A Cell-Based Assay for Detection of Anti-Fibrillarin Autoantibodies in Systemic Sclerosis

Objectives Anti-fibrillarin antibodies are useful for establishing diagnosis and predicting distinct clinical features in systemic sclerosis (SSc). Anti-fibrillarin produces a characteristic clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) that is useful to guide further testing in antigen-specific immunoassays (ASI). Immunoprecipitation (IP) is the gold standard ASI for anti-fibrillarin determination. We established a new anti-fibrillarin Cell-Based Assay (CBA) and compared its diagnostic performance with IP and ASI commercial kits. The clinical features of SSc patients with and without anti-fibrillarin antibodies were analyzed. Methods A TransMembrane Signal (TMS) was added to the human fibrillarin gene in order to drive the transgenic fibrillarin to the cytoplasmic membrane. HEp-2 cells transfected with a vector containing the TMS-fibrillarin were used as substrate for IFA in the CBA. Sixty-two samples with high-titer nucleolar pattern in HEp-2 IFA (41 clumpy and 21 homogeneous/punctate) were tested for anti-fibrillarin in the CBA, IP, line-blot and ELISA. Clinical SSc phenotype was evaluated in 106 patients according to positive/negative anti-fibrillarin result in the CBA. Results TMS-fibrillarin was properly located to the cytoplasmic membrane and recognized by human autoantibodies. Thirty-eight of 41 clumpy nucleolar samples (92.7%) and none of 21 samples with other nucleolar patterns were positive for anti-fibrillarin in the CBA. There was 100% agreement between the positive/negative results in the CBA and IP. Among the 38 CBA-positive samples, only 15 (39.5%) and 11 (29%) were considered positive for anti-fibrillarin in the line-blot and ELISA, respectively. Anti-fibrillarin was associated with higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028). Conclusion With an innovative strategy of targeting the transgenic autoantigen to the cell membrane, we developed a new straightforward assay for detection of anti-fibrillarin autoantibodies. This new CBA presented high sensitivity and specificity for the detection of anti-fibrillarin autoantibody, comparable to the gold standard IP. Moreover, anti-fibrillarin antibodies detected in the CBA identified patients with a higher frequency of dcSSc, cardiac and renal involvement.