Single‐center study of autoimmune encephalitis‐related autoantibody testing in Hungary

Abstract Objective Autoantibody detection is crucial for the early diagnosis of autoimmune encephalitis (AIE) since prompt therapy can determine the disease outcome. Here, we report a single‐center 6‐year retrospective study of autoantibody testing in AIE in the Hungarian population. Methods Serum and/or cerebrospinal fluid (CSF) autoantibody tests were performed using cell‐based indirect immunofluorescence assay for AIE diagnosis. Samples were provided by neurology clinics as part of a nationwide program. Test results were analyzed for samples received during the period from 2012 to 2018. Results We tested 1,247 samples from 1,034 patients with suspected AIE. Autoantibodies were present in 60 patients (5.8% of total). The distribution of patients with different autoantibodies by age and sex was as follows: NMDAR (70%), mostly in young females, LGI1 (15%) in middle‐aged males, GABABR (12%) in elderly males, and Caspr2 (7%) in males. Long‐term follow‐up was conducted in 30 patients with repeated test requests, of which 17 remained positive, and 13 switched to negative. Conclusion We report the most comprehensive clinical laboratory study of autoantibody testing in AIE in the Hungarian population. Our results show that the frequency of different autoantibody types in AIE corresponds to the data described in the literature.

the autoantibodies bind to the extracellular epitopes of the neuronal cell surface receptors or their associated proteins, which can lead to alteration of the structure and function of target antigens by different mechanisms. Thus in anti-NMDAR encephalitis, autoantibodies induce receptor cross-linking and internalization, in anti-LGI1 encephalitis autoantibodies interfere with protein-protein interactions, and in anti-GABA B R encephalitis autoantibodies may block the function of the target antigen (Hughes et al., 2010;Ohkawa et al., 2013). The autoantibodies cause reversible neuronal dysfunction, and immunotherapy (e.g., steroids, plasmapheresis, immunosuppression, and intravenous immunoglobulin) results in reduction of autoantibody levels and can lead to the improvement of patients (Hermetter, Fazekas, & Hochmeister, 2018). Patients can have a fatal outcome in case of lack of the proper therapy. This highlights the importance of early clinical diagnosis of AIE, in which the laboratory has a crucial role by providing accurate and reproducible testing of serum and/or cerebrospinal fluid (CSF) samples for the presence of autoantibodies.

| Samples
We carried out a retrospective statistical study of the results obtained by our laboratory based on serum and CSF analysis of patients with suspected AIE. Our laboratory was the first to introduce these tests in Hungary and has received samples from various neurological clinics and hospitals from 2012 through 2018 as part of a nationwide program. Serum and CSF samples were obtained with patients' informed consent. The study was approved by Regional Research Ethics Committee of the Medical Center, University of Pécs (RIKEB 6966/2017).

| Detection of AIE autoantibodies
For detection of AIE-related autoantibodies, a cell-based indirect immunofluorescence BIOCHIP assay was used (Euroimmun,

| Fluorescence imaging and evaluation
Fluoresce imaging was performed using a fluorescence microscope (Olympus BX61) coupled with Zeiss Axiocam 305 color microscope digital camera and image processing system. The BIOCHIPs were evaluated independently by at least two laboratory specialists.
Positive and negative controls were used, and reactions were graded as strong positive, positive, low positive, equivocal, and negative.

| Annual distribution and frequency of autoimmune encephalitis-related autoantibody types
Since the introduction of tests for AIE in 2012 at our institution, the number of test requests for diagnosing the disease has increased each year. Our laboratory has received 1,247 test requests (sera and/or CSF samples) from a total of 1,034 patients for detection of AIE-related autoantibodies ( Figure 1). We employed a cell-based indirect immunofluorescence BIOCHIP assay for the detection of six ion channel or their associated protein-specific autoantibodies and Caspr2, and the other against GABA B R and Caspr2). In 12 patients, the results obtained from sera were equivocal (11 NMDAR and one GABA B R); of which, five patients were negative upon simultaneous testing of CSF, and new samples from three patients were negative upon retesting.

| Characteristics of patients with positive autoantibodies
We investigated the distribution of AIE-related autoantibody subtypes by age and sex (Figure 3). Anti-NMDAR encephalitis mostly LGI1) turned to negative within the 3rd year; and one anti-NMDAR patient was found negative during the 5th year after the first positive test (Table 2).

| Influence of sample type on the laboratory test results
Among the 60 positive patients, in 34 cases (57% of autoantibody-

GABABR 12%
LGI1 15% NMDAR 70% autoantibodies in sera only, but not in the CSF. In four anti-GABA B R patients, positivity was detected in both serum and CSF, and in one case, serum showed higher level of antibodies than CSF. In one anti-Caspr2 patient, only the serum was positive. In one patient, both anti-LGI1 and anti-Caspr2 antibodies were detected in both sample types, although the anti-Caspr2 positivity was stronger in the serum than in CSF (Table 3).

| D ISCUSS I ON
AIE has been recognized during the past decade as a distinct disease entity (Dalmau & Graus, 2018;Venkatesan, Michael, Probasco, Geocadin, & Solomon, 2019). The discovery of AIE subtypes has changed the diagnostic and therapeutic approaches to many neurological disorders previously considered to be idiopathic. Our aim was to investigate the characteristics of autoantibody testing in patients with AIE, in which early and accurate clinical diagnosis plays a pivotal role. The current retrospective analysis included laboratory test results from 1,034 patients with suspected AIE, making it the most comprehensive study in Hungary to date. Our data confirmed the relative prevalence of AIE subtypes described previously (Dalmau & Graus, 2018). Anti-NMDAR encephalitis was the most common subtype, followed by anti-LGI1, anti-GABA B R, and anti-Caspr2 encephalitis, which is in agreement with previous reports (Gable, Sheriff, Dalmau, Tilley, & Glaser, 2012;van Sonderen et al., 2017). Our data regarding the age and sex of AIE patients agree with the data published in the literature (Ricken et al., 2018).
Highly sensitive and specific multiplex cell-based assay is

| CON CLUS ION
In conclusion, our data are in agreement with previous reports on the frequency and distribution of AIE-related autoantibodies, and detection of which can significantly aid the diagnosis of AIE and suggests treatment strategies. Early immunotherapy is often effective and can reduce the severity of AIE, promote recovery and decrease the risk of relapse (Crisp, Kullmann, & Vincent, 2016;Dalmau & Graus, 2018;Ricken et al., 2018;Varley, Taylor, & Irani, 2018 TA B L E 3 (Continued)

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
ZH, KaB, ZC, PB, ZK, and TB performed the tests and evaluated the results. ZH, KaB, and TB analyzed data. ZH and KoB performed imaging of indirect immunofluorescence staining of BIOCHIP using an Olympus BX61 fluorescence microscope. ZH, JN, and TB wrote the paper with editorial help from PB and ZK.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.