B-200 CORRELATION OF ANTI-DFS70 AUTOANTIBODY WITH HEP-20-10 CELL IMMUNOFLUORESCENCE ASSAY FOR NUCLEAR DENSE FINE SPECKLED PATTERN AC-2

Abstract

Over the past two decades, the identification and characterization of the dense fine speckled (DFS; ICAP AC-2) nuclear pattern in antinuclear antibody (ANA) testing-observed using HEp-2 or HEp-20-10 cells in indirect immunofluorescence assays (IFA)-has significantly influenced the landscape of ANA diagnostics. This specific AC-2 pattern is associated with antibodies targeting DFS70 (also known as lens epithelium-derived growth factor, LEDGF). Although the clinical significance of anti-DFS70 antibodies remains under investigation, emerging evidence suggests that high titers of these antibodies, particularly when present in isolation without other disease-specific autoantibodies, may be useful in excluding systemic autoimmune rheumatic diseases (SARDs) or ANA-associated rheumatic diseases (AARDs).(1,2) The correlation between the AC-2 pattern seen by IFA and the detection of anti-DFS70 antibodies through solid-phase assays continues to be an area of active research and clinical interest. This study analyzed a total of 514 serum samples, including 464 samples previously characterized as ANA-positive and 50 as ANA-negative based on indirect immunofluorescence assay (IFA) using HEp-20-10 cells (Sprinter XL, EUROIMMUN AG). All samples were further evaluated using both the Anti-DFS70 ELISA (EUROIMMUN AG) and the EUROLINE ANA Profile line blot immunoassay, which includes targets such as Mi-2, Ku, DFS70, nRNP/Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl100, Jo-1, CENP-B, PCNA, dsDNA, nucleosomes, histones, ribosomal P-proteins, and AMA M2 (EUROBlotOne, EUROIMMUN AG). These assays were employed to detect anti-DFS70 antibodies as well as a broad panel of other disease-associated autoantibodies. This is the first study to examine the correlation between anti-DFS70 antibodies, other SARD-associated autoantibodies, and ICAP-defined ANA patterns. Among samples exhibiting the AC-2 pattern, 99% were positive for anti-DFS70 antibodies as detected by either ELISA or EUROLINE. Anti-DFS70 antibodies were also identified in samples with other ANA patterns, including AC-1 (27.7%), AC-4/5/31 (15.9%), and AC-3 (18.2%). Notably, 4% of ANA-negative samples (AC-0) were positive for anti-DFS70 antibodies. Among all samples positive for anti-DFS70 by either assay, approximately 70% showed an exclusive AC-2 pattern on IFA. However, in samples where AC-2 was the sole ANA pattern observed, multiplex EUROLINE testing revealed that around 30% of patients also harbored additional SARD-associated autoantibodies-most commonly anti-dsDNA, anti-nucleosome, anti-Mi-2, anti-centromere B, anti-Scl-70, and anti-RNP-though these occurred at low frequency. In this study, we demonstrated that nearly all samples exhibiting the AC-2 pattern on HEp-20-10 IFA were positive for anti-DFS70 antibodies, as detected by either ELISA or EUROLINE. While other autoantibodies were occasionally associated with the AC-2 pattern, their frequency was low. Notably, only 70% of AC-2 pattern samples showed anti-DFS70 reactivity in the absence of other SARD-associated antibodies.