Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology

Impaired Cell Surface Expression of HLA-B Antigens on Mesenchymal Stem Cells and Muscle Cell Progenitors

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNγ stimulation for 48–72 h was required to induce full HLA–B protein expression. Quantitative real-time RT-PCR showed that IFNγ induced a 9–42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8+ T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types

Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

textabstractObjective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology

Multicentre comparison of a diagnostic assay : aquaporin-4 antibodies in neuromyelitis optica

Altres ajuts: This study was funded by the Eugene Devic European Network (EDEN) project (ERA-Net ERARE 2: http://www.erare.eu/financed-projects/eden). Euroimmun AG provided assay kits for groups from Denmark and Hungary at 50% discount, and at no cost to both Italian groups. The work in Oxford was supported by the National Health Service National Specialised Commissioning Group for Neuromyelitis Optica and the National Institute for Health Research Oxford Biomedical Research Centre. Competing interests: AS is funded by Fundació la Marató de TV3 (101610). FT is enrolled in the doctoral programme SPIN funded by the Austrian Science Fund (FWF; project W1206). ZI is funded by Lundbeckfonden and Scleroseforeningen of Denmark. AB has received a grant from Bayer Healthcare on NMO. LZ would like to thank RH for her excellent technical assistance with the assay. RH was supported by a Schrödinger fellowship funded by the Austrian Science Fund (FWF; project J3230). CC acknowledges funding from Fundació la Marató de TV3 (493/C/2012). FP acknowledges funding from the Bundesministerium für Bildung und Forschung (Competence Network Multiple Sclerosis KKNMS). BW acknowledges funding from the Dietmar-Hopp-Stiftung and from Merck Serono. RM and AR acknowledge Nathalie Dufay from NeuroBioTec-Banques (Hospices Civils de Lyon, France) and funding from Association pour la Recherche sur la Sclerose en Plaques (ARSEP). PW has received speaker honoraria from Biogen Idec and Euroimmun AG; holds a patent with Oxford University for LGI1/CASPR2 antibodies, licensed to Euroimmun AG; and for GABAAR antibodies. MR reports other from University Hospital of Innsbruck (TILAK), during the conduct of the study; personal fees from Euroimmun, outside the submitted work. AS has received compensation for consulting services and speaking from Bayer-Schering, Merck-Serono, Biogen-Idec, Sanofi-Aventis, Teva Pharmaceutical Industries Ltd and Novartis. HHN has received travel funding and speaker honoraria from Novartis Healthcare, Biogen Idec, Genzyme Denmark and Teva Denmark. ZI reports grants from Lundbeckfonden, Denmark; Scleroseforeningen, Denmark, during the conduct of the study; personal fees from compensation for consulting services and speaking from Bayer-Schering, Biogen-Idec, Merck-Serono, Novartis, Sanofi-Aventis, and Teva Pharmaceutical Industries Ltd, outside the submitted work. TB reports non-financial support from Euroimmun AG, during the conduct of the study. KR is shareholder and an employee of Euroimmun AG. AB reports grants from Bayer Heathcare, personal fees from Biogen Idec, personal fees from Merck Serono, personal fees from Teva, personal fees from Novartis, personal fees from Genzyme, other from Biogen Idec, other from Novartis, other from Genzyme, other from Roche, other from Alexion Pharmaceuticals, outside the submitted work. LG received congress and travel compensations from Euroimmun. AB received honoraria for serving in the scientific advisory boards of Almirall, Bayer, Biogen Idec, Genzyme, and received speaker honoraria from Biogen Idec, Genzyme, Novartis, TEVA with approval by the Director of AOU San Luigi University Hospital; his institution has received grant support from Bayer, BiogenIdec, Merck, Novartis, TEVA from the Italian Multiple Sclerosis Society, Associazione Ricerca Biomedica ONLUS and San Luigi ONLUS. LZ reports personal fees from Teva Pharmaceutical, personal fees from Novartis, outside the submitted work. MC has received compensation for consulting services and speaking honoraria from Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, Sanofi-Aventis, Genzyme, and Novartis. XM has received speaking honoraria and travel expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past years with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Phramaceuticals, Almirall and Roche. MT has received compensation for consulting services and speaking from Bayer-Schering, Merk-Serono, Biogen-Idec, Teva, Sanofi-Aventis, and Novartis. AS reports receiving grants from the Scientific and Technological Research Council of Turkey-Health Sciences Research (grants numbers 109S070 and 112S052); and also unrestricted grants from Bayer-Schering AG and Merck-Serono to their Neurology Department Clinical Neuroimmunology Unit. AS also reports receiving honoraria or consultation fees Biogen Idec, Novartis and Teva. He had received travel and registration coverage for attending several national or international congresses or symposia, from Merck Serono, Biogen Idec, Novartis, Teva, Bayer-Schering and Allergan. AA received grants to her department from The Scientific and Technological Research Institute of Turkey-Health Sciences (grants numbers 109S070 and 112S052), and she also received honoraria, research and travel grants from Teva, Merck-Serono, Gen Ilac, Bayer-Schering, Novartis. FP has received travel funding, speaker honoraria and personal compensation for activities on advisory boards and steering committees from Bayer, Biogen Idec, Alexion, Chugai, MedImmune, MerckSerono, Novartis, Teva, and Sanofi Genzyme. He has received grants from the Guthy Jackson Charitable Foundation and the National Multiple Sclerosis Society of the USA. OA reports grants from German Ministry for Education and Research (BMBF), during the conduct of the study; German Research Foundation (DFG); Hertie Foundation; Biogen; Novartis; Bayer; outside the submitted work; and Honoraria for consultancy and speaking by Biogen, Novartis, Bayer Schering, Teva, Chugai, Genzyme, MedImmune, and Merck Serono. BW reports personal fees from Bayer Healthcare, grants and personal fees from Biogen, Merck Serono; Genzyme, a Sanofi Company; Novartis Pharmaceuticals; TEVA Pharma; grants from Biotest, German Ministry of Education and Research; Dietmar Hopp Foundation; outside the submitted work. H-PH reports personal fees from MedImmune, outside the submitted work. MP is an employee of and holds stock in Euroimmun AG. PT reports non-financial support from Roche Pharmaceuticals, outside the submitted work. SS is an employee of Euroimmun AG. AV is on the Advisory Board for Neurology; was an associate editor for Brain; holds patents with Oxford University for MuSK, LGI1/CASPR2, and GABAAR antibodies, and receives a proportion of royalties. RM reports personal fees from MedImmune, personal fees and non-financial support from Biogen, non-financial support from Merck-Serono, non-financial support from Teva, non-financial support from Novartis, non-financial support from Sanofi, outside the submitted work.Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results of tests on 92 controls identified 12assays as highly specific (0-1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5-100%) of all 21 assays. The specificities (85.8-100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology