodius", Skopje, Republic of Macedonia.

2. The immunoserologic tests are of special significance in patients with few clinical criteria and help in the confirmation or dismissal of the diagnosis.

3. PUTTING TOGETHER THE DIAGNOSTIC MOSAIC IN AUTOIMMUNE DISEASES

4. PRINCIPLES OF AUTOANTIBODY DETECTION

5. ANTINUCLEAR ANTIBODIES (ANA) • Serologic marker for the diagnosis of autoimmune diseases • Part of the classification criteria for the diagnosis of Lupus erythematosussystemicus (SLE), MCTD, Sclerosis Systemica (SSc) and Sjogren’sSyndrome(SjS). • Present in other non-rheumatologic autoimmune diseases such as autoimmune hepatitis, autoimmune thyroiditis, drug-induced autoimmune syndrome. • Useful in the diagnosis of systemic diseases, but not in following disease activity.

6. The method of indirect immunofluorescence (IIF) on a HEp2 cell line is a gold standard in the detection of ANA.With the use of the Hep2 cell line, sensitivity of ANA detection is increased, whereas specificity is reduced.The use of ELISA for ANA detection is cheaper, but with a lower specificity. ANTINUCLEAR ANTIBODIES (ANA)

7. ANTINUCLEAR ANTIBODIES (ANA) • Patients with suspected autoimmune disease have significantly higher ANA titres (≥1: 160) compared to those without autoimmune disease . • In practice, only high-titre patients (≥1: 160) should be examined in the direction of autoimmune disease • In children's population of patients, the ANA profile works when the ANA titre is ≥1: 640 • ANA titres greater than 1: 1280 are associated with SLE in children

8. SPECIFIC АNА FOR THE DIAGNOSIS OF RHEUMATOID ARTHRITIS: Anti Cyclic Citrullinated Peptide antibody (Anti- CCP ) • Аnti – CCP absare a part of the revised criteria for the diagnosis of rheumatoid arthritis (RA) • Useful diagnostic biomarker for the early diagnosis of RA with nearly 95% specificity, and sensitivity similar to RF • Anti-CCP antibodies are present in children with JIA, PsA, SLE (Jaccoud arthritis), SjS, inflammatory myopathies and active tuberculosis, • Tested with ELISA.

9. Significance in disease activityfor these antibodies • No proof that anti-CCP levels correlate with disease activity Significance in disease prognosis • Anti-CCP are a useful prognostic test • Present few years before clinical symptoms • Marker for future disease (RA) • Indicate higher risk for aggressive joint damage

10. Elisys Duo-HUMANFully Automated ELISA Analyzer

11. SPECIFIC АNА FOR THE DIAGNOSIS OF LUPUS ERYTHEMATOSUS SYSTEMICUS (SLE)

12. SPECIFIC АNА FOR THE DIAGNOSIS OF LUPUS ERYTHEMATOSUS SYSTEMICUS (SLE) Significance of anti-dsDNA antibodies in disease activity • Anti-dsDNA abs are a specific serologic marker for SLE • Levels change with disease activity • High degree of association between the level of IgG anti-dsDNA and activity of patients with lupus nephritis • Anti-dsDNA can be positive in patients treated with minocycline, etanercept, infliximab and penicillamine Isenberg DA,MansonJJ,Ehrenstein MR,RahmanA.Fiftiyears of anti ds DNA antibodies:are we approaching journeiy’s and?Rheumatology (Oxford) 2007; 46: 1052

13. SPECIFIC АNА FOR THE DIAGNOSIS OF LUPUS ERYTHEMATOSUS SYSTEMICUS (SLE) Significance of anti-Sm antibodies in disease activity • Highly specific marker for SLE • Anti-Smstay positive in patients in remission • Significant in the detection of patients with low to no disease activity in SLE BeaufilsM, Kouki F, Mignon F, et al. Clinical significance of anti-Sm antibodies in systemic lupus erythematosus. Am J Med 1983; 74:201

