ORIGINAL CONTRIBUTION Early Differential Diagnosis of Multiple Sclerosis Using a New Oligoclonal Band Test Luisa M. Villar, PhD; Jaime Masjuan, MD; María C. Sádaba, BSc; Pedro González-Porqué, PhD; José Plaza, MD; Alfredo Bootello, MD; José C. Álvarez-Cermeño, MD Background: Intrathecal IgG synthesis (ITGS), in con- vous system infections, and 1 with motor neuron dis- junction with magnetic resonance imaging, can help in ease. Two patterns reflected ITGS. One pattern, showing the early diagnosis of multiple sclerosis (MS). Recently, OCGBs restricted to cerebrospinal fluid, was predomi- we developed a new oligoclonal IgG band (OCGB) test nantly found in MS. The other pattern, with OCGBs in for ITGS detection that is more sensitive and easier to serum and additional bands in cerebrospinal fluid, was interpret than previously described methods. mostly found in central nervous system infections. No patients with other inflammatory neurologic diseases Objective: To assess the accuracy of a new OCGB de- showed ITGS. These patients frequently displayed a mir- tection test in the diagnosis of MS. ror pattern, with identical bands in serum and cerebro- spinal fluid. Considering all patients, the sensitivity for Design: Prospective observational study. the diagnosis of MS was 96.2%, and the specificity was 92.5%. Excluding infections, which usually do not pre- Setting: A hospital neurology department. sent a differential diagnosis problem with MS, the sen- sitivity was still 96.2%, and the specificity increased to Patients: A total of 385 patients with various neuro- logic disorders. 99.5%. Main Outcome Measures: The sensitivity and speci- Conclusion: The accuracy of this OCGB method rein- ficity of the OCGB detection test for MS diagnosis. forces the value of cerebrospinal fluid studies in the early differential diagnosis of MS. Results: Intrathecal IgG synthesis was found in 127 pa- tients with MS (96.2%), 18 (35.3%) with central ner- Arch Neurol. 2005;62:574-577 ULTIPLE SCLEROSIS (MS) band (OCGB) detection is an important tool is the most frequent de- for MS diagnosis.5,6 We developed a novel myelinating disease of OCGB assay that consists of isoelectric fo- the central nervous sys- cusing (IEF) and IgG immunodetection by tem (CNS). The ratio- an alkaline phosphatase–labeled anti-IgG Mnale for MS treatment is to reduce the dis- antibody. It yields high sensitivity and a ease activity and to delay the progression sharp and strong pattern of bands that is of disability. Thus, an early and accurate di- easy to interpret.7 Alkaline phosphatase has agnosis of MS is important because it has proved to be useful in the detection of oli- been shown that early treatment has a ben- goclonal IgM bands8 that have an impor- Author Affiliations: eficial effect on disease evolution.1,2 tant role as prognostic markers in MS.9,10 Departments of Immunology Because no single test provides a defi- We herein analyze the presence of and Neurology, Hospital Ramón nite diagnosis, different criteria have been OCGBs using the alkaline phosphatase y Cajal (Drs Villar, Masjuan, used for MS diagnosis.3,4 Different para- method in 385 patients with different neu- González-Porqué, Plaza, clinical tests, such as detection of intrathe- rologic diseases to assess the validity of this Bootello, and Álvarez-Cermeño and Ms Sádaba), and cal IgG synthesis (ITGS) (present in most test for MS diagnosis. Special attention was Department of Medicine, patients with MS), evoked potentials, and paid to other inflammatory neurologic dis- University of Alcalá, Alcalá de imaging techniques, are used to support the eases of the CNS that may represent im- Henares (Dr Álvarez-Cermeño), diagnosis when necessary. Intrathecal IgG portant problems in the differential diag- Madrid, Spain. synthesis determined by oligoclonal IgG nosis of the disease. (REPRINTED) ARCH NEUROL / VOL 62, APR 2005 WWW.ARCHNEUROL.COM 574 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 METHODS IIIIIIIV PATIENTS A total of 385 patients studied in the Neurology Department of Ramón y Cajal Hospital were included in this study. Pa- tients were divided into 6 groups. Group 1 comprised 132 pa- tients with clinically definite MS according to the criteria of Poser et al.3 We further divided this group into 27 patients who un- derwent lumbar puncture during the first attack of the disease and converted to clinically definite MS during follow-up (group 1A) and 105 patients with a clinically definite MS diagnosis at lumbar puncture (group 1B). Group 2 comprised 37 patients with inflammatory diseases of the CNS different from MS, in- cluding 9 with myelitis, 8 with CNS vasculitis, 4 with neuro- SC SC SC SC lupus, 4 with paraneoplastic syndrome, 3 with Behçet disease, 3 with