Prevalence and Importance of Autoantibodies in Patients with Seronegative Rheumatoid Arthritis

antibodies attacking virus
3d illustration of antibodies attacking virus particles in the bloodstream.
The current case-control study aims to determine the frequency of additional autoantibodies and the clinical phenotypes among patients with seronegative RA.

Among patients traditionally defined as seronegative rheumatoid arthritis (RA), extended serology with additional screening for autoantibodies can identify patients with a similar pattern of activity as in traditionally defined seropositive RA, according to study results published in Arthritis Research & Therapy.

Seronegative RA is defined according to the absence of rheumatoid factor (RF), primarily immunoglobulin M (IgM) RF, and anti-citrullinated antibodies (ACPA). While there are known risk factors for seropositive RA, limited data are available on the risk factors and clinical phenotypes in seronegative RA patients.

The aim of the current case-control study was to determine the frequency of additional autoantibodies and the clinical phenotypes among patients with seronegative RA.

The study included newly diagnosed patients with RA, and age-, sex-, and residential area-matched controls from the Swedish population-based Epidemiological Investigation of RA cohort.

Using a custom-made microarray chip (Thermo Fisher Scientific, ImmunoDiagnostics), sera from 2755 RA cases and 370 controls were screened for anti-cyclic citrullinated peptide (CCP)-2 immunoglobulin G (IgG), 19 ACPA fine specificities, IgM/IgG/IgA RF, anti-carbamylated-protein (CarP) antibodies, and 17 other autoantibodies. Furthermore, the impact of RA-associated autoantibodies on disease course in “seronegative” RA was assessed during a 5-year follow-up period.

In a substantial proportion of the anti-CCP2-negative patients with RA there was evidence of ACPA fine-specificities and 11 out of 19 ACPA fine-specificities were detected in this subset, in frequencies higher than controls. ACPA levels were higher in anti-CCP2-negative RA patients than controls.

All RF isotypes were detected in the anti-CCP2-negative subset, the most common was IgM RF (23.8%), followed by IgG RF (17.4%) and IgA RF (10.6%).

Additionally, 17 autoantibodies primarily associated with other autoimmune rheumatic diseases, were detected at low frequencies in the study cohort, with minor differences between anti-CCP2-positive and anti-CCP2-negative patients.

When researchers combined ACPA, RF, and other autoantibodies, they detected autoantibodies in 59.8% of the anti-CCP2-negative patients with RA, as in a third of patients (34.5%) ACPA fine-specificities were found, RF isotypes were detected in 30.1%, and the other autoantibodies were detected in 30.3%. Interestingly, autoantibodies were also detected in 37% of the controls.

Data suggest an association between HLA-DRB1 shared epitope with the presence of ACPA in anti-CCP2-negative patients with RA, but no similar association was documented with the presence of RF or other autoantibodies in these patients.

In a subset of 534 seronegative patients with RA, defined as negative anti-CCP2 and negative RF autoantibodies, anti-CarP antibodies were identified in 15.9%, ACPA fine-specificities in 29.8% and IgA and/or IgG RF in 9.4%. The co-occurrence of ≥2 types of autoantibodies was reported in 9.6%. In fact, 43.6% of patients with seronegative RA were deemed to be seropositive according to ACPA, RF, and anti-CarP antibodies.

Compared to patients that were negative for all RA-associated autoantibodies, the presence of ACPA fine-specificities, IgG/IgA RF, or anti-CarP antibodies was associated with higher RA activity during the 5 year follow-up.

Limitations of the study include that the researchers did not investigate the presence of anti-CCP2 IgA nor did they analyze presence of ACPA using other commercial clinical tests. Another limitation to be considered is the loss of specificity when analyzing multiple autoantibody reactivities.

“Our study confirms that seronegative RA, defined only from the presence of anti-CCP2 IgG and IgM RF, is not truly a seronegative disease subset. Presence of ACPA and RF in the conventionally defined seronegative RA population defines a group of patients that resemble seropositive patients with respect to risk factors and clinical picture,” concluded the researchers.


Reed E, Hedström AK, Hansson M, et al. Presence of autoantibodies in “seronegative” rheumatoid arthritis associates with classical risk factors and high disease activity. Arthritis Res Ther. 2020;22(1):170. doi:10.1186/s13075-020-02191-2