Evaluation of Anti-dsDNA Antibodies in Laboratory Practice: Management of Different Analytical Methods and Correlation with Hep-2 Immunofluorescence Patterns

Background: Anti–double-stranded DNA (anti-dsDNA) antibodies are a key serological marker for Systemic Lupus Erythematosus (SLE) and are commonly assessed in con-junction with antinuclear antibody (ANA) testing by indirect immunofluorescence (IIF) on HEp-2 cells. However, their detection is influenced both by the heterogeneity of auto-immune response and by the characteristics of the analytical methods employed, com-plicating the diagnostic interpretation. Methods: In this retrospective single-center study, 3,090 patients undergoing anti-dsDNA analysis were screened, and 138 positive indi-viduals, with anti-dsDNA levels ≥15 IU/mL by fluoroenzyme immunoassay (FEIA), were included in the study. A control group of 29 anti-dsDNA–negative patients was also analyzed. Anti-dsDNA–positive patients were stratified by antibody levels (low, mild, high), and results were correlated with HEp-2 IIF titers and fluorescence patterns. In a subset of 30 positive patients, the anti-dsDNA antibodies had also been evaluated using immunoblot (IB) and Crithidia Luciliae indirect Immunofluorescence Test (CLIFT). Sta-tistical analyses assessed associations and concordance among methods. Results: Higher anti-dsDNA levels were generally associated with higher Hep-2 IIF titers. However, a considerable percentage (35%) of patients with positive anti-dsDNA were negative by HEp-2 IIF. Notably, high anti-dsDNA levels were detected in 19% of Hep-2 IIF–negative patients (titer 1:320). In the subset of 30 positive patients, FEIA analysis showed high concordance with immunoblot in both IIF positive (81%) and negative (100%) patients while CLIFT demonstrated lower agreement with both FEIA and IB independently from the IIF. Conclusions: Our findings indicate that anti-dsDNA an-tibody detection may occur independently of HEp-2 IIF positivity and that FEIA, par-ticularly when confirmed by immunoblot, represents a reliable approach for anti-dsDNA assessment. The observed results in this study likely reflect differences in epitope recognition and assay sensitivity among methods, suggesting the use of a multi-step diagnostic strategy in the serological evaluation of SLE.