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Objectives: Myositis-specific and associated autoantibodies are important biomarkers in routine clinical use. We assessed local testing performance for myositis autoantibodies by comparing line immunoassay (LIA) to protein radio-immunoprecipitation and identifying clinical characteristics associated with each myositis autoantibody in the MyoCite cohort., Methods: Serum samples from patients within the MyoCite cohort, a well-characterized retro-prospective dataset of adult and juvenile idiopathic inflammatory myopathy (IIM) patients in Lucknow, India (2017-2020), underwent LIA at Sanjay Gandhi Postgraduate Institute of Medical Science (SGPGIMS), Lucknow. Immunoprecipitation of 147 IIM patients' serum samples (125 adult-onset, 22 juvenile-onset) was conducted at the University of Bath, with researchers blind to LIA results. LIA performance was assessed against immunoprecipitation as the reference standard, measuring sensitivity, specificity and inter-rater agreement. Univariate and multivariate logistic regression determined clinical associations for specific myositis-specific autoantibodies., Results: Immunoprecipitation identified myositis autoantibodies in 56.5% (n = 83) of patient samples, with anti-Jo1 (n = 16; 10.9%) as the most common, followed by anti-MDA5 (n = 14, 9.5%). While LIA showed good agreement for anti-Jo1, anti-PL7 and anti-PL12 (Cohen's κ 0.79, 0.83 and 1, respectively), poor agreement was observed in other subgroups, notably anti-TIF1γ (Cohen's κ 0.21). Strongly positive samples, especially in myositis-specific autoantibodies, correlated more with immunoprecipitation results. Overall, 59 (40.1%) samples exhibited non-congruence on LIA and immunoprecipitation, and κ values for LIAs for anti-TIF1γ, anti-Ku, anti-PmScl, anti-Mi2 and anti-SAE ranged between 0.21 and 0.60., Conclusion: While LIA reliably detected anti-Jo1, anti-PL7, anti-PL12, anti-MDA5 and anti-NXP-2, it also displayed false positives and negatives. Its effectiveness in detecting other autoantibodies, such as anti-TIF1γ, was poor., (© Crown copyright 2024.)

Objectives: The CLASS (Classification Criteria of Anti-Synthetase Syndrome) project is a large international multicentre study that aims to create the first data-driven anti-synthetase syndrome (ASSD) classification criteria. Identifying anti-aminoacyl tRNA synthetase antibodies (anti-ARS) is crucial for diagnosis, and several commercial immunoassays are now available for this purpose. However, using these assays risks yielding false-positive or false-negative results, potentially leading to misdiagnosis. The established reference standard for detecting anti-ARS is immunoprecipitation (IP), typically employed in research rather than routine autoantibody testing. We gathered samples from participating centers and results from local anti-ARS testing. As an "ad-interim" study within the CLASS project, we aimed to assess how local immunoassays perform in real-world settings compared to our central definition of anti-ARS positivity., Methods: We collected 787 serum samples from participating centres for the CLASS project and their local anti-ARS test results. These samples underwent initial central testing using RNA-IP. Following this, the specificity of ARS was reconfirmed centrally through ELISA, line-blot assay (LIA), and, in cases of conflicting results, protein-IP. The sensitivity, specificity, positive likelihood ratio and positive and negative predictive values were evaluated. We also calculated the inter-rater agreement between central and local results using a weighted κ co-efficient., Results: Our analysis demonstrates that local, real-world detection of anti-Jo1 is reliable with high sensitivity and specificity with a very good level of agreement with our central definition of anti-Jo1 antibody positivity. However, the agreement between local immunoassay and central determination of anti-non-Jo1 antibodies varied, especially among results obtained using local LIA, ELISA and "other" methods., Conclusions: Our study evaluates the performance of real-world identification of anti-synthetase antibodies in a large cohort of multi-national patients with ASSD and controls. Our analysis reinforces the reliability of real-world anti-Jo1 detection methods. In contrast, challenges persist for anti-non-Jo1 identification, particularly anti-PL7 and rarer antibodies such as anti-OJ/KS. Clinicians should exercise caution when interpreting anti-synthetase antibodies, especially when commercial immunoassays test positive for non-anti-Jo1 antibodies.