Derivation of a new index to reduce the occurrence of multiple positivity in line immunoassay for myositis specific autoantibodies in inflammatory myopathies.

Myositis specific antibodies are clinically useful biomarkers in inflammatory myositis. Although immunoprecipitation is regarded as the gold standard, line immune assay (LIA) is widely used in practice. However, LIA is limited by multiple MSA positivity in up to 15% of patients, which limits its applicability in diagnosis and prognostication. We sought to interrogate the subset of patients with multiple antibody positivity on LIA to determine a new positive control band index with high specificity for the diagnosis of myositis. We retrospectively reviewed all samples tested for MSA (EUROLINE DL 1530-1601-4G) between April 2023 to April 2025. We calculated the PCBI (test reading/positive control) and compared it with optimal cutoffs as per EUROLINE and how it impacted multiple antibody positivity. Diagnosis of myositis subtype was confirmed by 2 rheumatologists. We retrieved records of 235 patients, 59.6% females with a mean age of 46.1 years. The underlying diagnosis was dermatomyositis (8.5%), juvenile dermatomyositis (2.1%), anti-synthetase syndrome (7.7%), necrotizing myopathy (2.6%), malignancy associated (4, 1.7%), MSA negative IIM or polymyositis (8, 3.4%) and CTD associated myositis (12, 5.1%) and ILD for IPAF evaluation (139, 59.1%). Multiple MSA positivity was seen in 25 patients (10.6%) with EuroLine, Using the revised PCBI cutoffs, we could discriminate between 14 of these MSA's and 11 remained positive for multiple antibodies. On correspondence analysis, both Euroline cutoffs and PCBI showed association between antibody positivity and clinical diagnosis (Chisq p values < 0.001). However, using the PCBI, 73.4% of the variability in the data compared to 69.13% with Euroline cutoffs. Rather than a single uniform cut off for all patients, a phenotype specific cut off for each clinical phenotype at the time of clinical assessment might offer a higher specificity for MSA with line immune assay.