Anti-NMDA-receptor antibody encephalitis: Performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay

Clinica Chimica Acta 421 (2013) 1–6

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Anti-NMDA-receptor antibody encephalitis: Performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay Brenda B. Suh-Lailam a, Thomas R. Haven a, Susan S. Copple b, Diana Knapp b, Troy D. Jaskowski a, Anne E. Tebo a, c,⁎ a b c

ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States ARUP Laboratories Inc., Salt Lake City, UT, United States Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, United States

a r t i c l e

i n f o

Article history: Received 9 November 2012 Received in revised form 28 January 2013 Accepted 4 February 2013 Available online 27 February 2013 Keywords: NMDAR Antibodies Diagnosis Encephalitis Autoimmune disease

a b s t r a c t Background: Antibodies targeting the NR1 subunit of the N-methyl-D-aspartate-receptor (NMDAR) are considered diagnostic for a novel form of autoimmune encephalitis. We report the validation of a qualitative indirect immunofluorescence antibody (IFA) test for the detection of anti-NMDAR IgG and describe the attributes of antibody-positive patients. Methods: The anti-NMDAR IgG assay (Euroimmun Diagnosika, Lübeck, Germany) was validated with serum and cerebrospinal fluid (CSF) specimens from 30 healthy and 50 disease controls as well as 5 anti-NMDAR IgG-positive individuals. Consecutive specimens (n = 1671) for anti-NMDAR IgG antibodies were evaluated and positive specimens titrated to end-point [starting dilutions: CSF; 1:1 and serum; 1:10]. In a subset of antibody-positive patients, we sought clinical information for correlation with diagnostic and treatment outcomes. Results: The assay demonstrated excellent performance characteristics in all groups evaluated. Of the 1671 specimens tested, 1389 were unique cases with a positivity rate of 9.0% (n = 123). For the antibody-positive samples, the female to male ratio was 2:1 with a prevalence of 46% in the pediatric population (≤17 years). Antibody titers were titrated to end-point for 106/123 specimens [45 CSF, 41 sera, and 20 CSF and serum pairs] with more than 75% having titers greater than 1:10 (CSF) and 1:20 (serum). Overall, high levels of these antibodies showed correlation to disease severity with variable response to treatment in the subset of patients evaluated. Conclusion: Our data suggests a high prevalence for anti-NMDAR antibody encephalitis irrespective of age and gender in our unselected disease cohort with support for measuring antibody titers in the evaluation of these patients. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Antibodies targeting the NR1 subunit of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) identify a newly-described encephalopathy associated with ovarian tumors in young women and severe psychiatric as well as neurologic deficits in previously healthy children and adults [1–8]. Although the general clinical presentation in pediatric and adult patients with anti-NMDAR encephalitis is similar, some reports suggest differences in frequency, disease phenotype and risk for tumors which may influence the diagnostic evaluation and management of these patients [2,4–7]. At onset, patients generally present with psychiatric symptoms, seizures, bizarre behavioral changes, memory deficit and confusion with subsequent ⁎ Corresponding author at: ARUP Laboratories, 500 Chipeta Way, Salt lake City, UT 84108, United States. Tel.: +1 801 583 2787x3138; fax: +1 801 584 5207. E-mail address: [email protected] (A.E. Tebo). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.02.010

development of movement disorders, decreased consciousness and autonomic instability in the late phase of disease [3,8]. Initially identified in young women with ovarian teratoma, anti-NMDAR encephalitis is also recognized to occur in children and men with or without a tumor [1–8]. The true prevalence of anti-NMDAR encephalitis remains unknown with a reported large variability in the female to male ratio, lack of epidemiological data, poor awareness and/or heterogeneity in disease presentation. In a consecutive patient population, Irani et al. reported a prevalence of 11.1% (50/450) [8]. Based on their clinical and laboratory experience, Dalmau and colleagues reported 400 cases in just 3 y [9]. A recent study of the California encephalitis project showed that the frequency of anti-NMDAR encephalitis rivaled viral etiologies in that cohort with a 65% prevalence in children ≤18 y [10]. In 203 patients with encephalitis, Ganerod and colleagues reported a prevalence of 21% cases with acute immune-mediated encephalitis [11]. Based on these studies, it would appear that the positive predictive value of the assay is largely dependent on the population investigated.

