Frequency of extractable nuclear antigen seropositivity among individuals seronegative for antinuclear antibodies on indirect immunofluorescence: a systematic review and meta-analysis

BackgroundThe frequency of extractable nuclear antigen (ENA) seropositivity in patients with negative antinuclear antibodies (ANA) by indirect immunofluorescence (IIF) remains insufficiently characterized. We aimed to estimate the frequency of ENA seropositivity among ANA-IIF–negative individuals (ENA+/IIF−), and to identify associated clinical and methodological determinants.

MethodsA systematic search of PubMed, EMBASE, Web of Science, and Scopus was conducted for studies published up to January 31, 2025. Studies evaluating ENA seropositivity in ANA-IIF–negative patients using HEp-2 or HEp-2000 substrates were included.

Meta-analysis was performed using the Freeman–Tukey double arcsine transformation and random-effects models.ResultsTwenty-eight studies, comprising 33 distinct patient groups and 28,552 ANA-IIF–negative samples, were included. The pooled proportion of ENA+/IIF− was 14.1% (95% CI: 10%–18.7%), with substantial heterogeneity (I² = 99%).

Subgroup analysis demonstrated significant variation according to clinical indication: 1.4% (95% CI: 1.1%–1.9%) in unspecified indications, 8.1% (95% CI: 6%–10.5%) in suspected connective tissue disease (CTD), and 44.1% (95% CI: 32.3%–54.6%) in confirmed CTD (p < 0.0001).