Verification of qualitative methods according to ISO 15189 for the diagnostics of PNS

Introduction: Paraneoplastic neurologic syndromes (PNS) are immune-mediated neurological disorders frequently associated with cancer and characterized by the presence of specific neuronal autoantibodies. Current diagnostic criteria (Graus criteria) recommend a combination of tissue-based indirect immunofluorescence testing (IIFT) and confirmatory immunoassays, such as line blot or cell-based assays (CBA). In ISO 15189–accredited laboratories, manufacturer-validated assays require performance verification before inclusion within the accredited scope. This study aimed to verify the analytical performance of line blot and CBA methods for neuronal antibody detection in a clinical diagnostic laboratory. Materials and methods: A total of 61 serum samples were included: 37 (VERPNS) for line blot verification and 24 (VERGAD) for CBA verification. Samples, derived from patients with suspected PNS or stiff person syndrome and from healthy donors, had been previously characterized by tissue-based IIFT and line blot assay in parallel. Screening was performed using BIOCHIP Mosaic IIFT, while the antigen-specific confirmation with line blot targeting 12 neuronal antigens. For verification purposes, VERPNS samples were analyzed by line blot for PNS-associated antibodies, whereas VERGAD samples were tested by CBA using transfected cells for anti-GAD65 detection. Results: Line blot analysis detected neuronal antibodies in 24/37 samples (64.9%) and showed complete concordance with pre-characterization results, with 100% diagnostic sensitivity and specificity for all verified antigens. CBA testing identified 12/24 anti-GAD65–positive samples and 12/24 negatives, also demonstrating full agreement and 100% positive and negative percent agreement. Both methods showed reliable and accurate performance. Discussion: Verification confirmed the suitability of line blot and CBA assays for routine diagnostic use and enabled the extension of the laboratory’s ISO 15189 accredited scope to additional neuronal antigens, supporting standardized and reliable serological diagnosis of PNS.