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For example, in the consecutive patient population described by Irani et al. [8], a prevalence of 11.1% is noted compared to 65% in children ≤18 y in the California encephalitis project [10]. NMDAR belongs to a group of glutamate receptors and is made up of GluN1, GluN2A–GluN2D, GluN3A, and GluN3B subunits. Functional NMDARs require assembly of two GluN1 subunits together with either 2 GluN2 subunits or a combination of GluN2 and GluN3 subunits as well as simultaneous binding of both glutamate and glycine for activation [reviewed in 12]. These receptors play an important role in many integrative brain functions as well as in neuronal development. Thus, it is not too surprising that autoantibodies which recognize different NMDAR subunits are implicated in a wide variety of neurological disorders including ischemic stroke, Rasmussen encephalitis, non-herpetic acute encephalitis, other focal epilepsies, paraneoplastic encephalopathies, as well as in systemic autoimmune disorders such as systemic lupus erythematosus (SLE) [13–16]. In SLE, antibodies directed against the NR2A subunit have been reported to play a role in neuropsychiatric manifestations [16]. However, antibodies directed to NR1 subunit have been demonstrated to be specific for anti-NMDAR encephalitis [2]. Until recently, testing for anti-NMDAR antibodies in the US was performed on a research basis. A qualitative cell-based indirect immunofluorescence antibody (IFA assay) that is approved for in vitro diagnostic use in human serum has recently become available.

Table 1 Characteristics of patient and healthy control samples used in anti-NMDAR IgG IFA evaluation. Study groups

Specimen # of F/M Age type samples ratio range (y)

Results

Healthy controls

Serum

30

25/5 25–44 Negative

NMDAR antibody-positive

Serum/ CSF

4/1

N/A

Disease controls (n = 50; F/M = 37/13)

Serum Serum Serum

13 10 9

10/3 15–76 Negative 8/2 19–60 Negative 7/2 24–39 Negative

Serum

11

7/4 27–80 Negative

Serum Serum

3 4

2/1 26–80 Negative 3/1 22–66 Negative

ANA IFA positive AQP4 positive AChR binding positive Oligoclonal band and MBP positive Hu/amphiphysin Antimitochondrial antibody positive Oligoclonal band and MBP (10 positive and 6 negative)

16a

CSF

N/A

7/9

Positive

3–63 Negative

Disease controls (age range 15–80 y) include samples positive for autoantibody markers which may interfere with cell-based IFA and/or suggestive diverse autoimmune diseases. Abbreviations; ANA: Antinuclear antibody; AQP4: Aquaporin 4; AChR: Acetylcholine receptor; MBP: Myelin basic protein; F/M ratio: Female to male ratio. a Of the CSF controls, 13 had a corresponding serum all of which were negative for NMDAR antibodies (data not shown).

2. Materials and methods

A Percentage of Patients (%)

