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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.

Ramberger M, Peschl P, Schanda K, Irschick R, Höftberger R, Deisenhammer F, Rostásy K, Berger T, Dalmau J, Reindl M - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.

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.

No MeSH data available.


Related in: MedlinePlus

NMDAR IgG antibody titers and ΔMFI values in the discovery group.(A) Using the CBA serum NMDAR IgG antibodies were exclusively detected in serum samples of patients with NMDAR encephalitis, but not in neurological and healthy controls. (B) In the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological and healthy controls, but one serum positive for NMDAR antibodies was missed with this method (shown in red). The cut-off ΔMFI value of 20,700 is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Kruskal Wallis test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = N-methyl-D-aspartate receptor encephalitis.
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pone.0122037.g002: NMDAR IgG antibody titers and ΔMFI values in the discovery group.(A) Using the CBA serum NMDAR IgG antibodies were exclusively detected in serum samples of patients with NMDAR encephalitis, but not in neurological and healthy controls. (B) In the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological and healthy controls, but one serum positive for NMDAR antibodies was missed with this method (shown in red). The cut-off ΔMFI value of 20,700 is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Kruskal Wallis test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = N-methyl-D-aspartate receptor encephalitis.

Mentions: With the CBA, in the discovery group NMDAR antibodies were detected in 7/7 (100%) patients with NMDAR encephalitis, 0/37 (0%) neurological controls and 0/32 (0%) healthy controls (Table 1). Sensitivity and specificity of the CBA were 100% (95% confidence intervals (CI) 59.0–100.0 and 94.8–100.0, respectively). Antibody titers in NMDAR encephalitis patients ranged from 1:640 to 1:20,480 (median 1:1,280) (Fig 2A).


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.

Ramberger M, Peschl P, Schanda K, Irschick R, Höftberger R, Deisenhammer F, Rostásy K, Berger T, Dalmau J, Reindl M - PLoS ONE (2015)

NMDAR IgG antibody titers and ΔMFI values in the discovery group.(A) Using the CBA serum NMDAR IgG antibodies were exclusively detected in serum samples of patients with NMDAR encephalitis, but not in neurological and healthy controls. (B) In the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological and healthy controls, but one serum positive for NMDAR antibodies was missed with this method (shown in red). The cut-off ΔMFI value of 20,700 is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Kruskal Wallis test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = N-methyl-D-aspartate receptor encephalitis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4376531&req=5

pone.0122037.g002: NMDAR IgG antibody titers and ΔMFI values in the discovery group.(A) Using the CBA serum NMDAR IgG antibodies were exclusively detected in serum samples of patients with NMDAR encephalitis, but not in neurological and healthy controls. (B) In the FACS assay serum NMDAR IgG ΔMFI levels were higher in patients with NMDAR encephalitis than in neurological and healthy controls, but one serum positive for NMDAR antibodies was missed with this method (shown in red). The cut-off ΔMFI value of 20,700 is indicated by a dashed horizontal line. Antibody titers and ΔMFI values were compared using a non-parametric test (Kruskal Wallis test) and overall p-values are shown in the graphs. Medians are indicated by horizontal bars. CBA = cell-based assay. ΔMFI = delta median fluorescence intensity. FACS = fluorescence activated cell sorting. HC = healthy controls. NC = neurological controls. NMDAR-E = N-methyl-D-aspartate receptor encephalitis.
Mentions: With the CBA, in the discovery group NMDAR antibodies were detected in 7/7 (100%) patients with NMDAR encephalitis, 0/37 (0%) neurological controls and 0/32 (0%) healthy controls (Table 1). Sensitivity and specificity of the CBA were 100% (95% confidence intervals (CI) 59.0–100.0 and 94.8–100.0, respectively). Antibody titers in NMDAR encephalitis patients ranged from 1:640 to 1:20,480 (median 1:1,280) (Fig 2A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.

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.

No MeSH data available.


Related in: MedlinePlus