Real-World Evaluation of ANA Testing Pathways: Serological Incompleteness and Comparison of Stepwise Versus Upfront Parallel Reflex Strategies

Background/Objectives: Antinuclear antibody testing by indirect immunofluorescence on HEp-2 cells (ANA-IIF) remains the reference test for systemic autoimmune rheumatic diseases (SARD). However, complete serological characterization requires integration with extractable nuclear antigen (ENA) screening, antigen-specific solid-phase assays (SPA), and confirmatory indirect immunofluorescence (IIF) tests. This study aimed to: (1) quantify real-world serological pathway incompleteness in ANA testing performed outside a structured reflex strategy; (2) compare simulated stepwise and upfront parallel reflex approaches in terms of serological yield, laboratory workload, and costs. Methods: We retrospectively analyzed 2207 consecutive patients referred for ANA-IIF testing between July 2025 and April 2026. Serological incompleteness was defined as non-completion of the predefined Regione Lombardia reflex framework. A fully characterized subgroup of 514 patients, defined by complete reflex-serological assessment, was used to compare a stepwise strategy, in which ANA-IIF positivity triggered downstream testing, with an upfront parallel strategy, in which ANA-IIF, ENA screening, and anti-double-stranded DNA (anti-dsDNA) testing were performed upfront. Results: ANA-IIF was positive in 913/2207 patients (41.4%). Overall, 508 patients (23.0%) had at least one missing follow-up assay expected under the reflex framework. In the fully characterized subgroup, the upfront parallel strategy increased the number of patients with at least one fully characterized autoantibody finding from 15 to 22 (+1.4%), but required 657 additional tests and an estimated incremental cost of approximately €2592. Conclusions: ANA-IIF testing outside a structured reflex pathway frequently resulted in incomplete serological assessment. Although the upfront parallel strategy increased serological yield, it required substantially greater laboratory workload and costs than the stepwise approach. Further studies are needed to define the optimal balance between serological yield, clinical relevance, and cost-effectiveness.