For investigating the performance characteristic of the anti-NMDAR IgG by IFA, serum samples from 30 healthy individuals (age range 25–44 y, female to male (F/M) ratio 25/5) and 50 disease controls (age range 15–80 y, F/M ratio 37/13) [13 antinuclear antibody (ANA) positive 31:80; 10 aquaporin 4 (AQP4) positive; 9 acetylcholine receptor (AChR) binding-positive; 11 oligoclonal band (OB)and myelin basic protein (MBP)-positive, 3 neuronal antibody positive (Hu/amphiphysin) and 4 mitochondrial antibody positive] as well as 16 CSF specimens [6 positive OB and MBP; and 4 OB-positive and MBP-negative; 3 OB-negative and MBP-positive and 4 OB-negative and MBP-negative] were evaluated. Of the 50 sera, 11 (OB-positive and MBP-positive) had a clinical diagnosis consistent with multiple sclerosis (MS). We chose to include the non-specific ANA due to its association with known autoimmune diseases such as SLE that may have neurologic manifestations. The other tests were also selected based on their presence in diseases with clinical symptoms similar to NMDAR encephalitis and/or inflammation of the central nervous system (CNS). No clinical information was available for the 16 CSF specimens, however, 13 of these had a serum pair to further evaluate the assay performance. As positive controls, 5 defined NMDAR antibodypositive samples [4 sera and 1 CSF] from a single investigator were also analyzed. In addition, 1671 consecutive serum and/or CSF patient specimens received at ARUP Laboratories for anti-NMDAR antibody testing were investigated for the presence of these antibodies. Samples referred for testing where received from facilities all over the US with no clinical indications for evaluation. For some of these specimens, requests for CSF evaluation (albumin, cell count, protein, oligoclonal band profile) and differential assessment of diverse autoimmune diseases as well as viral and bacterial infections were requested. Of the 1671 samples, 1389 were unique cases with 123 positive for antiNMDAR antibody. Anti-NMDAR antibody titers were determined in 106 and clinical information supporting a diagnosis of anti-NMDAR IgG encephalitis was sought and correlated with diagnostic and treatment outcomes in 25 cases from chart reports, emails or telephone conversations. This study was performed using a generalized approved study protocol for autoimmune disease research studies by the Institutional Review Board (IRB) of the University of Utah (IRB #29507).

100 80 60 40 20 0 Male

Female

0-17 years

Male

B

Female

18-50 years

NMDA Negative

Anti-NMDAR IgG Positive (%)

2.1. Study population

Male

Female

>50 years

NMDA Positive

50 40 30 20 10 0

0-17 years

18-50 years

>50 years

Age Range (Years) Fig. 1. Age and sex-distribution of anti-NMDAR IgG-positive patient samples in consecutive samples tested at ARUP Laboratories. (A) Percent (%) positive and negative for anti-NMDAR IgG by sex and specific age groups. (B) Comparable prevalence of anti-NMDAR IgG antibodies in patients ≤17 and 18–50 y.

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2.2. Determination and titration of anti-NMDAR IgG antibodies

serum and CSF. Intra- and inter-assays CV were established by demonstrating comparability in reading and interpreting results for samples that were negative [3 sera: b1:10; 3 CSF: b1:1] as well as multiple specimens with varying levels of antibodies in the previously titered samples (3 sera: ≥1:10 and ≤1:40; 3 sera: ≥:1:80 and 3 CSF: ≥1:1and ≤1:10; 3 CSF ≥1:20). Reading and interpretation were performed for within run and on 4 different days by at least 3 Medical Technologists. Equivalency was determined if results were within ±2 variation in end-point titers.

All specimens [serum and cerebrospinal fluid (CSF)] were evaluated for anti-NMDAR IgG antibodies by IFA using human embryonic kidney (HEK) 293 cells transfected with the NR1 subunit of the NMDAR complex and immobilized on BIOCHIPs (Euroimmun AG, Lübeck, Germany). Untransfected HEK 293 cells were used as negative control on the BIOCHIPs. This assay is currently cleared for in vitro diagnostic use by the FDA in the USA as a qualitative test with serum as the recommended specimen type at an initial dilution of 1:10. CSF specimens were evaluated and validated at a dilution of 1in 1 (neat or referred to as 1:1) and testing was performed following the manufacturer guidelines. Following incubation of samples with transfected and untransfected cell lines, slides were washed and stained with fluorescein-labeled anti-human IgG antibodies and visualized using a fluorescence microscope. Results were reported as positive or negative based on the manufacturer's suggested recommendations for reading and interpretation. To validate the determination of anti-NMDAR IgG antibody titers, positive specimens (72 sera and 74 CSF) were serially diluted to end-point titers. Some of the specimens evaluated were collected at different times following initial screening and therefore not unique samples. For serum samples, a two-fold dilution (1:10, 1:20, 1:40, 1:80, 1:160, 1:320 etc.) was employed while CSF samples were evaluated at 2, 5 and 10-fold dilutions (data not shown) and subsequently validated to be read at 1:1, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 etc. for increased reproducibility as well as comparability of titers in

