Antibody titres at diagnosis and during follow-up of anti-NMDA receptor encephalitis: a retrospective study

Abstract

Background: Anti-N-methyl-d-aspartate (NMDA) receptor encephalitis is a severe but treatable autoimmune disorder which diagnosis depends on sensitive and specific antibody testing. We aimed to assess the sensitivity and specificity of serum and CSF antibody testing in patients with anti-NMDA receptor encephalitis, and the relation between titres, relapses, outcome, and epitope repertoire.

Methods: In this observational study, we used rat brain immunohistochemistry and cell-based assays (CBA) with fixed or live NMDA receptor-expressing cells to determine the sensitivity and specificity of antibody testing in paired serum and CSF samples. Samples were obtained at diagnosis from patients with anti-NMDA receptor encephalitis and from control participants worldwide. We deemed a patient to be antibody positive if their serum, their CSF, or both tested positive with both immunohistochemistry and CBA techniques; we determined titres with serial sample dilution using brain immunohistochemistry. We examined samples from 45 patients (25 with good outcome [modified Rankin Scale, mRS 0-2], ten with poor outcome [mRS 3-6], and ten with relapses) at three or more timepoints. We determined the epitope repertoire in the samples of 23 patients with CBA expressing GluN1-NMDA receptor mutants.

Findings: We analysed samples from 250 patients with anti-NMDA receptor encephalitis and 100 control participants. All 250 patients had NMDA receptor antibodies in CSF but only 214 had antibodies in serum (sensitivity 100.0% [98.5-1000%] vs 85.6% [80.7-89.4%], p<0.0001). Serum immunohistochemistry testing was more often in agreement with CBA with fixed cells (77 [71%] of 108) than with CBA with live cells (63 [58%] of 108, p=0.0056). In multivariable analysis, CSF and serum titres were higher in patients with poor outcome than in those with good outcome (CSF dilution 340 vs 129, difference 211, [95% CI 1-421], p=0.049; serum dilution 7370 vs 1243, difference 6127 [2369-9885], p=0.0025), and in patients with teratoma than in those without teratoma (CSF 395 vs 110, difference 285 [134-437], p=0.0079; serum 5515 vs 1644, difference 3870 [548-7193], p=0.024). Over time there was a decrease of antibody titres in the 35 patients with good or poor outcome and samples followed at three timepoints regardless of outcome (from diagnosis to last follow-up: CSF 614 to 76, difference 538 [288-788]; serum 5460 to 1564, difference 3896 [2428-5362]; both p<0.0001). Relapses were associated with a change in titre more often in CSF than in serum (14 of 19 vs seven of 16, p=0.037). After recovery, 24 of 28 CSF samples and 17 of 23 serum samples from patients remained antibody positive. Patients' antibodies targeted a main epitope region at GluN1 aminoacid 369; the epitope repertoire did not differ between patients with different outcomes, and did not change during relapses.

Interpretation: The sensitivity of NMDA receptor antibody testing is higher in CSF than in serum. Antibody titres in CSF and serum were higher in patients with poor outcome or teratoma than in patients with good outcome or no tumour. The titre change in CSF was more closely related with relapses than was that in serum. These findings emphasise the importance of including CSF in antibody studies, and that antibody titres can complement clinical assessments.

Funding: Dutch Cancer Society, National Institutes of Health, McKnight Neuroscience of Brain Disorders award, the Fondo de Investigaciones Sanitarias, ErasmusMC fellowship, and Fundació la Marató de TV3.

Copyright © 2014 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Determination of antibody titers using rat brain immunohistochemistry

Brain reactivity of serial dilutions of serum and CSF of a patient with good outcome (A) and a patient with poor outcome (C). Samples were obtained at the time of diagnosis (first sample) and at the last follow-up (last sample); the intermediate samples are not shown. During the interval between first and last samples, the modified Ranking Scale (mRS) of the patient with good outcome improved from 5 to 1, and the CSF titers decreased substantially; however, the serum titers were only mildly changed, showing a poor correlation between serum titers and clinical improvement. During a similar interval, the mRS of the patient with poor outcome did not improve, and the CSF and serum titers remained unchanged. Scale bars: 500µm. Panels B and D show a graphic representation of the change of titers in CSF (blue line) and serum (red), and the change of mRS (green) during the three time points studied. CSF titer and serum titer (× 100) are represented in the left Y axis and mRS is represented in the right Y axis.

Figure 2. Change of antibody titers in patients with good and poor outcome

A difference of magnitude of at least two dilutions up is colored in red, while a decrease of at least two dilutions down is colored in blue; minor changes are colored in grey. Each patient has a unique symbol which facilitates the comparison of patient’s serum and CSF. Patients with a tumor are depicted by circles or hexagons. Clinical details are provided in Table S2. The median decrease of antibody titers in CSF and serum between the indicated time points (originating from the initial median titer at time point 1) are shown in panels E and F; in these two panels patients with poor outcome are represented with dark colored lines, and patients with good outcome with light colored lines. Note that the median titer decrease was greater and occurred faster in CSF of patients with good outcome than in CSF of patients with poor outcome, or in serum of patients regardless of outcome.

Figure 3. Change of antibody titers in patients with clinical relapses

A difference of magnitude of at least two dilutions in the expected direction (decrease at remission, and increase at relapse) is colored in blue, smaller change of titers are shown in grey. Each patient has a unique symbol which facilitates the comparison of patient’s serum and CSF. Patients with an underlying tumor are depicted by circles. Clinical details are provided in Table S3.

Figure 4. Typical pattern of reactivity of patients’ antibodies with GluN1 deletion constructs

Schematic representation of GluN1 and GluN1 mutants (A). For all studies in this manuscript the wild type (WT) form of GluN1was used; the other GluN1 constructs were used to determine changes in the pattern of epitope recognition. The typical pattern of reactivity of a patient’s CSF is shown in row B, which demonstrates recognition of all constructs except G369I. The reactivity of the indicated GluN1 rabbit polyclonal antibody with the same mutants is shown in row C, and the merged reactivities in row D. Mutation G369I abolished the reactivity of 27/36 samples (Table 3) and substantially decreased the reactivity of the other 9 (Table S4 and Figure S2). Scale bar: 10µm.