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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

Immunofluorescence CBA with HEK293A cells transiently overexpressing functional NMDAR tagged with green fluorescent proteins.Staining pattern with NMDAR antibody positive (A-C) and negative (D) serum. HEK293A cells were transiently transfected to overexpress EmGFP-tagged NR1, NR2A and GFP-tagged NR2B, incubated with diluted human serum and NMDAR antibodies were visualized by a Cy3-conjugated secondary antibody and counter-stained with DAPI to detect dead cells (left column: green fluorescence/EmGFP+GFP; middle column: red fluorescence/Cy3; right column: overlay of EmGFP/GFP, Cy3 and DAPI (A+D)). (B)+(C) Images show colocalization of NMDAR and serum NMDAR antibodies at high magnification (scale bars: 10 μm). (B) NMDAR antibodies bound to surface of cells. (C) Bound NMDAR antibodies internalized by the cells. CBA = cell-based assay. DAPI = 4’,6-diamidino-2-phenylindole. (Em)GFP = (emerald) green fluorescent protein. NMDAR = N-methyl-D-aspartate receptor.
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pone.0122037.g001: Immunofluorescence CBA with HEK293A cells transiently overexpressing functional NMDAR tagged with green fluorescent proteins.Staining pattern with NMDAR antibody positive (A-C) and negative (D) serum. HEK293A cells were transiently transfected to overexpress EmGFP-tagged NR1, NR2A and GFP-tagged NR2B, incubated with diluted human serum and NMDAR antibodies were visualized by a Cy3-conjugated secondary antibody and counter-stained with DAPI to detect dead cells (left column: green fluorescence/EmGFP+GFP; middle column: red fluorescence/Cy3; right column: overlay of EmGFP/GFP, Cy3 and DAPI (A+D)). (B)+(C) Images show colocalization of NMDAR and serum NMDAR antibodies at high magnification (scale bars: 10 μm). (B) NMDAR antibodies bound to surface of cells. (C) Bound NMDAR antibodies internalized by the cells. CBA = cell-based assay. DAPI = 4’,6-diamidino-2-phenylindole. (Em)GFP = (emerald) green fluorescent protein. NMDAR = N-methyl-D-aspartate receptor.

Mentions: Fig 1 shows the typical antigen distribution of HEK293A cells overexpressing NMDAR tagged with green fluorescent proteins and the staining pattern with serum IgG of an NMDAR encephalitis patient at low magnification and higher magnification using a confocal microscope. It clearly shows the colocalization of membrane-associated NMDAR with serum antibodies of the patient but no colocalization with intracellular NMDAR probably residing within the endoplasmic reticulum (Fig 1B). Internalization of NMDAR in response to antibody binding observed in some but not all cells in the live CBA is shown in Fig 1C.


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)

Immunofluorescence CBA with HEK293A cells transiently overexpressing functional NMDAR tagged with green fluorescent proteins.Staining pattern with NMDAR antibody positive (A-C) and negative (D) serum. HEK293A cells were transiently transfected to overexpress EmGFP-tagged NR1, NR2A and GFP-tagged NR2B, incubated with diluted human serum and NMDAR antibodies were visualized by a Cy3-conjugated secondary antibody and counter-stained with DAPI to detect dead cells (left column: green fluorescence/EmGFP+GFP; middle column: red fluorescence/Cy3; right column: overlay of EmGFP/GFP, Cy3 and DAPI (A+D)). (B)+(C) Images show colocalization of NMDAR and serum NMDAR antibodies at high magnification (scale bars: 10 μm). (B) NMDAR antibodies bound to surface of cells. (C) Bound NMDAR antibodies internalized by the cells. CBA = cell-based assay. DAPI = 4’,6-diamidino-2-phenylindole. (Em)GFP = (emerald) green fluorescent protein. NMDAR = N-methyl-D-aspartate receptor.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122037.g001: Immunofluorescence CBA with HEK293A cells transiently overexpressing functional NMDAR tagged with green fluorescent proteins.Staining pattern with NMDAR antibody positive (A-C) and negative (D) serum. HEK293A cells were transiently transfected to overexpress EmGFP-tagged NR1, NR2A and GFP-tagged NR2B, incubated with diluted human serum and NMDAR antibodies were visualized by a Cy3-conjugated secondary antibody and counter-stained with DAPI to detect dead cells (left column: green fluorescence/EmGFP+GFP; middle column: red fluorescence/Cy3; right column: overlay of EmGFP/GFP, Cy3 and DAPI (A+D)). (B)+(C) Images show colocalization of NMDAR and serum NMDAR antibodies at high magnification (scale bars: 10 μm). (B) NMDAR antibodies bound to surface of cells. (C) Bound NMDAR antibodies internalized by the cells. CBA = cell-based assay. DAPI = 4’,6-diamidino-2-phenylindole. (Em)GFP = (emerald) green fluorescent protein. NMDAR = N-methyl-D-aspartate receptor.
Mentions: Fig 1 shows the typical antigen distribution of HEK293A cells overexpressing NMDAR tagged with green fluorescent proteins and the staining pattern with serum IgG of an NMDAR encephalitis patient at low magnification and higher magnification using a confocal microscope. It clearly shows the colocalization of membrane-associated NMDAR with serum antibodies of the patient but no colocalization with intracellular NMDAR probably residing within the endoplasmic reticulum (Fig 1B). Internalization of NMDAR in response to antibody binding observed in some but not all cells in the live CBA is shown in Fig 1C.

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