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

Abstract

N-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.

NMDA receptor antibodies

“…Serum samples from 366 individuals were included: 51 patients with ADEM, 41 patients with NMOSD, 34 patients with clinically isolated syndrome (CIS), and 89 patients with MS. Nine patients with NMDAR encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were included as controls. Most of the patients have previously been included in a study on NMDAR antibody assay validation 6 and in other studies on MOG and/or AQP4 antibodies. 7 , 8 NMOSDs were diagnosed as described by Wingerchuk et al in 2007, ADEM was diagnosed according to the criteria of the International Pediatric MS Study Group, and MS and CIS were diagnosed according to the 2005 revisions to the McDonald criteria, as previously described.…”

“… 7 , 8 NMOSDs were diagnosed as described by Wingerchuk et al in 2007, ADEM was diagnosed according to the criteria of the International Pediatric MS Study Group, and MS and CIS were diagnosed according to the 2005 revisions to the McDonald criteria, as previously described. 6 – 8 Diagnosis of NMDAR encephalitis was based on clinical assessment (e.g., new onset of neuropsychiatric symptoms) and demonstration of antibodies in serum or CSF, as recommended recently. 9 The relative frequency of symptoms suggestive of NMDAR encephalitis was selectively increased in our cohort of patients with demyelinating diseases ( figure ).…”

“…For the detection of autoantibodies against NMDAR, AQP4, and MOG we used live HEK293A cells transfected with the respective complementary DNA, as described previously. 6 , 7 The following cutoff values were used: ≥1:20 for NMDAR antibodies, ≥1:160 for MOG antibodies, and ≥1:20 for AQP4 antibodies.…”

Serum anti-NMDA-receptor antibodies and cognitive function after ischemic stroke (PROSCIS-B)

“…Details on the procedure have been described elsewhere. (15,16) Titer levels started from a dilution of 1:10, which defined All rights reserved. No reuse allowed without permission.…”

Autoantibodies to central nervous system neuronal surface antigens: psychiatric symptoms and psychopharmacological implications

“…Most CBAs ascertain titre by performing serial dilutions and ascertaining the lowest dilution at which immunofluorescence is detected. More finely grained quantitative CBA results are possible using fluorescence-activated cell sorting (Amatoury et al 2013 ), but so far, this is limited to a small number of laboratories and the sensitivity of the method has in one study been shown to be inferior to microscopy (Ramberger et al 2015 ). (4) Since NMDAR antibody encephalitis is associated with IgG antibodies, it had been assumed that only these antibodies had functional effects and hence could cause disease.…”