NMDAR antibody ΔMFI at different serum dilutions in NMDAR antibody positive and negative sera.

<p>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 ΔMFI values are indicated by dashed horizontal lines. The table shows cut-off ΔMFI and numbers of samples tested positive for NMDAR antibodies by the FACS assay at different serum dilutions. (B) Correlation of ΔMFI 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, ΔMFI values below 1,000 were set to 1,000. Correlation of exact ΔMFI values were calculated using non-parametric Spearman correlation. Correlation coefficient (R) and the p-value are shown in the graph. Here, the cut-off at the 1:100 dilution differs from the original cut-off (20,700), since a different batch of cells was used for analysis of this subsample. ¹ Data were analyzed using ROC analysis. CBA = cell-based assay. CTRL = control sample. ΔMFI = delta median fluorescence intensity. NMDAR-E = <i>N</i>-methyl-D-aspartate receptor encephalitis. ROC = receiver operating characteristic.</p

Comparison of Diagnostic Accuracy of Microscopy and Flow Cytometry in Evaluating <i>N</i>-Methyl-D-Aspartate Receptor Antibodies in Serum Using a Live Cell-Based Assay

<div><p><i>N</i>-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1–100.0 and 95.7–100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7–100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4–97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, p<0.0001) and correlation (r = 0.697, p<0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.</p></div

Immunofluorescence CBA with HEK293A cells transiently overexpressing functional NMDAR tagged with green fluorescent proteins.

<p>Staining pattern with NMDAR antibody positive (A-C) and negative (D) serum. HEK293A cells were transiently transfected to overexpress EmGFP-tagged NR1, NR2A and GFP-tagged NR2B, incubated with diluted human serum and NMDAR antibodies were visualized by a Cy3-conjugated secondary antibody and counter-stained with DAPI to detect dead cells (left column: green fluorescence/EmGFP+GFP; middle column: red fluorescence/Cy3; right column: overlay of EmGFP/GFP, Cy3 and DAPI (A+D)). (B)+(C) Images show colocalization of NMDAR and serum NMDAR antibodies at high magnification (scale bars: 10 μm). (B) NMDAR antibodies bound to surface of cells. (C) Bound NMDAR antibodies internalized by the cells. CBA = cell-based assay. DAPI = 4’,6-diamidino-2-phenylindole. (Em)GFP = (emerald) green fluorescent protein. NMDAR = <i>N</i>-methyl-D-aspartate receptor.</p

NMDAR IgG antibody titers and ΔMFI values in the discovery group.

<p>(A) Using the CBA serum NMDAR IgG antibodies were exclusively detected in serum samples of patients with NMDAR encephalitis, but not in neurological and healthy controls. (B) In the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological and healthy controls, but one serum positive for NMDAR antibodies was missed with this method (shown in red). The cut-off ΔMFI value of 20,700 is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Kruskal Wallis test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = <i>N</i>-methyl-D-aspartate receptor encephalitis.</p

Demographic data of patients and controls.

<p>CBA = cell-based assay. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = <i>N</i>-methyl-D-aspartate receptor encephalitis.</p><p>¹ Data are shown as median (range), p-value: groups were compared using</p><p>² Chi-Square test and</p><p>³ Kruskal-Wallis test,</p><p>⁴ Fisher’s exact test and</p><p>⁵ Mann-Whitney <i>U</i> test.</p><p>Demographic data of patients and controls.</p

NMDAR IgG antibody titers and ΔMFI values in the validation group.

<p>(A) With the CBA all sera of NMDAR encephalitis patients were positive for NMDAR IgG antibodies, but none of the neurological controls. (B) Using the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological controls, but again two sera positive for NMDAR antibodies were missed with this method (shown in red). The cut-off ΔMFI value of 20,700 as determined in the discovery group is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Mann-Whitney <i>U</i> test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. NC = neurological controls. NMDAR-E = <i>N</i>-methyl-D-aspartate receptor encephalitis.</p

Gating and analysis strategy for NMDAR and CD2 expressing HEK293A cells for FACS based analysis.

<p>(A) Gating of the HEK293A main cell population. (B) Exclusion of 7-AAD^pos cells. (C)+(D) Gating of (Em)GFP^pos7-AAD^neg NMDAR and CD2 expressing cells, respectively. The green (“(Em)GFP-A”) channel detects both, EmGFP and GFP-tagged proteins. Binding of patient‘s antibodies to NMDAR results in a difference (arrow) of the APC signal obtained with NMDAR-transfected cells (red line) when compared to the APC signal of CD2-transfected cells (black line), which results in ΔMFI (E). This difference is absent in the serum of a healthy control (F). 7-AAD-A = 7-amino-actinomycin D (area). APC-A = allophycocyanin (area). (Em)GFP-A = (emerald) green fluorescent protein (area). ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. FSC-H = forward scatter (height). NMDAR = <i>N</i>-methyl-D-aspartate receptor. SSC-H = side scatter (height).</p