For the analysis of the accuracy of the FACS assay, all 1123 serum samples were used. To compare the accuracy of the FACS assay with the C-CBA kit, 225 serum samples (54 FACS-positive, 6 FACS-borderline, and 165 FACS-negative) were used. These 225 samples were further classified into groups 1–5 according to the MFIi of the FACS assay: Group 1 = AQP-4 Ab positive sera with high titer of MFIi (≥ 50), Group 2 = AQP-4 Ab positive sera with medium titer of MFIi (10 ≤ MFIi < 50), Group 3 = AQP-4 Ab positive sera with low titer of MFIi (3.5 ≤ MFIi < 10), Group 4 = sera with borderline MFIi (2 ≤ MFIi < 3.5), Group 5 = AQP-4 Ab negative sera (MFI < 2). MFIi values were considered to be significant only when the MFI ratio is ≥2). The researchers who performed the C-CBA were blind to the results of the FACS assay. Abbreviations: AQP4-Ab = aquaporin-4 antibody, C-CBA = commercial cell-based assay, FACS = fluorescence-activated cell sorting, FACS-positive = tested positive in the FACS assay, FACS-negative = tested negative in the FACS assay, FACS-borderline = tested borderline in the FACS assay, IIDD = idiopathic inflammatory demyelinating disease, MFIi = mean fluorescence intensity index.