Anti-dsDNA antikorlarının saptanmasında üç ELISA yönteminin CLIF testiyle karşılaştırılması

Yıl 2015, Cilt: 72 Sayı: 2, 103 - 108, 01.06.2015

Feyza Çetin Alparslan Toyran Özlem Aytaç Feride Alaca Coşkun İpek Mumcuoğlu Feyza Alp Altan Aksoy

Öz

Amaç: Sistemik lupus eritematozus SLE hücre çekirdeğindeki antijenlere karşı antikorların oluşturduğu otoimmün bir hastalıktır. Amerikan Romatoloji Derneği ACR kriterlerine göre SLE tanısında kullanılan immünolojik parametreler anti nükleer antikor ANA ve anti-dsDNA otoantikorlarıdır. Bu çalışmada anti-dsDNA antikorların saptanmasında, CLIF yöntemi referans yöntem kabul edilerek, üç farklı ELISA kitinin özgüllüğünün araştırılması amaçlanmıştır. Yöntemler: Çalışmaya 1 Mayıs – 31 Temmuz 2013 tarihleri arasında hastanemiz mikrobiyoloji laboratuvarına gönderilen klinik olarak SLE tanısı almış olan 81 hastanın serum örnekleri dahil edilmiştir. Bu serumlarda ANA varlığı öncelikle immünfloresan antikor IFA yöntemiyle araştırılmıştır. Pozitif serum örnekleri -80 OC’de prospektif analiz için saklanmış ve aynı gün dört farklı anti-dsDNA testi ile çalışılmıştır. Bu testler; üç adet anti-dsDNA ELISA kiti; CHORUS dsDNA-G DIESSE Diagnostica Senese, İtalya , Anti-dsDNA-Ncx ELISA IgG EUROİMMUN, Almanya , QUANTA Lite dsDNA SC ELISA INOVA Diagnostics, Kaliforniya ve CLIF testi Crithidia luciliae anti-dsDNA, EUROIMMUN, Almanya idi. Bulgular: Hastaların IFA yöntemi ile ANA paternleri; %35,8 homojen patern, %22,2 homojen+diğer patern, %24,7 granüler patern, %8,7 granüler + diğer paternler, %7,4 nükleolar patern ve %1,2 sentromer patern olarak belirlenmiştir. Bu sonuçlara göre SLE hastalarında en sık rastlanan ANA paterni homojen patern olmuştur. Çalışılan 81 serum örneğinin anti-dsDNA pozitiflikleri; CLIF yöntemi ile 22 %27 , INOVA ile 46 %56 , EUROIMMUN ile 34 %41 , CHORUS ile 50 %61 saptanmıştır. CLIF referans yöntem kabul edildiğinde ELISA kitlerinin özgüllükleri ve pozitif prediktif değerleri PPD sırasıyla; CHORUS ile %50, %42; INOVA ile %54, %41; EUROIMMUN ile %71, %50’dir.Sonuç: ANA pozitif olgularda incelenen yöntemler içinde Anti-dsDNA-Ncx ELISA IgG yöntemi diğer yöntemlere göre daha yüksek özgüllük ve PPD’e sahiptir. Bu çalışmanın CLIF yöntemi kullanılmadığında seçilecek ELISA yöntemi açısından laboratuvarlara yol gösterici olacağı düşünülmektedir

Anahtar Kelimeler

Sistemik lupus eritematozus, SLE, anti-dsDNA, CLIF, ELISA

Comparison of three ELISA methods with CLIF test for detection of Anti-dsDNA antibody

Öz

Objective: Systemic lupus erythematosus SLE is an autoimmune disease caused by antibodies which produced against antigens commonly on cell nuclei. According to the criteria of American College of Rheumatology ACR , the immunological parameters which are used for diagnosis of SLE, are anti- nuclear antibodies ANA and anti- dsDNA autoantibodies. In this study it is aimed to investigate the specifity of three different ELISA tests by comparing with CLIF test, as a reference method, for the determination of anti-dsDNA antibodies. Methods: Sera of 81 patients who were diagnosed as SLE and sent to the Microbiology Department of our hospital between 1 May - 31 July 2013, were included in the study. In these sera, ANA positivity was firstly investigated by immunofluorescence antibody test IFA . Positive sera were stored at -80 OC for prospective analysis and processed with four different anti-dsDNA assays at the same day. These assays were three antidsDNA ELISA kits including; CHORUS dsDNA-G DIESSE Diagnostica Senese, Italy , anti-dsDNA-Ncx ELISA IgG EUROIMMUN, Germany, California , QUANTA Lite dsDNA SC ELISA INOVA Diagnostics and CLIF test Crithidia luciliae anti-dsDNA, EUROIMMUN, Germany . Results: ANA patterns of patients defined by IFA were determined as; 35.8% homogeneous pattern, 22.2% homogeneous and other patterns, 24.7% granular pattern, 8.7% granular and other patterns, 7.4% nucleolar pattern and 1.2% centromere pattern. According to these results, most common ANA pattern in SLE patients was found as homogeneous pattern. AntidsDNA positiveness of 81 sera samples were 22 27% with CLIF test, 46 56% with INOVA, 34 41% with EUROIMMUN and 50 61% with CHORUS. When CLIF test was considered as reference method, specificity, and positive predictive value PPV of ELISA kits were respectively; 50%, 42% for CHORUS; 54%, 41%for INOVA and 71%, 50% for EUROIMMUN. Conclusion: Anti-ds DNA-NcX IgG ELISA method had higher specifity and PPV than other tested methods in ANA positive cases. It is thought that this study may guide the laboratories to choose ELISA method in lack of CLIF method.

