Abstract
AIMS: Dysferlin, a 237-kDa protein crucial for muscle function, is deficient in dysferlinopathy, a rare form of muscular dystrophy caused by recessive mutations in the DYSF gene. While genetic analysis is the primary diagnostic approach, protein-based studies remain essential for confirming pathogenicity, particularly for variants of uncertain significance. This study aims to validate an immunohistochemical assay for dysferlin detection in neutrophils from peripheral blood films (PBF) and to evaluate how different storage conditions affect its performance, with particular focus on refining variables that may affect assay reliability and reproducibility. METHODS: We performed a follow-up assessment of a new immunoassay to detect dysferlin expression in neutrophils from PBFs, using samples from genetically confirmed dysferlinopathy patients and healthy controls in an Indian cohort. RESULTS: The assay exhibited very high sensitivity, but its specificity was affected by sample quality and storage conditions. Protein degradation occurred in samples stored at room temperature in environments exceeding 30°C, while storage at 4°C preserved dysferlin immunoreactivity for up to 20 weeks. CONCLUSIONS: The PBF-based dysferlin assay is a rapid, cost-effective and minimally invasive screening tool, well-suited for use in resource-limited settings. It provides a viable approach for initial screening and variant assessment. As the assay's reliability is influenced by sample quality and storage conditions, proper training in collection techniques and strict adherence to storage protocols are essential for ensuring diagnostic accuracy and enabling broader clinical application.