Using an aquaporin-4 (AQP4) M1-isoform–specific enzyme-linked immunosorbent assay (ELISA) and a fixed transfected cell-based assay (CBA), we tested AQP4-IgG in a northern California population representative cohort of 3293 potential cases with multiple sclerosis (MS). Seropositive cases were tested additionally by fluorescence-activated cell sorting, a live transfected cell-based assay.
Sera samples were available in 1040 cases; 7 yielded positive results, 4 by ELISA alone and 3 by both ELISA and CBA. Clinical data (episodes of optic neuritis and longitudinally extensive transverse myelitis [reported on at least 1 magnetic resonance imaging spine]) supported the alternative diagnosis of neuromyelitis optica for 2 patients as seropositive by both ELISA and CBA. These 2 patients alone tested positive by a fluorescence-activated cell-sorting assay. The diagnosis of MS was considered correct in the other 5 patients. Thus, 5 ELISA results and 1 fixed CBA result were false positive.
Conclusions and Relevance
Sensitive serological evaluation for AQP4-IgG in this large population-representative cohort of predominantly white non-Hispanic patients with MS reveals that neuromyelitis optica spectrum disorder is rarely misdiagnosed as MS in contemporary US neurological practice (0.2%). The frequency of a false-positive result for ELISA and CBA in this MS cohort were 0.5% and 0.1%, respectively. This finding reflects the superior specificity of CBA and justifies caution in interpreting AQP4-IgG results obtained by ELISA.
Antibodies targeting the astrocytic aquaporin-4 (AQP4-IgG) water channel are clinically validated biomarkers of a spectrum of relapsing autoimmune inflammatory central nervous system disorders termed neuromyelitis optica spectrum disorders (NMOSDs).1,2 The recurrent episodes of optic neuritis and transverse myelitis that are characteristic of NMO cause cumulative disability with high risk of blindness and paraplegia.2
Since its introduction to clinical practice in 2005, AQP4-IgG testing has allowed early distinction of most NMO cases and its partial or inaugural forms (NMOSDs) from multiple sclerosis (MS) and, thus, early initiation of NMO-appropriate treatment.2
Without this test, NMOSDs tend to be misdiagnosed as MS. Early detection of the antibody marker is important because the prognosis and optimal treatments for the 2 diseases differ.
In 2012, a multicenter blinded study of AQP4-IgG assays determined that second-generation recombinant antigen-based assays were most sensitive and highly specific in aiding the diagnosis of NMOSDs.3 This and other studies investigating specificity have been limited by small numbers of disease control patients. In this study, we tested sera samples from a large predominantly white non-Hispanic population representative cohort of patients with MS. We used the 2 AQP4-IgG assays used most commonly for clinical service testing in the United States to address what the rate of AQP4-IgG false positivity is in MS and how frequently NMO is misdiagnosed as MS.
The study protocol was approved by the institutional review boards of Kaiser Permanente Division of Research; the University of California, Berkeley; and the Mayo Clinic. All participants provided written consent. The study population consisted of self-reported white non-Hispanic and African American patients with MS identified through electronic medical records among 3.2 million members of the Kaiser Permanente Medical Care Plan, Northern California Region (KPNC), comprising 30% of the population.4 Eligibility criteria for MS cases included 1 or more outpatient diagnoses of MS by a neurologist, patients aged 18 through 69 years, and membership in KPNC at initial contact. At the time of the study, 98 patients had a diagnosis of NMO or NMOSD in the entire KPNC membership; none were included in the MS cohort.
