AB1108 ANALYSIS OF HOMOGENOUS PATTERN IN ANTI-NUCLEAR ANTIBODY-INDIRECT IMMUNOFLUORESCENCE ASSAY

Abstract

Background:Autoantibodies that produce the homogenous pattern on anti-nuclear antibody-indirect immunofluorescence (ANA-IIF) assay using human epithelial cell (HEp-2) substrate are histones, dsDNA and nucleosome. Homogenous pattern may be seen in patients with many different systemic autoimmune diseases as well as organ-specific autoimmune diseases. Homogenous pattern is difficult to distinguish from dense fine speckled (DFS) pattern and other staining pattern may be masked by homogenous pattern.Objectives:The purpose of this study was to analyze the profile of autoantibodies in patients with homogenous pattern on IIF-ANA assay and to find out the clinical significance of homogenous pattern.Methods:A total of 103 sera samples with homogenous pattern on IIF-ANA assay were obtained. The IIF-ANA assay was performed using the PhD system (Bio-Rad Laboratories, Hercules, CA, USA) with Kallestad HEp-2 slides (Bio-Rad Laboratories). EliA CTD Screen and EliA dsDNA (Thermo Fisher Scientific, Freiburg, Germany) were performed using the Phadia 100 system (Thermo Fisher Scientific). EliA CTD Screen has following specific antigens: U1RNP (RNP70, A, C), SS-A/Ro (60 kDa, 52 kDa), SS-B/La, Centromere B, Scl-70, Jo-1, fibrillarin, RNA Pol III, ribosomal P-protein, PM-Scl, PCNA, Mi-2, Sm, and native purified DNA. Specific autoantibody tests against histone and nucleosome assay were performed using Euroimmun microplate ELISA (Euroimmun AG, Luebeck, Germany). Western blot (WB) assay was performed to confirm the presence of anti-DFS70 using HeLa whole-cell lysates as previously described. Clinical information regarding the presence of autoimmune diseases and other clinical conditions of individual patients was obtained from a retrospective review of clinical records.Results:Of the 103 patients with homogenous pattern on IIF-ANA assay, 21 were diagnosed as systemic autoimmune rheumatic disease (SARD) or organ-specific autoimmune diseases (autoimmune group), whereas 82 were not patients with autoimmune diseases (non-autoimmune group). Among 103 patients, 51 patients (49.5%) were negative on all autoantibody tests performed in this study: CTD screening assay and specific autoantibody tests against anti-DFS70, dsDNA, histone, and nucleosome. The detection rates of autoantibodies were 10.7% for dsDNA, 15.5% for histone, and 19.4% for nucleosome. Total of 32 patients (31%) were positive in at least one of dsDNA, histone, and nucleosome autoantibodies. The detection rate of CTD screening assay was 31.1% (32/103). Of 32 CTD screening positive patients, 18 (56%) were positive and the remaining 14 (44%) were negative on dsDNA, histone or nucleosome. Anti-DFS70 antibody was detected in 6 patients (5.8%) and they have no other autoantibodies. At least one of dsDNA, histone, and nucleosome autoantibodies were positive in 42.9% (9/21) of autoimmune group and 28% (23/82) of non-autoimmune group. Positive result on CTD screening was found in 38.1% (8/21) of autoimmune group and 23.2% (19/82) of non-autoimmune group.Conclusion:It is generally accepted that homogenous pattern is caused by antibodies against dsDNA, histone and nucleosome and have a clinical relevance with SLE, chronic autoimmune hepatitis, and juvenile idiopathic arthritis. However, the results of this study found that the majority (79.6%) of patients with homogenous pattern had no autoimmune diseases and only 31% had autoantibodies to dsDNA, histone or nucleosome. Especially 49.5% of patients were all negative for autoantibodies included in CTD screening assay as well as autoantibodies responsible for homogenous pattern such as autoantibodies against dsDNA, histone, and nucleosome. This suggests that homogenous pattern may be originated by autoantibodies that cannot be identified by conventional test methods.Disclosure of Interests:None declared