A

3

2.3. Statistical analysis We used GraphPad Prism version 5.04 for statistical evaluation. Anti-NMDAR IgG antibodies were log-transformed for a better fit and correlations between serum and CSF paired samples were performed using Wilcoxon matched-pairs signed rank test. Results with p b 0.05 were considered significant. 3. Results The anti-NMDAR IgG assay performance was determined using samples with defined autoantibodies for classical autoimmune diseases which may be considered in the differential evaluation of anti-NMDAR antibody encephalitis and/or may potentially interfere with the analytical functioning of the test. In all, sera from 30 healthy individuals as well as 50 disease controls with a variety of autoimmune diseases

25

Number of Patients

20

15

10

5

0 Neat - 1:5

1:10 - 1:20

1:40 - 1:80

1:160 - 1:320

1:640 - 1:1280

Antibody Titer of Initial Specimen Female - CSF

B

Male - CSF

Female - Serum

Male - Serum

C

Fig. 2. Anti-NMDAR IgG antibodies levels in positive patient specimens. (A) Distribution of anti-NMDAR IgG antibody titers in unique patient specimens evaluated at ARUP Laboratories. (B) Anti-NMDAR IgG titers for paired serum and CSF samples. Paired samples with either a negative serum or CSF could not be plotted. (C) Significantly higher anti-NMDAR IgG antibody levels in serum than in CSF (Wilcoxon matched-pairs signed rank test, p value = 0.02). Note that the antibody titers were log-transformed for a better fit.

