Clinical association of intrathecal and mirrored oligoclonal bands in paediatric neurology

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1 DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY ORIGINAL ARTICLE Clinical association of intrathecal and mirrored oligoclonal bands in paediatric neurology ADRIANE J SINCLAIR 1 LOUISE WIENHOLT 2 ESTHER TANTSIS 3 FABIENNE BRILOT 3 RUSSELL C DALE 3 1 Department of Child Neurology, Sydney Children's Hospital, Sydney; 2 Immunology laboratory, Royal Prince Alfred Hospital, Sydney; 3 Neuroimmunology Group, Institute for Neuroscience and Muscle Research, the Children's Hospital at Westmead, University of Sydney, Australia. Correspondence to Professor Russell C Dale at Clinical School, the Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia. russell.dale@health.nsw.gov.au This article is commented on by Lim on pages of this issue. PUBLICATION DATA Accepted for publication 21st July Published online 24th October ABBREVIATIONS ADEM Acute disseminated encephalomyelitis CSF Cerebrospinal fluid HSV Herpes simplex virus NMDAR N-methyl-D-aspartate receptor OCB Oligoclonal band VGKC Voltage-gated potassium channel AIM Biomarkers such as autoantibodies, neopterin, and oligoclonal bands (OCBs) are increasingly used for the diagnosis of treatable inflammatory central nervous system (CNS) disorders. We investigated the correlation between the results of OCB testing and clinical diagnoses in a large contemporary cohort of children with a broad range of neurological conditions. METHOD Cerebrospinal fluid (CSF) and serum from 200 children (94 females, 106 males; age range 2mo 15y 10mo, mean age 6y 9mo, SD ±4.9) who underwent CSF investigation for their neurological condition were tested for OCBs using isoelectric focusing. RESULTS The patients were divided into those with inflammatory (n=58) and non-inflammatory (n=142) CNS disorders. Intrathecal OCBs (OCBs restricted to the CSF) were found in 11 out of 58 (19%) of those with inflammatory CNS disorders compared with none of the 142 patients with non-inflammatory CNS disorders (p<0.001). Diseases associated with intrathecal OCB were multiple sclerosis, Rasmussen encephalitis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, voltage-gated potassium channel (VGKC) encephalopathy, herpes (HSV) encephalitis, other encephalitides, acute cerebellar ataxia, and aseptic meningitis. Mirrored OCBs (identical OCBs in the serum and CSF) were less specific but were still found in 14 out of 58 (24%) children with inflammatory CNS disorders compared with only 6 out of 142 (4%) children with non-inflammatory CNS disorders (p<0.001). Diseases associated with mirrored OCBs included acute disseminated encephalomyelitis (ADEM), VGKC encephalopathy, West syndrome, NMDAR encephalitis, other encephalitides, polio-like illness, Rasmussen encephalitis, cerebral vasculitis, metachromatic leukodystrophy, and bacterial meningitis. Intrathecal OCBs and mirrored OCBs had a positive predictive value for inflammatory CNS disease of 1 (95% confidence interval [CI] ) and 0.7 (95% CI ) respectively. CONCLUSION Intrathecal OCBs were restricted to patients with inflammatory CNS disorders. They are a useful, but non-specific, biomarker of CNS inflammation of multiple causes. Mirrored OCBs are less specific, but still support a possible inflammatory CNS disorder. The presence of either intrathecal or mirrored OCBs should raise suspicion of an inflammatory CNS disorder. There is increasing interest in biomarkers that diagnose potentially treatable inflammatory central nervous system (CNS) disorders. 1 Some serum and cerebrospinal fluid (CSF) autoantibodies are specific biomarkers associated with autoimmune syndromes such as N-methyl-D-aspartate receptor (NMDAR) encephalitis and voltage-gated potassium channel (VGKC) encephalopathy. Other biomarkers, including CSF neopterin and oligoclonal bands (OCBs), are less specific, but they are still useful markers of immune activation or inflammation in the CNS. 2 Before the discovery of the NMDAR antibody biomarker, OCBs were used to support the hypothesis that encephalitis lethargica and immunemediated chorea encephalopathy syndrome were autoimmune diseases; these syndromes have subsequently been shown to be NMDAR encephalitis. 