Anahtar Kelimeler

Systemic lupus eriythematozus, SLE, anti-dsDNA, CLIF, ELISA

Kaynakça

• 1. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol, 2000; 53 (6): 424–32.
• 2. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum, 1982; 25 (11): 1271–7.
• 3. Hochberg MC. Updating the American College of Rheumatology Revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum, 1997 ; 40 (9): 1725.
• 4. Gniewek RA, Stites DP, McHugh TM, Hilton JF, Nakagawa M. Comparison of antibody testing methods: immunofluorescence assay versus enzyme immunoassay. Clin Diag Lab Immunol, 1997; 4 (2): 185-8.
• 5. Tan EM, Feltkamp TE, Smolen JS, Butcher B, Dawkins R, Fritzler MJ et al. Range of antinuclear antibodies in “healthy” individuals. Arthritis Rheum, 1997; 40 (9): 1601–11.
• 6. Juby AG, Davis P. Prevalence and disease associations of certain autoantibodies in elderly patients. Clin Invest Med, 1998; 21 (1): 4–11.
• 7. Sheil WC Jr, Jason M. Diagnostic associations of patients with antinuclear antibodies referred to community rheumatologists. J Rheumatol, 1989; 16 (6): 782–5.
• 8. Hughes R, UI-Hassan S. Anti-dsDNA antibodies: their role in the detection and monitoring of SLE. CLI, 2006; 7: 12-7.
• 9. Isenberg DA, Manson JJ, Ehrenstein MR, Rahman A. Fifty years of anti-dsDNA antibodies: are we approaching journey’s end? Rheumatology, 2007; 46(7): 1052-6.
• 10. Hahn BH. Antibodies to DNA. N Engl J Med, 1998; 338(19): 1359-68.
• 11. Biesen R, Dahnrich C, Rosemann A, Barkhudarova F, Rose T, Jakop O et al. Anti-dsDNA-NcX ELISA: dsDNA-loaded nucleosomes improve diagnosis and monitoring of disease activity in systemic lupus erythematosus. Arthritis Res Ther, 2011; 13(1): 26.
• 12. Werle E, Blazek M, Fiehn W. The clinical significance of measuring different anti-dsDNA antibodies by using the Farr assay, an enzyme immunoassay and a Crithidia luciliae immunofluorescence test. Lupus, 1992; 1: 369–77.
• 13. Hodinka L, Meretey K, Jancso A, Falus A, Korda J, Bozsoky S. Antibodies to native DNA in connective tissue disease. A comparison of radioimmunoassay, counter immunoelectrophoresis and indirect immunofluorescence on Crithidia luciliae substrate. ArchImmunol Ther Exp, 1979; 27: 641–6.
• 14. Holman HR, Kunkel HG. Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science, 1957; 126: 162–3.
• 15. Friou GJ. Antinuclear antibodies: diagnostic significance and methods. Arthritis Rheum, 1967; 10: 151–9.
• 16. Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis, 2010; 69(8): 1420–2.
• 17. Solomon DH, Kavanaugh AJ, Schur PH. Evidencebased guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum, 2002; 47(4): 434–44.
• 18. Von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin Arthritis Rheum, 1995; 24(5): 323-58.
• 19. Fritzler MJ. Clinical relevance of autoantibodies in systemic rheumatic diseases. Mol Biol Rep, 1996; 23(3-4): 133–45.
• 20. Sjöwall C, Sturm M, Dahle C, Bengtsson AA, Jönsen A, Sturfelt G et al. Abnormal antinuclear antibody titer are less common than generally assumed in established cases of systemic lupus erythematosus. J Rheumatol, 2008; 35(10): 1994–2000.
• 21. Frodlund M, Dahlström O, Kastbom A, Skogh T, Sjöwall C. Associations between antinuclear antibody staining patterns and clinical features of systemic lupus erythematosus: analysis of a regional Swedish register. BMJ, 2013; 3(10): e003608.
• 22. Suh-Lailam B, Chiaro TR, Davis KW, Wilson AR, Tebo AE. Evaluation of a high avidity anti-dsDNA IgG enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus. Int J Clin Exp Pathol, 2011; 4(8): 748-54.
• 23. Venner AA, Ibanez D, Gladman DD, Urowitz MB, MacKinnon A, Blasutig IM et al. Comparison of three anti-dsDNA assays: Performance and correlation with systemic lupus erythematosus disease activity. Clinical Biochemistry, 2013; 46: 317-20.
• 24. Chiaro TR, Davis KW, Wilson A, Suh-Lailam B, Tebo AE. Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for thediagnosis of systemic lupus erythematosus--implications for interlaboratorytesting. Clin Chim Acta. 2011; 412 (11- 12): 1076-80.
• 25. Buzzulini F, Rigon A, Soda P, Onofri L, Infantino M, Arcarese L et al. The classification of Crithidia luciliae immunofluorescence test (CLIFT) using a novel automated system. Arthritis Research Therapy, 2014; 16: 71.
• 26. Ghirardello A, Villalta D, Morozzi G, Afeltra A, Galeazzi M, Gerli R et al. Diagnostic accuracy of currently available anti-double-stranded DNA antibody assays. Clin Exp Rheumatol, 2011; 29(1): 50-6.
• 27. Andrejevic S, Jeremic I, Bukilica MS, Nikolic M, Stojimirovic B, Nikolic BB. Immunoserological parameters in SLE: high-avidity anti-dsDNA detected by ELISA are the most closely associated with the disease activity. Clin Rheumatol, 2013; 32: 1619-26.
• 28. Compagno M, Jacobsen S, Rekvig OP, Truedsson L, Heegaard NH, Nossent J et al. Low diagnostic and predictive value of anti-dsDNA antibodies in unselected patients with recent onset of rheumatic symptoms: results from a long-term follow-up Scandinavian multicentre study. Scand J Rheumatol, 2013; 42(4): 311-6.