Ix healthy and neurological controls from the discovery group) forPLOS ONE

Ix healthy and neurological controls from the discovery group) forPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,10 /A Live Cell Based Assay for Detection of NMDAR AntibodiesFig 5. ZM241385 supplier Correlation of NMDAR IgG titers and MFI values determined by CBA and FACS assays. The TSA chemical information cut-off value (20,700 MFI, determined by the FACS assay) is indicated by the dashed horizontal line. Correlation of antibody titers and MFI values were calculated using non-parametric Spearman correlation. Correlation coefficient (R) and the p-value are shown in the graph. False negative samples in the FACS assay are depicted in red. In total, 49 samples had a MFI value <1,000, which were all negative in the CBA. CBA = cell-based assay. MFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. NMDAR = N-methyl-D-aspartate receptor. doi:10.1371/journal.pone.0122037.gNMDAR antibodies, and compared the previously used 1:100 dilution to a dilution of 1:20. For this comparison, we focused on samples that were false negative or close to the cut-off value during the initial antibody testing with the FACS assay. Using either dilution 8/9 (89 ) NMDAR antibody positive and 0/12 (0 ) antibody negative samples were detected by the FACS assay. Sensitivity and specificity of both dilutions were therefore comparable to previously obtained results. Interestingly, the cut-off MFI was lower with this set of experiments using the 1:100 dilution compared to previously obtained results (Fig 6), underlining the high interassay variation of the FACS based assay. Correlation of MFI at both dilutions was 0.9558 (Spearman's ; p<0.0001; Fig 6B). Analysis of the re-evaluation group further demonstrated the high variability of the testing system. The inter-assay variation after including new data from the re-evaluation group increased considerably with coefficients of variation of up to 36 . The variability was not correlated with CBA titers (R = 0.3024; Spearman's ; p = 0.4306; S6 Fig).DiscussionAlthough NMDAR encephalitis is considered a rare disease, there is an increasing number of studies identifying this disorder [6, 8, 11?4]. The exact frequency is unknown, but several recent studies with large series of patients [4, 6] and studies focusing on the causes of encephalitis [21, 22] suggest this disorder to be the second most common autoimmune encephalitis afterPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,11 /A Live Cell Based Assay for Detection of NMDAR AntibodiesPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,12 /A Live Cell Based Assay for Detection of NMDAR AntibodiesFig 6. NMDAR antibody MFI at different serum dilutions in NMDAR antibody positive and negative sera. NMDAR antibody positive (n = 9) and negative (n = 12) serum samples have been determined by CBA. (A) Serum dilutions of 1:100 and 1:20 are shown. Respective cut-off MFI values are indicated by dashed horizontal lines. The table shows cut-off MFI and numbers of samples tested positive for NMDAR antibodies by the FACS assay at different serum dilutions. (B) Correlation of MFI obtained by using 1:100 and 1:20 dilution in the re-evaluation group of NMDAR positive samples in the CBA. Respective cut-off values are indicated by dashed lines. The one false negative sample at both dilutions is shown in red. For a better graphical presentation, MFI values below 1,000 were set to 1,000. Correlation of exact MFI values were calculated using non-parametric Spearman correlation. Correlation coefficie.Ix healthy and neurological controls from the discovery group) forPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,10 /A Live Cell Based Assay for Detection of NMDAR AntibodiesFig 5. Correlation of NMDAR IgG titers and MFI values determined by CBA and FACS assays. The cut-off value (20,700 MFI, determined by the FACS assay) is indicated by the dashed horizontal line. Correlation of antibody titers and MFI values were calculated using non-parametric Spearman correlation. Correlation coefficient (R) and the p-value are shown in the graph. False negative samples in the FACS assay are depicted in red. In total, 49 samples had a MFI value <1,000, which were all negative in the CBA. CBA = cell-based assay. MFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. NMDAR = N-methyl-D-aspartate receptor. doi:10.1371/journal.pone.0122037.gNMDAR antibodies, and compared the previously used 1:100 dilution to a dilution of 1:20. For this comparison, we focused on samples that were false negative or close to the cut-off value during the initial antibody testing with the FACS assay. Using either dilution 8/9 (89 ) NMDAR antibody positive and 0/12 (0 ) antibody negative samples were detected by the FACS assay. Sensitivity and specificity of both dilutions were therefore comparable to previously obtained results. Interestingly, the cut-off MFI was lower with this set of experiments using the 1:100 dilution compared to previously obtained results (Fig 6), underlining the high interassay variation of the FACS based assay. Correlation of MFI at both dilutions was 0.9558 (Spearman's ; p<0.0001; Fig 6B). Analysis of the re-evaluation group further demonstrated the high variability of the testing system. The inter-assay variation after including new data from the re-evaluation group increased considerably with coefficients of variation of up to 36 . The variability was not correlated with CBA titers (R = 0.3024; Spearman's ; p = 0.4306; S6 Fig).DiscussionAlthough NMDAR encephalitis is considered a rare disease, there is an increasing number of studies identifying this disorder [6, 8, 11?4]. The exact frequency is unknown, but several recent studies with large series of patients [4, 6] and studies focusing on the causes of encephalitis [21, 22] suggest this disorder to be the second most common autoimmune encephalitis afterPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,11 /A Live Cell Based Assay for Detection of NMDAR AntibodiesPLOS ONE | DOI:10.1371/journal.pone.0122037 March 27,12 /A Live Cell Based Assay for Detection of NMDAR AntibodiesFig 6. NMDAR antibody MFI at different serum dilutions in NMDAR antibody positive and negative sera. NMDAR antibody positive (n = 9) and negative (n = 12) serum samples have been determined by CBA. (A) Serum dilutions of 1:100 and 1:20 are shown. Respective cut-off MFI values are indicated by dashed horizontal lines. The table shows cut-off MFI and numbers of samples tested positive for NMDAR antibodies by the FACS assay at different serum dilutions. (B) Correlation of MFI obtained by using 1:100 and 1:20 dilution in the re-evaluation group of NMDAR positive samples in the CBA. Respective cut-off values are indicated by dashed lines. The one false negative sample at both dilutions is shown in red. For a better graphical presentation, MFI values below 1,000 were set to 1,000. Correlation of exact MFI values were calculated using non-parametric Spearman correlation. Correlation coefficie.