At commencement of the current study, Kaiser neurologists had reviewed a total of 3293 potential MS cases and approved contact with 2823 (86%) of them. Each neurologist was asked to disqualify potential cases who did not have MS, were too ill or impaired to participate, or had left KPNC. Patients were then contacted by mail and study procedures were explained in a follow-up telephone call by project staff. The telephone call included several questions concerning the self-identified race/ethnicity of potential participants and their diagnosis of MS to screen for eligibility. Those who were eligible and agreed to participate made an appointment to complete a computer-assisted telephone interview. After the interview, the participants were mailed a consent form and blood-sample collection kit. The participant was asked to return the signed consent form by mail and to take the blood collection kit to a KPNC phlebotomy laboratory where 3 tubes of blood (one 10-mL serum tube and two 10-mL ethylenediaminetetraacetic acid tubes) were drawn by a KPNC phlebotomist. Blood samples were shipped for arrival within 24 hours of the blood draw for processing and then stored in a −80° C freezer. Sera was available for testing from a total of 1040 MS cases (91% white and 9% African American). Published diagnostic criteria were used.5,6 Disease subtypes were relapsing-remitting (68.4%), secondary progressive (15%), and primary progressive or relapsing progressive (9.8%) MS, as well as unspecified (6.8%).
All serum samples were tested at the Mayo Clinic Neuroimmunology Laboratory for AQP4-IgG under blinded conditions by 2 clinically validated assays; human AQP4-M1 isoform was used as an antigen. Assays were an enzyme-linked immunosorbent assay (ELISA; RSR/Kronus Ltd; ≥5 IU/mL was considered positive) and visual fluorescence observation in a fixed transfected cell-based assay (CBA; EUROIMMUN).7,8 All specimens yielding a positive result by either assay were tested additionally by live AQP4-M1–transfected CBA (in-house validated quantitative flow cytometry fluorescence-activated cell sorting). Methods were previously described.9 Full electronic medical record review was conducted by 1 study neurologist (N.B.) using a standardized approach developed by study investigators (S.J.P., L.F.B., A.L.B., and C.A.S.) for all MS cases who initially tested seropositive. Based on Wingerchuk criteria,10 our record review included a systematic search for evidence of optic neuritis and myelitis and magnetic resonance imaging (MRI) evidence for contiguous spinal cord lesion (≥3 segments in length). All available MRI reports were reviewed for each MS case independently by 2 study neurologists (N.B. and S.J.P.). It was determined whether the onset brain MRI was nondiagnostic for MS. Any additional evidence for NMO-IgG seropositivity was noted. Following review, each case was categorized based on clinical and MRI data by 2 study neurologists (N.B. and S.J.P.) as definite NMO, suggestive NMO, definite MS, or uncertain NMO vs MS.
The 1040 MS cases whose sera samples were tested for AQP4-IgG are described in Table 1. Seven sera samples yielded positive results, 4 by ELISA alone and 3 by ELISA and CBA. After review of clinical and neuroimaging data, the diagnosis of MS was considered correct in 5 of these patients, indicating 5 ELISA false positives and 1 CBA false positive (Table 2). The results of the 2 patients who met clinical criteria for a revised diagnosis of NMO were seropositive CBA and ELISA. Both patients had histories of optic neuritis and longitudinally extensive transverse myelitis (≥1 spine MRI). They were the only 2 patients whose results were seropositive by fluorescence-activated cell sorting.
Sensitive serological evaluation for AQP4-IgG in this large cohort of patients with MS reveals that NMOSD is rarely misdiagnosed as MS in contemporary US neurological practice (0.2%). False seronegativity attributable to immunosuppressant therapy is unlikely because less than 4% of patients had received immunosuppressant medications in the 30 days preceding blood draw.
A major strength of this investigation was the size of the MS case cohort that allows for a well-powered study. Members of KPNC are representative of the population of northern California, with the exception of extremes of the socioeconomic spectrum.4 Second, cases were stringently ascertained using an internally validated algorithm based on electronic medical record data, rigorous participant screening, and the application of published diagnostic criteria.5,6
The generalizability of our findings may be limited to white individuals and not necessarily to the greater MS population in the United States. This is an important consideration because NMO disproportionately affects African and Asian races/ethnicities.2 Despite a preponderance of white individuals (91%) in this MS cohort, the ratio of white to African American patients with false-positive AQP4-IgG results was only 3 to 2. This suggests that African American patients may be more prone to false-positive AQP4-IgG results (more so by ELISA). The positive results yielded by AQP4-IgG ELISA in 4 patients with MS whose results were seronegative by CBA and fluorescence-activated cell-sorting assays reflect the superior specificity of cell-binding assays for detecting AQP4-IgG and justifies caution in interpreting low positive AQP4-IgG results obtained by ELISA.