3 5 OCBs have also been useful in the identification of unusual variants of known autoimmune encephalopathy. 6 OCBs are clones of immunoglobulin (typically IgG) that can be detected in the CSF and or serum. The qualitative method of isoelectric focusing on agarose gels followed by immunoblotting is the accepted standard method for OCB detection. The IgG index (the ratio of CSF serum IgG to CSF serum albumin), a quantitative method of analysis, is a less sensitive test, and when elevated is suggestive of a CNS B-cell response. There are limited reports of use of the IgG index in paediatric studies, but in adult studies the IgG index has been found to be only rarely elevated in patients with multiple sclerosis who have negative OCBs Alper et al., 7 in a paediatric cohort, found that the IgG index was less frequently elevated in those with acute disseminated encephalo- ª The Authors. Developmental Medicine & Child Neurology ª 2012 Mac Keith Press DOI: /j x 71

2 myelitis (ADEM) than in those with multiple sclerosis. 7 Interpretation of OCBs is dependent on the comparison of CSF and serum samples, with a number of patterns described (Fig. 1). 8 WhenOCBsarepresentintheCSFandabsentfromthe serum, this is termed an intrathecal pattern and is indicative of local antibody production and hence a humoral immune response within the CNS. When identical clones of IgG are present in the CSF and serum, this is termed a mirrored pattern and suggests that IgG has entered the CNS from the systemic circulation, such as occurs in blood brain barrier damage. A combination of mirrored and intrathecal patterns can occur, in which identical clones of IgG are present in the CSF and serum; however, the CSF also contains additional OCBs. This is consistent with both CNS contamination and local (intrathecal) IgG synthesis. Intrathecal OCBs are accepted to be of greater clinical significance than mirrored OCBs in the diagnosis of inflammatory CNS disorders. 10 Data pertaining to OCBs in paediatric patients are largely restricted to specific diseases, for example multiple sclerosis, 12 ADEM, 13 and opsoclonus myoclonus syndrome. 14 In 1986, Kostulas et al. 9 reported OCB findings in a paediatric cohort with varying neurological diagnoses. They found OCBs most commonly in patients with inflammatory disorders; however, they also reported finding OCBs in children with presumed non-inflammatory disorders such as epilepsy and migraine. In recent years, the ability to diagnose disorders of inflammatory aetiology has greatly improved. We studied the diagnostic What this paper adds Intrathecal OCBs have a strong positive predictive value of inflammatory CNS disease in children. Although less specific, mirrored OCBs are also found more commonly in children with inflammatory CNS disease. associations and sensitivity specificity of intrathecal and mirrored OCB in a contemporary cohort of children with neurological diseases. METHOD We identified 205 paediatric neurology patients who had OCB testing between January 2006 and February During that time, OCB testing was routinely requested for all paediatric neurology patients at the Children s Hospital at Westmead undergoing CSF analysis. This study was part of ethically approved studies of inflammatory and autoimmune disorders of the CNS at the hospital. We excluded five patients in whom paired CSF and serum samples or clinical information was unavailable. Patients were aged 2 months to 15 years 10 months, with a mean age of 6 years 9 months SD ±4.9. There were 94 females and 106 males. All CSF and sera specimens were analysed by isoelectric focusing at a single laboratory. In the case of those patients with acquired disorders, analysis was performed on samples taken during the acute period of illness, and in the first episode if the disorder was relapsing. The study also included patients with chronic illnesses who were undergoing CSF analysis in an elective manner. Patient case notes were reviewed and patients were categorized by pathophysiology (inflammatory vs non-inflam- 1. CSF No OCB 2. Serum 3. CSF 4. Serum Intrathecal 5. CSF Mirrored 6. Serum 7. CSF Mirrored and intrathecal 8. Serum Figure 1: Isoelectric focusing on agarose gels followed by immunoblotting. Lanes 1 and 2: no oligoclonal bands (OCBs) detected in cerebrospinal fluid (CSF) or serum. Lanes 3 and 4: OCB detected in CSF but not in serum indicative of intrathecal immunoglobulin G (IgG) production. Lanes 5 and 6: identical OCB detected in both CSF and serum suggestive of an abnormal CSF-blood barrier (mirrored pattern). Lanes 7 and 8: combination of mirrored and intrathecal IgG production, a pattern not observed in this study but observed in subacute sclerosing panencephalitis. 72 Developmental Medicine & Child Neurology 2013, 55: 71 75

3 matory CNS disease), disease group, and diagnosis (Table I). Case note review and patient categorization were performed by a neurology registrar (AJS), with verification provided by a paediatric neurologist (RCD). Assigned diagnoses were based on those documented in the case notes by the treating paediatric neurologist. The inflammatory CNS group (n=58; 29%) included patients with diagnoses with an established infectious, autoimmune, immune-mediated, or inflammatory cause. The term inflammatory CNS will be used to describe infectious, autoimmune, inflammatory, or immune-mediated CNS disorders. The non-inflammatory CNS group (n=142; 71%) included patients with diagnoses for which there was no established inflammatory pathophysiology and patients in whom we were unable to assign a diagnosis. We subdivided the inflammatory CNS group into demyelinating diseases (n=26), infection-mediated diseases (n=11), autoantibody-associated diseases (n=7), and other immunemediated diseases (n=14). Diagnoses within these subgroups are presented in Table I. Within the infection-mediated group, six patients were categorized as having other encephalitides. These patients had evidence of encephalitis (acute or chronic) fulfilling criteria for encephalitis but without definite evidence of a specific infectious agent. 15 The non-inflammatory CNS group included patients with very diverse diagnoses (Table I) along with patients in whom the diagnosis was unknown (n=15). The two-tailed Fisher s exact test was used to calculate p-values, and a p-value <0.05 was considered significant; 95% confidence intervals (CI) are presented for positive predictive value and negative predictive value. RESULTS Intrathecal OCBs were detected in patients with inflammatory CNS disorders (11 58; 19%), but not in those with noninflammatory CNS disorders (0 142; 0%; p<0.001; Table II). Diagnoses associated with intrathecal OCBs were multiple sclerosis (n=3), Rasmussen encephalitides (n=2), NMDAR encephalitis (n=1), VGKC encephalopathy (n=1), acute cerebellar ataxia (n=1), aseptic meningitis (n=1), herpes simplex virus (HSV) encephalitis (n=1), and other encephalitis (n=1; Table II). Mirrored OCBs were found more frequently in those with inflammatory CNS disorders (14 58, 24%) than in those with non-inflammatory CNS disorders (6 142; 4%; p<0.001; Table II). Mirrored OCBs were detected in patients with ADEM (n=4), VGKC encephalopathy (n=1), NMDAR encephalitis (n=2), basal ganglia encephalitis (n=1), other encephalitides (n=1), polio-like illness (n=2), Rasmussen encephalitis (n=1), varicella zoster-associated cerebral vasculitis (n=1), bacterial meningitis (n=1), West syndrome (n=2), and metachromatic leukodystrophy (n=1), and in several patients with unknown diagnoses (n=3). No patient showed a combination of mirrored and intrathecal patterns. Intrathecal OCBs had a sensitivity of 0.19 (95% CI ), a specificity of 1 (95% CI ), a positive predictive value of 1 (95% CI ), and a negative predictive value of 0.75 (95% CI ) for the presence of inflammatory CNS disease (Table II). Mirrored OCBs had a sensitivity of 0.24 (95% CI ), a specificity of 0.96 (95% CI ), a positive predictive value of 0.7 (95% CI ), and a negative predictive value of 0.76 (95% CI ; Table II). Table I: Disease groups and diagnoses Disease group (n) Non-inflammatory CNS (n=142) Epilepsy (n=58) Static encephalopathy (n=15) Progressive genetic or metabolic (n=8) Other (n=61) Inflammatory CNS (n=58) Demyelinating (n=26) Infection-mediated (n=11) Autoantibody-associated (n=7) Other immune-mediated (n=14) Diagnoses (n) Electroclinical syndromes: West syndrome (n=6), Dravet syndrome (n=1), febrile seizures plus (n=1), epilepsy with myoclonic astatic atonic seizures (n=2), juvenile absence epilepsy (n=1) Structural or metabolic: perinatal brain injury (n=1), traumatic brain injury (n=1), hypoglycaemia (n=1), malformation of cortical development (n=4), previous CNS infection (n=3) Genetic: PCDH19 mutation (n=1), SCN1A (n=2) Other or unknown (n=34) Cerebral palsy (n=3), developmental delay (n=4), chromosomal disorders (n=6), unknown (n=2) Metachromic leukodystrophy (n=1), Menkes disease (n=1), mitochondrial probable mitochondrial (n=2), hereditary spastic paraplegia (n=2), unknown (n=2) Functional disorders (n=10), movement disorders (n=10), Guillain Barré syndrome (n=5), headache disorders (n=4), stroke (n=4), miscellaneous a (n=13), unknown (n=15) MS (n=6), ADEM (n=11), clinically isolated syndrome (n=9): optic neuritis (n=3), transverse myelitis (n=3), other (n=3) Bacterial meningitis (n=1), aseptic meningitis (n=1), other encephalitides (n=6), acute necrotizing encephalopathy (n=2), HSV encephalitis (n=1) NMDAR encephalitis (n=3), VGKC encephalopathy (n=3), basal ganglia encephalitis (n=1) Rasmussen encephalitis (n=3), acute cerebellar ataxia (n=4), Polio-like illness (n=2), cerebral vasculitis (n=1), opsoclonus myoclonus syndrome (n=2), Sydenham chorea (n=1), Aicardi Goutières syndrome (n=1) a Miscellaneous: paroxysmal tonic upgaze of infancy, idiopathic intracranial hypertension, isolated cranial nerve palsy, acquired brain injury, pseudoparalysis secondary to vitamin C deficiency, posterior reversible encephalopathy syndrome, autism spectrum disorder with behavioural disturbance, hydrocephalus with ventriculoperitoneal shunt dysfunction, myasthenia gravis, hereditary motor sensory neuropathy, urea cycle disorder, syncope, malformation of cortical development. CNS, central nervous system; MS, multiple sclerosis; ADEM, acute disseminated encephalomyelitis; HSV, herpes simplex virus; NMDAR, N-methyl-D aspartate receptor; VGKC, voltage-gated potassium channel. Intrathecal and Mirrored OCBs in Paediatric Neurology Adriane Sinclair et al. 73

4 Table II: Clinical correlation of intrathecal and mirrored oligoclonal bands (OCBs) OCB result Disease groups and diagnoses Intrathecal, n (%) Mirrored, n (%) Total (6) (10) Non-inflammatory CNS (0) (4) Epilepsy 0 58 (0) 2 58 (3) Static encephalopathy 0 15 (0) 0 15 (0) Progressive genetic 0 8(0) 1 8(13) or metabolic Other 0 61 (0) 3 61 (5) Inflammatory CNS (19) (24) Demyelinating 3 26 (12) 4 26 (15) ADEM 0 11 (0) 4 11 (36) Clinically isolated syndrome 0 9(0) 0 9(0) Multiple sclerosis 3 6(50) 0 6(0) Infection-mediated 3 11 (27) 2 11 (18) Bacterial meningitis Aseptic meningitis HSV encephalitis Other encephalitides Acute necrotizing encephalopathy Autoantibody-associated 2 7(29) 4 7(57) NMDAR encephalitis VGKC encephalopathy Basal ganglia encephalitis Other immune-mediated 3 14 (21) 4 14 (29) Rasmussen encephalitis Acute cerebellar ataxia Polio-like illness Opsoclonus myoclonus syndrome Varicella zoster vasculitis Aicardi Goutières syndrome Sydenham chorea CNS, central nervous system; ADEM, acute disseminated encephalomyelitis; HSV, herpes simplex virus; NMDAR, N-methyl-D aspartate receptor; VGKC, voltage-gated potassium channel. Those with autoantibody-associated disorders were likely to have either intrathecal or mirrored OCBs (6 7; 86%). In both patients with NMDAR encephalitis whose initial samples showed mirrored OCBs, CSF samples taken 59 and 70 days after the first samples were found to contain intrathecal OCBs. Among those with demyelinating disorders, intrathecal OCBs were detected only in patients with multiple sclerosis (3 6; 50%), whereas mirrored OCBs were detected only in those patients with ADEM (4 11; 36%). All three patients with Rasmussen encephalitis had either intrathecal or mirrored OCBs. Patients with infection-mediated disorders (n=11) had either intrathecal (3 11; 27%) or mirrored OCBs (2 11; 18%). DISCUSSION We sought to investigate the utility of OCBs