Diagnostic value of anti-carbamylated protein combined with Anti-RA33 antibodies in seronegative rheumatoid arthritis: a retrospective cohort study

Abstract

Seronegative rheumatoid arthritis (SNRA), defined by the absence of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA), poses a persistent diagnostic challenge. Anti-carbamylated protein antibodies (Anti-CarP-IgG) and anti-RA33 antibodies targeting heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP-A2/B1) have been proposed as supplementary biomarkers, yet their combined diagnostic utility in SNRA remains insufficiently characterized. A retrospective cohort study enrolled 550 RA patients (2010 ACR/EULAR criteria) at a single tertiary center (2024–2025). Patients were stratified into SNRA (RF-IgM < 14 IU/mL and ACPA < 5 U/mL; n = 189) and seropositive RA (SPRA; n = 361). Anti-CarP-IgG and Anti-RA33-IgG were measured by ELISA (positivity cutoff > 20 U/mL). Disease activity was assessed by DAS28-ESR/CRP; radiographic damage by modified Sharp/van der Heijde score and Larsen staging. Odds ratios (OR) with 95% confidence intervals (CI) were computed; Bonferroni correction was applied for multiple comparisons. Anti-RA33-IgG positivity was significantly lower in SNRA versus SPRA (15.9% vs. 29.9%, p < 0.001; OR 2.26, 95% CI 1.44–3.55), as was Anti-CarP-IgG positivity (10.6% vs. 23.0%, p = 0.001; OR 2.52, 95% CI 1.49–4.26). Combined testing identified at least one novel antibody in 23.8% of SNRA patients versus 45.2% of SPRA patients ( p < 0.001). Within SNRA, Anti-CarP-IgG-positive patients showed significantly more advanced radiographic staging: Stage IV prevalence was 25.0% vs. 7.1% in Anti-CarP-IgG-negative patients (OR 4.36, 95% CI 1.35–14.05, p = 0.021), despite equivalent DAS28-ESR ( p = 0.902). Combined Anti-CarP-IgG and Anti-RA33-IgG testing captures serological evidence in nearly one-quarter of SNRA patients. Anti-CarP-IgG positivity associates with a 4.4-fold increased odds of Stage IV radiographic disease, supporting its role as a prognostic stratification tool in SNRA.