Rasmussen disease, 2 with Hashimoto encephalitis, Figure. Four different patterns were found in IgG immunoblots. Pattern I 2 with gluten ataxia, 1 with neurosarcoidosis, and 1 with Sjögren shows a polyclonal response in serum (S) and cerebrospinal fluid (CSF) (C) disease. Group 3 was composed of 26 patients with inflam- in a patient with nonspecific headache. Pattern II shows the same oligoclonal matory diseases of the peripheral nervous system: 15 with Guil- bands in serum and CSF (mirror pattern) in a patient with Guillain-Barré lain-Barré syndrome and 11 with other inflammatory polyneu- syndrome. Pattern III shows oligoclonal IgG bands detected in serum, with ropathies (9 with chronic inflammatory demyelinating more than 2 additional bands present in CSF, in a patient with viral polyneuropathy and 2 with monoclonal IgM-associated poly- encephalitis. Pattern IV shows more than 2 oligoclonal bands in CSF with a polyclonal distribution in the paired serum sample in a patient with multiple neuropathy). sclerosis. Group 4 comprised 100 patients with noninflammatory neu- rologic diseases: 31 with neurodegenerative disorders (16 with Alzheimer disease, 4 with frontotemporal dementia, 1 with de- brane was blocked in 2% dry milk in saline serum for 30 min- mentia with Lewy bodies, 9 with motor neuron disease, and 1 utes and then incubated with alkaline phosphatase– with Parkinson disease), 24 with stroke, 12 with neoplastic men- conjugated rabbit anti-human IgG (The Jackson Laboratory, ingitis, 11 with pseudotumor cerebri, 7 with normal pressure Bar Harbor, Me) diluted 1:5000 in 0.2% dry milk in saline for hydrocephalus, 6 with diabetic polyneuropathy, 4 with olivo- 1 hour at room temperature in a platform shaker. The mem- pontocerebellar atrophy, 2 with arteriovenous malformation, brane was then washed 25 times with tap water and once with 2 with idiopathic epilepsy, and 1 with Wernicke disease. Group saline serum for 10 minutes. Finally, the membrane was stained 5 consisted of 51 patients with CNS infections: 30 with acute using nitro blue tetrazolium and bromo-chloroindolyl phos- viral meningoencephalitis, 4 with acute bacterial meningitis, phate. 4 with tuberculous meningitis, 1 with neurosyphilis, 1 with spi- nal cord schistosomiasis, 1 with progressive multifocal leuko- TEST VALIDITY CALCULATION encephalopathy, and 10 with human immunodeficiency virus infection, 3 of them with superimposed tuberculous meningi- The following ratios were used: tis and 3 others with progressive multifocal leukoencephalopa- Sensitivity=[TP/(TPϩFN)]ϫ100 thy. Group 6 comprised 39 patients with nonspecific head- ϩ ϫ aches without any neurologic abnormalities. Specificity=[TN/(TN FP)] 100 Positive predictive value=[TP/(TPϩFP)]ϫ100 SAMPLES AND LABORATORY TESTS Negative predictive value=[TN/(TNϩFN)]ϫ100 Paired serum and cerebrospinal fluid (CSF) samples were col- Accuracy=[(TPϩTN)/(TPϩTNϩFPϩFN)]ϫ100 lected for diagnostic purposes after informed consent was ob- tained. The samples were stored at –40°C until oligoclonal band STATISTICS testing was performed. Serum and CSF IgG concentrations were analyzed in a neph- Results were analyzed using a statistical software package (Prism elometer (BNII; Behring, Marburg, Germany). Oligoclonal IgG 3.0; GraphPad, San Diego, Calif). We used the Fisher exact test detection was performed by IEF and immunodetection as pre- to compare percentages. viously described.7 Before IEF, serum samples were diluted in deionized water to reach the same IgG concentration as that of parallel CSF samples. Isoelectric focusing was performed on RESULTS agarose gels prepared with agarose IEF, Pharmalyte pH 3-10, and Pharmalyte pH 8-10.5 (all from Amersham Biosciences, IgG PATTERNS Uppsala, Sweden). Focusing was performed on a Multiphor II apparatus (Amersham Biosciences) cooled to 10°C. The elec- All results were interpreted by immunologists (L.M.V. trode strips were soaked with 1M sodium hydroxide (catho- and P.G.-P.) masked to diagnosis. Four different pat- lyte) and 0.05M sulfuric acid (anolyte). Three-microliter paired terns were found in IgG immunoblots (Figure). In pat- samples were applied on the anodic side of the gel. The IEF was run at 10 W for 30 minutes and then at 20 W until focus- tern I, a polyclonal response (no individual bands pres- ing was completed after 1500 volt hours. Voltage was limited ent) was detected in serum and CSF. In pattern II, the at 1250 V. The run time was approximately 90 minutes. same OCGBs were detected in serum and CSF (mirror After IEF, the proteins were transferred to a nitrocellulose pattern). In pattern III, OCGBs were detected in serum membrane (Millipore, Billerica, Mass). The nitrocellulose mem- and CSF, with more than 2 additional bands present in