Evaluation of anti-nuclear antibody patterns and their associations with autoimmune disease biomarkers

Objective Anti-nuclear antibody (ANA) testing is widely used in the evaluation of systemic autoimmune rheumatic diseases (SARD). We evaluated ANA testing performed at Labcorp between 2015 and 2024 and examined serological associations between ANA titers and HEp-2 indirect immunofluorescence assay (IFA) patterns and commonly used SARD-associated autoantibodies in a large real-world laboratory population. Methods We conducted a retrospective review of de-identified results from 8,807,628 ANA tests performed at Labcorp between 2015 and 2024. The primary unit of analysis was the ANA test/specimen; repeated testing from the same individual may be represented. ANA positivity was assessed by IFA at titers of 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. Reference-laboratory tests used a positivity threshold of >=1:80, whereas the rheumatology-specialty laboratory used >=1:40. Among ANA-positive specimens, 696,456 underwent testing for one or more SARD-associated autoantibodies using routine reflex or ordered testing panels. Results ANA positivity decreased progressively with increasing titer thresholds, and positivity rates differed substantially between the reference and rheumatology-specialty laboratory settings. Speckled and homogeneous patterns were the most frequent patterns. Centromere patterns were generally detected at higher titers and were strongly associated with centromere antibody positivity. Among ANA-positive specimens that underwent additional autoantibody testing, the proportion with one or more positive SARD-associated autoantibodies generally increased with ANA titer. Several less common patterns showed associations with individual autoantibodies; these findings are exploratory and require independent validation. Conclusion This large real-world laboratory dataset quantifies relationships among ANA titer, HEp-2 IFA pattern, and SARD-associated autoantibody positivity. Higher ANA titers were associated with a greater frequency of disease-associated serological markers, but these data do not establish an optimal diagnostic ANA cutoff or a clinical diagnosis of SARD. Common pattern-autoantibody relationships were confirmed at scale, while associations involving uncommon ANA patterns and anti-DFS70 should be interpreted cautiously and validated in clinically characterized cohorts.