Evaluating the consistency of ANA results: A cross-method study of three immunodiagnostic techniques

Objectives: Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for antinuclear antibody (ANA) screening, followed by antigen-specific confirmation using immunoassays such as immunoblot (IB) and ELISA. This study aimed to compare antigen-specific ANA/ENA results obtained by these three methods. Materials and methods: Serum samples from 100 individuals (mean age 47 years; 12 men, 88 women) were analyzed. ANA screening was performed by IIF on HEp-2 cells (ANA-HEp-2, ByoSystems) using a EUROStar III Plus fluorescence microscope; titers >1:80 were considered positive. IB was performed using ANA Profile 3 plus DFS70 (EUROIMMUN). ELISA testing included SS-A, SS-B, Scl-70, and dsDNA-NcX (EUROIMMUN), and RNP/Sm, Jo-1, and AMA-M2 (Orgentec). Results: Based on IIF, participants were classified as ANA-positive (n = 48), borderline (n = 26), or ANA-negative (n = 26). The most frequent IB reactivities were SS-A, Ro-52, centromere B, and AMA-M2. In ANA-negative and borderline groups, IB signal intensity >20 was detected for SS-A (n = 2), Ro-52 (n = 4), and Scl-70 (n = 3). Among ANA-positive individuals, ICAP pattern interpretation showed concordance with IB in 40 cases and discordance in two cases. Agreement between ELISA and IB varied by antigen (50%–88%), with the highest concordance for SS-A. Correlation between IB and ELISA ranged from moderate to very strong (r = 0.5–>0.9). Conclusion: IIF, IB, and ELISA provide complementary information, with variable sensitivities and clinically relevant agreement, underscoring the need for a structured diagnostic algorithm combined with clinical context.