B-141 Antinuclear Antibody (ANA) HEp-2 Immunofluorescence Pattern Distribution in a Large Brazilian Laboratory Cohort: A Ten-Year Retrospective Study

Abstract

Antinuclear antibody (ANA) testing by indirect immunofluorescence (IIF) on HEp-2 cells remains the reference screening method for suspected systemic autoimmune rheumatic diseases (SARD). Beyond semi-quantitative titers, HEp-2 IIF provides clinically informative fluorescence patterns that support differential diagnosis and guide reflex testing. However, ANA positivity is common in individuals without SARD and varies by age and sex, reinforcing the need for region-specific epidemiologic data aligned with International Consensus on ANA Patterns (ICAP) standards. We aimed to characterize the long-term distribution of ANA titers and HEp-2 patterns in a large Brazilian laboratory cohort. We conducted a retrospective, cross-sectional analysis of laboratory information system records from a private reference laboratory in Fortaleza (Ceará, Brazil), including 147,774 ANA HEp-2 IIF encounters (2015–2024). Titers were extracted from structured fields and report text, and the highest titer per encounter (denominator of 1:xxx) was used for analysis. The official cutoff titer =1:80 defined positivity. Positive encounters were summarized by sex, age (predefined strata), and standardized pattern categories consolidated via a dictionary aligned with ICAP-style nomenclature. Among positives, highest titers were compared by sex (Welch t-test and Mann–Whitney U) and across age strata (one-way ANOVA and Kruskal–Wallis), with Tukey HSD post-hoc for pairwise age contrasts (a=0.05). Of 147,774 encounters, 36,943 were positive (=1:80; 25.0%). Positivity was strongly female-predominant (31,411/36,943; 85.0%) versus males (5,529/36,943; 15.0%). Among positives, age was 53.0±18.8 years (median 53.0). Nuclear speckled phenotypes predominated: dense fine speckled 14,216/36,943 (38.5%) and fine speckled 13,926/36,943 (37.7%), together accounting for ∼76% of positive encounters. Other frequent patterns included nucleolar 3,340/36,943 (9.0%) and homogeneous nuclear 2,264/36,943 (6.1%). Titers were mostly moderate: 1:160–<1:320 in 25,265/36,943 (68.4%), and =1:640 in 2,428/36,943 (6.6%). Highest titers differed statistically by sex (Welch t=15.69, p<0,001; Cohen’s d=0.19, small effect; Mann–Whitney p<0,001). Titers also differed across age strata (ANOVA F=15.34,001; Kruskal–Wallis H=137.35,001). Tukey HSD localized these differences, showing lower mean titers in younger adults (18–29 and 30–44) versus older groups (45–59 and 60–74) (all p<0,001), with no difference between 45–59 vs 60–74 (p=0.131). In this large routine ANA HEp-2 IIF cohort, reactivity was frequent and strongly female-predominant, with a stable profile characterized by persistent predominance of nuclear speckled phenotypes. Fine speckled and dense fine speckled nuclear patterns (particularly at 1:160–1:320) accounted for most classified positives, supporting their use as local reference benchmarks for reporting consistency and quality monitoring. With access to the full ICAP-level pattern distribution rather than aggregated categories, less frequent and mixed phenotypes can be quantified, improving epidemiologic resolution and enabling more granular, locally informed reflex-testing and clinical triage strategies.