14. SPECIFIC АNА FOR THE DIAGNOSIS OF SYSTEMIC SCLEROSIS (SSc)

15. Nihtyanova SI, Denton CP. Autoantibodies as predictive tools in systemic sclerosis. Nat Rev Rheumatol 2010; 6:112.

16. SPECIFIC АNА FOR THE DIAGNOSIS OF SYSTEMIC SCLEROSIS (SSc) Clinical significance • Routinely most used antibodies for the diagnosis of SSc are anti-scl70 and anti-centromere (ACA) with high specificity (>99.5%) and moderate sensitivity (20-50%) • Useful in the confirmation, but not in the dismissal of diagnosis • Follow-up of these abs is necessary in patients suspected for SSc

17. SPECIFIC АNА FOR THE DIAGNOSIS OF Polymyositis/Dermatomyositis (PM/DM) Gunawardena H, Betteridge ZE, McHugh NJ.Myositis-specific autoantibodies: theirclinical and pathogenic significance in disease expression. Rheumatology (Oxford). 2009; 48(6):607

18. Anti-Neutrophil Cytoplasmic Antibodies (ANCA) FOR THE DIAGNOSIS OF VASCULITIS John H Stone, MD, MPHRichard J Glassock, MD, MACP,Gerald B Appel, MD, Clinical spectrum of antineutrophil cytoplasmic antibodies.Aug 2013. | This topic last updated: Jan 02, 2013.

19. DISEASES WITH STRONG ASSOCIATION WITH ANCA Jennette JC. Nomenclature and classification of vasculitis: lessons learned fromgranulomatosiswith polyangiitis (Wegener's granulomatosis). Clin Exp Immunol2011;164: 710.

20. DISEASES WITH STRONG ASSOCIATION WITH ANCA • Predictive value of the test for these ANCA positive diseases is 98% • Higher levels of ANCA cannot be the only parameter for the diagnosis of disease relapse and justify the use of immunosupressants • If the patient during disease activity was ANCA positive, and during treatment becomes ANCA negative, that does not mean disease remission by itself

21. SPECIFIC АNА FOR THE DIAGNOSIS OF SJOGREN`S SYNDROME (SjS) Reveille JD, Solomon DH, Schur PH, Kavanaugh A. Evidence based guidelines for the use of immunologic laboratory tests: Anti-Ro (SS-A) and La (SS-B). Arthritis Care Res 2013;

22. SPECIFIC АNА FOR THE DIAGNOSIS OF SJOGREN`S SYNDROME (SjS) Anti-SSA/Ro abscan be present in patients with: • Lupus erythematosus systemicus (SLE)( 32%) • Dermatomyositis or polymyositis (19%) • Sclerosis systemica (21%) • Cirrhosis biliaris prim. • Rheumatoid arthritis (15%)

23. SPECIFIC АNА FOR THE DIAGNOSIS OF SJOGREN`S SYNDROME (SjS) • Presence of antiSS-A/Ro with or without antiSS-B/La are one of the six criteria for the diagnosis of SjS • High levels of these abs correlates with a higher incidence of extraglandular manifestations, especially purpura, skin vasculitis and demyelinating syndrome similar to Multiple Sclerosis (MS) • Levels of these abs do not correlate with disease activity

24. SPECIFIC АNА FOR THE DIAGNOSIS OF SJOGREN`S SYNDROME (SjS) • Can be detected before the diagnosis of SLE and are positive in half of ANA negative patients with SLE • Present in certain subsets of patients with SLE (subcutaneous lupus erythematosus) • Move thorough the placental barrier and cause transitory photosensitive rash and/or congenital heart block in the fetus

25. SPECIFIC АNА FOR THE DIAGNOSIS OF MIXED CONNECTIVE TISSUE DISEASE (MCTD) Anti-U1RNP antibodies are present in patients with SLE and SSc, while in patients with MCTD this is sine qua non for the diagnosis of this disease. Bennett RM. Overlap Syndromes. In: Textbook ofRheumatology 8th, WB Saunders Co, Philadelphia 2009. p.1381.

26. CONCLUSION • Autoantibodies have a significant diagnostic potential and a discriminatory ability for patients with different autoimmune diseases • Autoantibodies together with other serologic markers are important in the monitoring of disease activity

27. The presantion is sponsored and initated by Biotec- Macedonia and is intended for education only for Healthcare profesionals.