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Post-marketing evaluation of a multiparametric immunofluorescence assay for the detection of anti-Aquaporin 4 antibodies in the diagnosis of neuromyelitis optica

Background: Neuromyelitis Optica (NMO) serology is a powerful tool for differential diagnosis from Multiple Sclerosis (MS). This study aims to perform a post-marketing evaluation of the first indirect immunofluorescence assay (IIFA) commercially available for NMO serology. Methods: Sera obtained from 20 NMO patients (according to the Wingerchuk’s criteria without considering the NMO IgG status) and 77 controls (47 MS patients and 30 healthy subjects) were tested using the BIOCHIP Mosaic (Euroimmun, Germany), containing 5 substrates: primate cerebellum, cerebrum and optic nerve; human embryonic kidney cells (HEK) transfected and non transfected with Aquaporin 4 (AQP4). Two positive controls (goat anti-human AQP4 H19, Santa Cruz and positive control serum provided by the kit) were tested, too. Sensitivity and specificity analyses were performed for each substrate and considering different combinations of substrates. Inter and intra-laboratory reproducibility were analysed. Results: Five fluorescent staining patterns were identified by incubating serum samples and controls (A, B, C, D, E). Pattern A was characterized by the staining of AQP4 transfected cells, absence of staining in non transfected cells, and “typical staining” of the white matter in primate tissues; Patterns B, C and D showed different combinations of “typical” and “atypical” staining patterns on the substrates; the absence of fluorescent staining was identified as Pattern E. Both positive controls showed the Pattern A, thus it was considered the typical pattern of anti-AQP4 antibodies. Pattern A was found in 19/97 serum samples, all diagnosed as NMO; Pattern B in 1/97 (MS patient), Pattern C in 4/97 (2 MS patients and 2 healthy subjects), Pattern D in 6/97 (5 MS patients and 1 healthy subject) and Pattern E in 68/97 ( 1 NMO patient, 40 MS patients and 27 healthy subjects) analyzed samples. The clinical diagnosis of NMO has been considered as the “gold standard” for sensitivity and specificity analyses: by the combination of all substrates the assay showed 95% sensitivity and 100% specificity for NMO, whereas lower sensitivity and specificity values were obtained if single substrates were considered. A 100% agreement was found for both intra and inter- laboratory reproducibility analyses (K=1.0). Conclusion: the Biochip Mosaic is a powerful tool for NMO serology, fast to perform, high sensitive and specific for NMO, highly reproducible, suitable for inter-laboratory standardization.

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