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were analyzed for analytical and clinical specificity. Based on these two control cohorts, excellent specificity of the anti-NMDAR antibody cell based assay (Table 1) was observed. The diagnostic sensitivity of the assay was verified using a limited number of positive samples provided by a single investigator. These samples were also used in training and documenting the necessary expertise to perform testing (data not shown). Due to the encephalopathology associated with anti-NMDAR IgG antibodies as well as reports suggesting a possible differential presence of these autoantibodies in serum and CSF, there was a clinical need to validate CSF as an alternate specimen type [3,8,9]. To achieve this, CSF specimens leftover from laboratory evaluation of multiple sclerosis with and without evidence of CNS inflammation were employed (Table 1). Compared to serum samples which are screened at 1:10, CSF specimens were screened neat and qualitative results reported at this dilution. Upon implementation of the anti-NMDAR IgG assay, we monitored the positivity rate as well as distribution by antibody-positive cases by gender and age. Of the 1389 unique patient specimens tested at our facility for this antibody marker, 123 were positive with a positivity rate of 9%. Anti-NMDAR antibodies were detected in 81 females (median age 21 y, range 2–53 y) and 42 males (median 19 y, range 0–78 y), with ~ 2:1 female to male ratio. Fig. 1A shows the distribution of antibody-positive patients based on gender and specific-age groups. In the pediatric cases (≤ 17 y), there appears to be no gender bias in the % of anti-NMDAR IgG antibody-positivity as seen in patients in the 18–50 y age group. Based on the age-specific cohorts defined (Fig. 1B), there was also no difference in antibody prevalence in pediatric cases (≤17 y) and individuals between 18 and 50 y. However, in patients >50 y the prevalence of these antibodies was found to be much lower (Fig. 1B). Overall, the frequency of antibodies observed in our consecutive population is similar to that described by Irani et al. [8]; 50/450 (11.1%) versus 123/1389 (~ 9%). In addition, the number of positive cases identified in about 14 months (n = 123) is in line with the 419 patients in 3 y. as described by Dalmau et al. [9]. The anti-NMDAR IgG is a qualitative test with results reported as negative or positive; 2 independent studies suggest that determination of antibody levels in the CSF may be important in the evaluation and management of positive patients [3,8]. These investigations showed that individuals with relatively higher levels of antibodies particularly in the CSF did present with more severe manifestations and that if these antibodies remained stable following treatment, the outcome was not usually favorable. We determined end-points titers for 106 unique patients (45 CSF, 41sera and 20 CSF/serum pairs). The titers for these unique specimens (first time tested in our laboratory) based on sex and age are shown in Fig. 2A. In addition, we compared antibody levels in the serum and CSF pairs for 20 patients collected within a 24-h period (Fig. 2B and C). Of the 20 paired samples, 14 were positive for both serum and CSF, 4 were CSF positive and serum negative while 2 were serum positive and CSF negative. One of the CSF positive (1:1) and serum negative specimen was subsequently confirmed to have a different diagnosis from anti-NMDAR encephalitis (P066; Table 2). For the limited paired specimens analyzed, the serum antibody levels seem to be significantly higher than that of the CSF specimens (Fig. 2B and C). This finding appears to be consistent with the observation by Irani et al. [8] but contrary to that of Dalmau and colleagues [3] which may be reflective of the test methods and their analytical principles. Of the 106 antibody-positive patients with titers, a diagnostic confirmation could be obtained for 25 cases. These included 7 females with classical anti-NMDAR encephalitis with ovarian tumor, 15 cases without tumor (11 females and 4 males), 2 females who expired shortly after disease confirmation and1 patient whose result was determined to be false-positive (CSF titer of 1:1). Of the 25 patients with clinical information, 3 (12%) had a previous diagnosis of anti-NMDAR antibody encephalitis and testing was done to determine relapse (P041, P055 and P065; Table 2). In one patient (P041), reappearance of antibodies was associated with identification of a teratoma. Removal of ovaries resulted

Table 2 Anti-NMDAR IgG antibody titers at onset and follow-up for confirmed clinical patients. Study Sex/ ID age

Onsetζ

Titers at diagnosis

Anti-NMDAR encephalitis without tumor P002 F/12 N/A 1:320 (S); 1:20 (C) P042 M/8 ~1 week 1:160 (C) P053 F/12 N/A Positive§ (S) P055 F/12 >2 years 1:1 (C) P065 F/25 >2 years 1:160 (S); 1:20 (C) P070 M/18 N/A 1:640 (S); 1:80 (C) P085 F/22 ~2 weeks 1:160 (S) & 1:10 (C) P088 M/4 N/A 1:160 (S); 1:5 (C) P089 F/13 ~1 week 1:160 (S) P090 M/18 1 month 1:640 (S) P096 F/18 N/A 1:160 (S) P104 P107 P110 P111

F/9 F/13 F/31 F/19

~1 week ~2 weeks N/A ~1 week

1:20 (S) 1:160 (S) 1:80 (C) 1:320 (S); 1:80 (C)

Anti-NMDAR encephalitis with tumor P032 F/22 N/A 1:40 (S) P041 F/24 2 years 1:80 (S) P062 F/26 N/A 1:160 (S); 1:40 (C) P076 F/21 N/A 1:80 (S); 1:160 (C) P098 F/25 1 month 1:40 (C) P105 F/10 ~2 weeks 1:1280 (S) & 1:20 (C) P143 F/31 ~1 week 1:80 (S)

