Optimizing line blot assays: impact of band intensity adjustment on diagnostic performance in myositis autoantibodies testing

INTRODUCTION: Line blot assays (LBA) have several limitations regarding diagnostic validity, as sensitivity and specificity vary considerably among antigens. OBJECTIVE: The aim of this study was to assess whether the adjustment of each specific band intensity (BI) relative to the positive control band (PCB), combined with increasing the cut-off value from 15 to 20, improves the diagnostic performance of the LBA for detecting autoantibodies related to idiopathic inflammatory myopathies in our patient cohort. METHODS: We included all serum samples tested for myositis‑specific antibodies (MSA) and myositis‑associated antibodies (MAA) between January 2022 and April 2023 at the Immunology Department of Hospital Clínic of Barcelona. Two analyses were performed: Analysis A, using the manufacturer's recommended cut-off (BI ≥ 15), and Analysis B, applying the proposed BI adjustment relative to the PCB and raising the cut-off to 20 in samples that were positive in Analysis A. RESULTS: A total of 939 patients were included. Using Analysis A, 280/939 (29.8%) patients had at least one positive result, whereas 659/939 (70.2%) were negative for all MSA and MAA. After applying Analysis B, 151/280 (53.9%) remained positive, while 129/280 (46.1%) became negative. With Analysis A, sensitivity was 76%, specificity 75%, positive predictive value (PPV) 24%, and negative predictive value (NPV) 96%. Under Analysis B, sensitivity was 70%, specificity 89%, PPV 41%, and NPV 96%. CONCLUSIONS: Applying the proposed BI adjustment effectively removes weakly positive results with low specificity in an objective and reproducible manner, thereby increasing the overall specificity of the LBA. Key Points • Applying the proposed band intensity removes weakly positive results. • This approach enhances the specificity, and positive predictive value of a commercial line blot assay.