Abstract
Detection of neuronal surface antibodies (NSAb) is important for the diagnosis of autoimmune encephalitis (AE). Although most clinical laboratories use a commercial diagnostic kit (Euroimmun, Lübeck, Germany) based on indirect immunofluorescence on transfected cells (IIFA), clinical experience suggests diagnostic limitations. Here, we assessed the performance of the commercial IIFA in serum and CSF samples of patients with suspected AE previously examined by rat brain immunohistochemistry (Cohort A). Of 6213 samples, 404 (6.5%) showed brain immunostaining suggestive of NSAb: 163 (40%) were positive by commercial IIFA and 241 (60%) were negative. When these 241 samples were re-assessed with in-house IIFA, 42 (18%) were positive: 21 (9%) had NSAb against antigens not included in the commercial IIFA and the other 21 (9%) had NSAb against antigens included in the commercial kit (false negative results). False negative results occurred more frequently with CSF (29% vs 10% in serum) and predominantly affected GABABR (39%), LGI1 (17%) and AMPAR (11%) antibodies. Results were reproduced in a separate cohort (B) of 54 AE patients with LGI1, GABABR or AMPAR antibodies in CSF which were missed in 30% by commercial IIFA. Patients with discordant GABABR antibody results (positive in-house but negative commercial IIFA) were less likely to develop full-blown clinical syndrome; no significant clinical differences were noted for the other antibodies. Overall, NSAb testing by commercial IIFA led to false negative results in a substantial number of patients, mainly those affected by anti-LG1, GABABR or AMPAR encephalitis. If these disorders are suspected and commercial IIFA is negative, more comprehensive antibody studies are recommended.
Highlights
Detection of antibodies to neuronal surface proteins and synaptic receptors is important to establish a definitive diagnosis of autoimmune encephalitis [1]
These 404 samples were analyzed by the commercial indirect immunofluorescence assay (IIFA) and 163 (40%) resulted positive for IgG against one of the included antigens: 68 (42%) for NMDAR [27 sera/41 cerebrospinal fluid (CSF)], 52 (32%) for LGI1 [26 sera/26 CSF], 16 (10%) for AMPAR [11 sera/5 CSF], 15 (9%) for CASPR2 [9 sera/6 CSF], 11 (7%) for GABABR [6 sera/5 CSF] and 1 (1%) for DPPX [serum] antibodies (Figure 1)
21 (9%) of the 241 commercial IIFA-negative samples showed a positive result on the in-house IIFA for antigens included in the commercial kit: 11 LGI1 (4 sera/7 CSF), 7 GABABR (1 serum/6 CSF), 2 AMPAR (2 CSF), and 1 NMDAR (Figures 1 and 2)
Summary
Detection of antibodies to neuronal surface proteins and synaptic receptors is important to establish a definitive diagnosis of autoimmune encephalitis [1]. Samples that produced a neuropil immunostaining on rat brain immunohistochemistry were subsequently examined with two types of IIFAs: 1) the Autoimmune Encephalitis Mosaic 6 kit (Euroimmun, Lübeck Germany), following manufacturer’s instructions and recommended dilutions (undiluted CSF and 1:10 serum), to test IgG antibodies against N-methyl-D-aspartate (NMDA) receptor (GluN1), a-amino-3-hydroxyl-5-methyl-4isoxazole-propionate (AMPA) receptor (GluA1, GluA2), gamma-aminobutyric (GABA) B receptor (B1 and B2 subunits), contactin-associated protein-like 2 (CASPR2), leucine-rich glioma-inactivated protein 1 (LGI1) and dipeptidyl-peptidase 6 (DPPX), 2) in-house IIFAs in which HEK293 cells were transfected with DNA constructs to express the following antigens: NMDA receptor (GluN1, GluN2), AMPA receptor (GluA1, GluA2), GABAB receptor (B1, B2), CASPR2, LGI1 (with and without disintegrin and metalloproteinase domain-containing protein 23 [ADAM23] co-transfection), GABAA receptor (a1, b3), metabotropic glutamate receptors mGluR1, mGluR2, mGluR5, Ig-Like Domain-Containing Protein 5 (IgLON5) or Seizure 6-like protein 2 (SEZ6L2) as previously described [7,8,9,10,11,12,13,14,15,16,17]. Written inform consent was not required as the study was observational, and the detection of neuronal surface antibodies was requested as part of the routine diagnostic work-up
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