Pre-test probability determines optimal ANA screening strategy: a risk-stratified comparison of indirect immunofluorescence and CTD screen assay

Objectives: Indirect immunofluorescence (IIF) and CTD screen exhibit divergent performance characteristics for antinuclear antibody (ANA) detection, yet their relative utility across different clinical contexts remains poorly defined. We compared the diagnostic performance of ANA-IIF and CTD screen assay across stratified levels of pre-test probability. Methods: We evaluated 822 consecutive patients referred to a specialist rheumatology service, tested in parallel by both ANA-IIF and CTD screen. Patients were stratified by pre-test probability based on clinical, laboratory, and imaging risk factors: high risk (≥2 major factors), moderate risk (1 major factor), and low risk (minor factors only). Primary outcome was CTD diagnosis according to international classification criteria. Diagnostic accuracy parameters, including sensitivity, specificity, predictive values, and likelihood ratios, were calculated for each risk stratum. Results: ANA-IIF demonstrated higher sensitivity than CTD screen across all risk groups (0.76–1.00 vs. 0.76–0.83), whilst CTD screen exhibited superior specificity (0.89–0.96 vs. 0.43–0.81). In high-risk patients (CTD prevalence 47.1 %), both methods achieved clinically acceptable performance. In low-risk patients (CTD prevalence 12.9 %), CTD screen’s enhanced specificity yielded markedly superior diagnostic accuracy (0.88 vs. 0.61–0.76 for ANA-IIF), with higher positive predictive value. Discordant ANA-IIF-positive/CTD screen-negative CTD cases comprised predominantly mild undifferentiated disease or patients with clinically recognisable features mandating specialist referral regardless of ANA results. Conclusions: Optimal ANA screening methodology depends on pre-test probability. ANA-IIF remains preferred in high-risk populations, whilst CTD screen provides superior performance in low-risk settings through reduced false-positive rates without clinically significant diagnostic loss.