in a contemporary group of child neurology patients and make diagnostic associations. We found intrathecal OCBs only in patients with evidence of inflammatory CNS disease; however, a variety of autoantibody-associated, demyelinating, infection-mediated, and other immune-mediated diseases were associated with intrathecal OCBs. Mirrored OCBs were less specific than intrathecal OCBs for CNS inflammation, but were still more likely to be found in patients with inflammatory CNS disease than in those with non-inflammatory CNS disease (24% vs 4%; p<0.001) and therefore can also be of clinical utility. Mirrored OCBs imply the presence of clonal IgG in both CSF and serum. Indeed, many of the autoantibody biomarkers that are useful in CNS inflammation, such as antibodies against neuromyelitis optica aquaporin-4, VGKC, leucinerich glioma-inactivated 1, and myelin oligodendrocyte glycoprotein are measured in the serum, rather than in the CSF. In the case of many autoantibody-associated CNS disorders, the production of autoantibody might be first triggered in the periphery, rather than in the CNS, and the importance of serum versus CSF antibody production is a central emerging theme in neuroimmunology. 16,17 For these reasons, we believe that mirrored OCBs should not be ignored but may be an important biomarker in inflammatory CNS disorders. Many of the patients in this report with mirrored OCBs had autoantibody-associated disorders, such as NMDAR encephalitis and VGKC encephalopathy. Interestingly, we noted that the patients with NMDAR encephalitis who initially had mirrored OCBs subsequently developed intrathecal OCBs, suggesting that patients initially have a systemic autoimmune response, which subsequently becomes localized intrathecally. No patient was found to have a combination of both a mirrored and intrathecal OCB pattern, suggesting that this may be an uncommon finding in paediatric populations, or a pattern that develops in chronic disease, such as in subacute sclerosing panencephalitis. Specific diseases associated with OCBs were generally consistent with findings previously reported by others. Among our patients with demyelinating disorders (n=26), intrathecal OCBs were detected only in patients with multiple sclerosis, and mirrored OCBs were found only in patients with ADEM. Our numbers were small; however, studies specifically of paediatric patients with demyelinating diseases have also reported a higher frequency of intrathecal OCBs in children with multiple sclerosis than with ADEM or CIS. 7,12,13,18 The presence of mirrored OCBs in ADEM has also been reported in other studies. 19,20 Intrathecal OCBs were also found in patients with Rasmussen encephalitis and acute cerebellar ataxia, as previously reported. 21,22 Although OCBs can be useful, we failed to detect OCBs in many patients with recognized inflammatory CNS diseases. The absence of OCBs should not preclude consideration of an inflammatory CNS process, or testing for specific autoantibodies. There is emerging evidence that autoantibodies against VGKC and NMDAR are specific in autoimmune encephalopathy and it could be argued that these specific biomarkers will increasingly replace non-specific markers of inflammation or immune activation, such as OCBs and CSF neopterin. 1,2 However, non-specific biomarkers are likely continue to be of clinical utility as they can alert the clinician to atypical variants of autoimmune encephalopathy or to potential autoimmune diseases yet to be fully defined. 6 Additionally, specific autoantibody tests are less widely available than OCBs and may have a longer processing time; given that early diagnosis and treat- 74 Developmental Medicine & Child Neurology 2013, 55: 71 75