Sensitive serological evaluation for AQP4-IgG in this large population representative cohort of predominantly white non-Hispanic patients with MS reveals that the NMOSDs are rarely misdiagnosed as MS in contemporary US neurological practice (0.2%). The frequency of a false-positive result for ELISA and CBA in this MS cohort were 0.5% and 0.1%, respectively. This finding reflects the superior specificity of CBA and justifies caution in interpreting low positive AQP4-IgG results obtained by ELISA.
Accepted for Publication: May 8, 2014.
Corresponding Author: Sean J. Pittock, MD, Department of Neurology, Mayo Clinic, 200 First St SW, Rochester, MN 55905 (firstname.lastname@example.org).
Published Online: September 1, 2014. doi:10.1001/jamaneurol.2014.1581.
Author Contributions: Drs Pittock and Barcellos had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Pittock, Lennon, Barcellos.
Acquisition, analysis, or interpretation of data: Lennon, Bakshi, Shen, McKeon, Quach, Briggs, Bernstein, Schaefer, Barcellos.
Drafting of the manuscript: Pittock, Bernstein, Barcellos.
Critical revision of the manuscript for important intellectual content: Lennon, Bakshi, Shen, McKeon, Quach, Briggs, Schaefer, Barcellos.
Obtained funding: Pittock, Schaefer, Barcellos.
Administrative, technical, or material support: Lennon, Bakshi, Briggs, Bernstein.
Study supervision: Pittock, Schaefer, Barcellos.
Conflict of Interest Disclosures: Dr Pittock receives support from Alexion Pharmaceutical Inc and is a named inventor on patents that relate to functional aquaporin-4/neuromyelitis optica antibody assays and neuromyelitis optica antibodies as a cancer marker. Dr Pittock has provided consultation to Alexion Pharmaceutical, MedImmune LLC, and Chugai Pharma but has received no personal fees or compensation for these consulting activities. Dr Lennon is a named inventor on 2 patent applications filed by the Mayo Foundation for Medical Education and Research that relate to the neuromyelitis optica aquaporin-4 antibody, its application to cancer, and functional assays for detecting aquaporin-4 antibodies. Dr Lennon and the Mayo Clinic have received royalties that exceed the federal threshold for significant financial interest from licensing of the above listed technology and have rights to receive future royalties. Serological testing for neural autoantibodies is offered on a service basis by Mayo Collaborative Service Inc, an agency of the Mayo Foundation. Neither Dr Lennon nor her laboratory benefit financially from this testing. Dr McKeon receives support from MedImmun Inc. No other disclosures were reported.
Funding/Support: This work was supported by grants RO1 NS065829, R01 NS049510, and R01 ES017080 from the National Institutes of Health/National Institute of Neurological Disorders and Stroke, grant R01 AI076544 from the National Institutes of Health/National Institute of Allergy and Infectious Diseases, and the Guthy-Jackson Charitable Foundation. Dr Pittock receives research support from the Guthy-Jackson Charitable Foundation and grant NS065829 from the National Institutes of Health. Dr Briggs is a postdoctoral fellow at the National Multiple Sclerosis Society. Dr McKeon receives research support from the Guthy-Jackson Charitable Foundation.
Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: We thank John Schmeling, BS, Mayo Clinic, for performing aquaporin-4-IgG serological assays and for assistance with data analysis, Katie Cornelius, BS, Mayo Clinic, for technical assistance, and Jessica Sagen, BA, Mayo Clinic. None received financial compensation.
Pittock SJ, Lennon VA, Bakshi N, Shen L, McKeon A, Quach H, Briggs FBS, Bernstein AL, Schaefer CA, Barcellos LF. Seroprevalence of Aquaporin-4–IgG in a Northern California Population Representative Cohort of Multiple Sclerosis. JAMA Neurol. 2014;71(11):1433-1436. doi:10.1001/jamaneurol.2014.1581