Follow-up titers; time after

None 1:20 (C); 2 months 1:80 (C); 6 weeks 1:1 (C); >2 months 1:5 (C); 3 months None 1:80 (C); >4 months after None 1:80 (S); 4 months 1:320 (S); 6 weeks b1:10 (S) & 1:1 (C); > 4 months 1:20 (S); >3 months 1:10 (S); 1 month 1:5 (C); 1 month None

1:320 (S); >1 year b1:10 (S) 1:160 (S) & 1:10 (C); 2 weeks 1:160 (S); 1 month None None None

Anti-NMDAR encephalitis; expired shortly after diagnosis P052 F/13 N/A 1:320 (S) None P100 F/28 ~1 month 1:640 (S) None Onsetζ: refers to estimated time of disease onset (N/A: not available). S and C represent serum and CSF used for evaluation respectively. Positive§: no titer available for this patient. A patient with false positive CSF anti-NMDA result (at 1:1) and a subsequent diagnosis of Klebsiella infection is not shown.

in a negative serum within less than 6 weeks (P041; Table 2). Table 2 shows the age, gender, time of disease onset, antibody titers at evaluation and follow-up where available for each patient. For some of these patients, multiple specimens taken at different periods during their management were available to monitor treatment outcome. Based on the data obtained at evaluation and for limited follow-up for some of these patients (Fig. 3), determination of antibody levels appears to be of clinical relevance particularly in the CSF. Of the patients represented in Fig. 3, three patients with CSF specimens evaluated for anti-NMDAR antibodies with a confirmed diagnosis of autoimmune encephalitis without tumor had a rapid decline in antibody titers following treatment (Fig. 3A–C), albeit for a very short period of follow-up. Patients in panels 3D and 3E had persistent levels of serum antibodies which did not reflect improvement in clinical outcome, especially for the case in 3D. The patient represented in 3E (P032) had rising antibody titers which were associated with the emergence of a second tumor about a year following diagnosis and the identification of the first tumor. 4. Discussion This study was initiated to validate a test that was recently approved by the Food and Drug Administration (FDA) for the diagnosis of antiNMDAR encephalitis for which little data regarding the assay performance as well as the disease characteristics was known outside research groups familiar with the clinical entity. To avoid misclassification of patients, it was important to demonstrate excellent specificity of this test as well as document the necessary competence in the reading and interpreting the cell based assay. In addition, given the paucity of data related to the disease prevalence, we sought to monitor the characteristics of antibody-positive patients in consecutive specimens referred for testing at our institution, a national esoteric reference laboratory. A major

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B

1000

1000

Antibody titer

Antibody Titer

A

100

10

100

10

1

1 0

5

10

15

0

20

6

12

Days

18

24

30

Days

C

D 1000 Antibody titer

1000

Antibody titer

5

100

10

1

100

10

1 0

15

30

45

60

75

0

Days

25

50

75

100

125

Days

E Antibody titer

1000

100

10

1 0

100

200

300

400

Days Fig. 3. Anti-NMDAR IgG antibody profiles in serum (▲) and CSF (–○–) in select patients at different time points following evaluation and initiation of treatment. Panels A–C (P062, P110 and P042; Table 2) show rapid decline in CSF following treatment with persistently high serum anti-NMDAR IgG antibody titers in patient P062 (panel A) following oophorectomy (downward arrow). Reduction in CSF anti-NMDAR IgG antibody titers in treated patients correlated with clinical outcome. (D) Persistently high serum antibody titers despite clinical response to treatment. (E) Shows increasing titers in serum anti-NMDAR IgG antibodies in a patient with a previous diagnosis of LE with ovarian tumor who subsequently developed another tumor in the second ovary.