5 ment can improve outcomes, OCBs are likely to continue to play a useful role. Assessing a diagnostic test such as OCBs ideally requires comparison with a criterion standard, but unfortunately there is no practical criterion standard test for CNS inflammation as brain biopsy is too invasive. The group with CNS inflammatory disorders in this study is a heterogeneous group in which the precision of diagnosis and the certainty regarding the inflammatory basis of the disease varies substantially. A prospective study with an extended follow-up time would allow for collection of all pertinent data (such as auxiliary evidence of inflammation) and would increase the level of accuracy for the less precise diagnoses and would perhaps reduce the numbers of patients in whom the diagnosis is unknown. ACKNOWLEDGEMENTS The authors have funding from the National Health Medical Research Council, the University of Sydney, Multiple Sclerosis Research Australia, and the Star Scientific Foundation. REFERENCES 1. Dale RC, Brilot F. Biomarkers of inflammatory and autoimmune central nervous system disorders. Curr Opin Pediatr 2010; 22: Dale RC, Brilot F, Fagan E, Earl J. Cerebrospinal fluid neopterin in paediatric neurology: a marker of active central nervous system inflammation. Dev Med Child Neurol 2009; 51: Dale RC, Church AJ, Surtees RA, et al. Encephalitis lethargica syndrome: 20 new cases and evidence of basal ganglia autoimmunity. Brain 2004; 127: Dale RC, Irani SR, Brilot F, et al. N-methyl-D-aspartate receptor antibodies in pediatric dyskinetic encephalitis lethargica. Ann Neurol 2009; 66: Hartley LM, Ng SY, Dale RC, Church AJ, Martinez A, de Sousa C. Immune mediated chorea encephalopathy syndrome in childhood. Dev Med Child Neurol 2002; 44: Suleiman J, Brenner T, Gill D, et al. Immune-mediated steroid-responsive epileptic spasms and epileptic encephalopathy associated with VGKC-complex antibodies. Dev Med Child Neurol 2011; 53: Alper G, Heyman R, Wang L. Multiple sclerosis and acute disseminated encephalomyelitis diagnosed in children after long-term follow-up: comparison of presenting features. Dev Med Child Neurol 2009; 51: Freedman MS, Thompson EJ, Deisenhammer F, et al. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus statement. Arch Neurol 2005; 62: Kostulas V, Eeg-Olofsson O, Olsson T, Link H. Demonstration in children of oligoclonal IgG bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling. Pediatr Neurol 1986; 2: Link H, Huang YM. Oligoclonal bands in multiple sclerosis cerebrospinal fluid: an update on methodology and clinical usefulness. J Neuroimmunol 2006; 180: Mayringer I, Timeltaler B, Deisenhammer F. Correlation between the IgG index, oligoclonal bands in CSF, and the diagnosis of demyelinating diseases. Eur J Neurol 2005; 12: Banwell B, Bar-Or A, Arnold DL, et al. Clinical, environmental, and genetic determinants of multiple sclerosis in children with acute demyelination: a prospective national cohort study. Lancet Neurol 2011; 10: Tenembaum S, Chamoles N, Fejerman N. Acute disseminated encephalomyelitis: a long-term follow-up study of 84 pediatric patients. Neurology 2002; 59: Pranzatelli MR, Slev PR, Tate ED, Travelstead AL, Colliver JA, Joseph SA. Cerebrospinal fluid oligoclonal bands in childhood opsoclonus myoclonus. Paediatr Neurol 2011; 45: Granerod J, Cunningham R, Zuckerman M, et al. Causality in acute encephalitis: defining aetiologies. Epidemiol Infect 2010; 138: Irani SR, Vincent A. Autoimmune encephalitis new awareness, challenging questions. Discov Med 2011; 11: Vincent A, Bien CG, Irani SR, Waters P. Autoantibodies associated with diseases of the CNS: new developments and future challenges. Lancet Neurol 2011; 10: Dale RC, Brilot F, Banwell B. Pediatric central nervous system inflammatory demyelination: acute disseminated encephalomyelitis, clinically isolated syndromes, neuromyelitis optica, and multiple sclerosis. Curr Opin Neurol 2009; 22: Dale RC, de Sousa C, Chong WK, Cox TC, Harding B, Neville BG. Acute disseminated encephalomyelitis, multiphasic disseminated encephalomyelitis and multiple sclerosis in children. Brain 2000; 123: Franciotta D, Columba-Cabezas S, Andreoni L, et al. Oligoclonal IgG band patterns in inflammatory demyelinating human and mouse diseases. J Neuroimmunol 2008; 200: Bien CG, Granata T, Antozzi C, et al. Pathogenesis, diagnosis and treatment of Rasmussen encephalitis: a European consensus statement. Brain 2005; 128: Connolly AM, Dodson WE, Prensky AL, Rust RS. Course and outcome of acute cerebellar ataxia. Ann Neurol 1994; 35: Intrathecal and Mirrored OCBs in Paediatric Neurology Adriane Sinclair et al. 75