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 ... -  《-》

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 ... -  《-》

Anti-DFS70 antibodies detected by immunoblot methods: A reliable tool to confirm the dense fine speckles ANA pattern.

Autoantibodies to the DFS70 (dense fine speckles 70) protein have been identified among the antinuclear antibodies (ANA) in patients with various disorders. However, the ANA test in indirect immunofluorescence (IIF) is not a reliable method to identify anti-DFS70 antibodies. We undertook this study to evaluate the diagnostic performance of two new immunoblot methods for the detection of anti-DFS70 antibodies and to investigate whether their different DFS70 antigen composition could affect diagnostic accuracy in detecting anti-DFS70 antibodies. 62 samples showing a DFS70 staining pattern by IIF were tested by dot blot (Alphadia) and line blot (Euroimmun) methods. The dot blot method employs a truncated sequence of the DFS70 antigen (residues 349-435), while the line blot uses the full-length protein (aa 1-530). The 62 samples were previously assayed by a chemoluminescent (CLIA) method also using a truncated antigen (aa 349-435): 27 were CLIA positive and 35 were CLIA negative. 120 sera from subjects with infectious diseases were used as controls. Both immunoblot methods were positive in the 27 IIF/CLIA positive samples; in addition, the Alphadia dot blot identified another seven DFS70 samples and the Euroimmun line blot was positive in five samples that were negative by CLIA. Among the 120 control samples, two false positives were recorded for the CLIA method, six for the Alphadia method and four for the Euroimmun method. Therefore, in this selected series of samples, sensitivity and specificity were 43.5% and 98.3% for the CLIA method, 54.8% and 95% for the dot blot and 51.6% and 96.6% for the line blot, respectively. Because of great inconsistency in assessing the DFS70 pattern using the ANA-IIF test, specific assays should be used to confirm anti-DFS70 antibodies. The results of this study show that there is no difference in the overall diagnostic accuracy among methods that use the truncated or the full-length DFS70 antigenic sequence and that it is likely that antibodies directed against antigens other than DFS70 may be responsible for producing a DFS70-like ANA-IIF pattern.

Bizzaro N ，Pesente F ，Cucchiaro F ，Infantino M ，Tampoia M ，Villalta D ，Fabris M ，Tonutti E ... -  《-》

Clinical use of anti-DFS70 autoantibodies.

Kang SY ，Lee WI ，Kim MH ，La Jeon Y ... -  《-》

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》