finding of our investigation is the prevalence of anti-NMDAR IgG antibodies in unique patient samples which is comparable to results obtained under similar settings [8,9]. Our data corroborate earlier published observations by others and highlights the importance of raising awareness regarding the prevalence of this immune form of encephalitis which is treatment responsive in a majority of patients if diagnosed early [4–9]. In addition, our results also indicate that testing for these antibodies in both serum and CSF may improve diagnostic sensitivity. Furthermore, reporting of test results in titers may be helpful both in predicting disease risk and management. However, more follow-up studies are required to provide guidelines regarding the use of antibody testing in monitoring treatment and/or long term management of anti-NMDAR IgG-positive patients. Our observation that NMDAR IgG antibody levels were higher in the serum compared to CSF in paired specimens has also been reported by others [6,8,17]. Irani et al. [18], noted that serum levels of NMDAR antibodies are almost always higher in the CSF suggesting that the disease may originate in the peripheral and not in the brain. While this explanation is plausible, this difference may also be due to the methodologies and their inherent analytical principles.

Our investigation is not without shortcomings, most notably the lack of clinical information for a majority of the antibody-positive patients. Due to intricate nature of referral testing, direct access to clinical information for all patients was impractical. Thus, it was impossible to determine under our setting if all antibody-positive patients had syndromes consistent with described features of this form of encephalitis. For the 25 cases with clinical information, the presence of these antibodies in 24 patients was concordant with the typical NMDAR antibody encephalitis. Another limitation of our study was the absence of an alternate methodology such as rodent/primate brain (hippocampus or cerebellum) immunohistochemistry or fluorescent immunoprecipitation assays [8,19] for anti-NMDAR IgG antibodies to confirm discrepant results particularly between paired CSF and serum specimens. We recently exchanged 50 blinded specimens for evaluation with another facility for detecting these antibodies using the same methodology and observed unequivocal concordance (Tebo, unpublished data). This provides some reassurance that the analytical and diagnostic performance of this assay is consistent with expected clinical outcome. Thus, while confirmation by an alternative methodology seems ideal, it is important

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to note that qualitative and quantitative differences have been reported in intra-laboratory studies [8]. NMDAR encephalitis is a form of autoimmune-mediated encephalitis and may be paraneoplastic or non-paraneoplastic [1–8]. Paraneoplastic encephalitis is seldom treatment-responsive and mainly characterized by autoantibodies directed to intracellular neuronal antigens such as Hu, Ma2, CV2/CRMP5 and ampiphysin. Autoantibodies to neuronal cell membrane antigens are usually associated with non-paraneoplastic encephalitis and are typically treatment-responsive [18,20]. In addition to anti-NMDAR antibodies, autoantibodies against leucine-rich glioma inactivated 1 protein (LGI1), contactin associated protein 2(CASPR2), contactin-2 (VGKC-complex antibodies), α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptor (AMPAR), and γ-aminobutyric acid type B receptor (GABABR) may not be associated with cancers [18,20–22]. These antibodies are usually associated with autoimmune encephalitis and should be considered in its differential diagnosis. Our study reflects the successful implementation of a new immunologic test for the diagnosis of anti-NMDAR encephalitis in a reference laboratory setting. Based on the excellent performance of the anti-NMDAR IgG by IFA, testing for these antibodies in patients with encephalitis of unknown origin should be sought early in disease evaluation. Identification of biomarkers that may help stratify anti-NMDAR IgG-positive patients based on risk of a neoplasm would be beneficial for long term disease management. Acknowledgments We acknowledge the contributions of the Autoimmune Immunology technical staffs of ARUP Laboratories Inc., especially Rashelle Giles for the assistance in coordinating the testing, interpretation and reporting of anti-NMDAR IgG antibody titers. Kits for evaluation and validation were provided free-of-charge by EUROIMMUN US. References [1] Vitaliani R, Mason W, Ances B, Zwerdling T, Jiang Z, Dalmau J. Paraneoplastic encephalitis, psychiatric symptoms, and hypoventilation in ovarian teratoma. Ann Neurol 2005;58(4):594–604. [2] Dalmau J, Tuzun E, Wu HY, et al. Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma. Ann Neurol 2007;61:25–36. [3] Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-nmda-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008;7:1091–8.

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