Diagnostic performance and cutoff optimisation of the CTD Screen assay for ANA detection compared with HEp-2 IFA

Aim: Antinuclear antibodies (ANAs) are important in diagnosing systemic autoimmune rheumatic diseases (SARDs), and the HEp-2 cell indirect immunofluorescence assay (HEp-2 IFA) remains the gold standard for their detection. This study evaluates the utility and optimal cutoff of the EliA connective tissue disease Screen (CTD Screen), an enzyme immunoassay, as a complementary automated method for ANA detection. Methods: A total of 694 samples (347 HEp-2 IFA positive and 347 HEp-2 IFA negative) were analysed using the CTD Screen assay. Samples that were positive by both methods were further tested using a line immunoassay (LIA) to detect specific ANAs. Diagnostic accuracy was assessed with a 95% confidence interval (95% CI) using sensitivity, specificity, predictive values, Cohen’s kappa (κ), and receiver operating characteristic (ROC) analysis. Results: With the manufacturer’s cutoff, CTD Screen sensitivity and specificity were 73.9% (95% CI 69.0–78.3) and 92.6% (95% CI 89.2–95.0), respectively. The optimized in-house cutoff improved sensitivity to 81.5% (95% CI 77.1–85.3) and specificity to 85.6% (95% CI 81.4–89.0). The assay demonstrated substantial agreement with HEp-2 IFA (κ = 0.683) and an area under the ROC curve of 0.845 (95% CI 0.814–0.876). LIA confirmed the presence of antibodies in 89.9% of CTD Screen-positive samples. Disease-specific analysis demonstrated the highest sensitivity for systemic sclerosis (97.4%) and the best accuracy for mixed CTD [area under the curve (AUC) = 0.900]. Conclusions: CTD Screen shows substantial agreement with HEp-2 IFA and good diagnostic accuracy; however, it is best used within a tiered diagnostic approach that incorporates confirmatory assays and clinical correlation rather than as a standalone test.