Development and multi-center evaluation of a novel immunoadsorption method for anti-DFS70 antibodies.

Antinuclear antibodies (ANA) represent a hallmark in the diagnosis of ANA-associated rheumatic diseases (AARD). However, anti-DFS70 antibodies are present in a higher portion of the healthy individuals (HI) than in patients with AARD. Consequently, we developed a novel, highly specific indirect immunofluorescence (IIF) method that blocks anti-DFS70 antibodies from binding to HEp-2 cells and to evaluate the method in a multi-center study. A total of 18 samples from systemic lupus erythematosus patients (SLE, n = 7) and HI (n = 11) were used for the initial development of the immunoadsorption method. For the multi-center evaluation, samples with a dense fine speckled (DFS) pattern (n = 99) were collected at three different sites based on their established IIF screening procedure at the respective laboratories. Additionally, four characterized samples with established clinically relevant IIF patterns (centromere, nucleolar, speckled, homogeneous) were blended in five different ratios (10%, 25%, 50%, 75%, 90%) with a sample positive for anti-DFS70 antibodies, which by itself showed a dense fine speckled (DFS) IIF pattern. All samples were tested by IIF with NOVA Lite HEp-2 ANA and NOVA Lite HEp-2 Select on the NOVA View® instrument, and also tested by QUANTA Flash DFS70 chemiluminescent immunoassay (CIA) for confirmation of anti-DFS70 antibodies (Inova Diagnostics, San Diego, CA, USA). For the development of the immunoadsorption method, only 1/7 ANA-positive samples from SLE patients, but 8/10 ANA-positive samples from healthy individuals turned negative using the immunoadsorption. Subsequently, 73/99 (73.7%) of the DFS pattern samples were positive by CIA for anti-DFS70 antibodies showing a strong quantitative Spearman's correlation (rho = 0.57 (95% CI, 0.39-0.71, p < 0.0001)) between light intensity units (LIU) measured by NOVA View and CIA. Intensities measured with NOVA Lite HEp-2 and NOVA Lite HEp-2 Select demonstrated significantly lower intensity values after inhibition with DFS70 antigen (p < 0.0001). When samples were processed to mimic samples with mixed patterns (DFS + clinically relevant pattern), the new immunoadsorption method demonstrated that all clinically relevant patterns remained unchanged whereas the LIUs from NOVA View analysis significantly decreased after inhibition (p < 0.0001). The data showed that the NOVA Lite HEp-2 Select kit effectively inhibits anti-DFS70 antibody binding to its cellular target antigen.

Bentow C ，Rosenblum R ，Correia P ，Karayev E ，Karayev D ，Williams D ，Kulczycka J ，Fritzler MJ ，Mahler M ... -  《-》

Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases.

Shovman O ，Gilburd B ，Chayat C ，Amital H ，Langevitz P ，Watad A ，Guy A ，Perez D ，Azoulay D ，Blank M ，Segal Y ，Bentow C ，Mahler M ，Shoenfeld Y ... -  《CLINICAL AND EXPERIMENTAL RHEUMATOLOGY》

Application of anti-DFS70 antibody and specific autoantibody test algorithms to patients with the dense fine speckled pattern on HEp-2 cells.

Lee H ，Kim Y ，Han K ，Oh EJ ... -  《-》

A better definition of the anti-DFS70 antibody screening by IIF methods.

Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

Infantino M ，Shovman O ，Pérez D ，Grossi V ，Manfredi M ，Benucci M ，Damiani A ，Gilburd B ，Azoulay D ，Serrano A ，Shoenfeld Y ... -  《-》

Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases.

Mahler M ，Parker T ，Peebles CL ，Andrade LE ，Swart A ，Carbone Y ，Ferguson DJ ，Villalta D ，Bizzaro N ，Hanly JG ，Fritzler MJ ... -  《-》