Gustavo Miguel da Rocha Rodrigues

The role of biomarkers in the diagnosis of Multiple sclerosis

2011/2012

março, 2012

Gustavo Miguel da Rocha Rodrigues

The role of biomarkers in the diagnosis of Multiple Sclerosis

Mestrado Integrado em Medicina

Área: Neurologia

Trabalho efetuado sob a Orientação de:

Dra. Joana da Cruz Guimarães Ferreira de Almeida

Trabalho organizado de acordo com as normas da revista:

Multiple Sclerosis and related Disorders

março, 2012

IIIPORTO Projeto de Opção do 6° ano - DECLARAÇÃO DE INTEGRIDADE

!FMUP! FACULDADE DE MEDICINAUNIVERSIDADE DO PORTO

UNIDADE CURRICUlARPROJETO DE o e c z oDISSERTAÇÃO f MONOGRAFIA

Eu, Gustavo Miguel da Rocha Rodrigues, abaixo assinado, nO mecanográfico 060801225, estudante do

60 ano do Mestrado Integrado em Medicina, na Faculdade de Medicina da Universidade do Porto,

declaro ter atuado com absoluta integridade na elaboração deste projeto de opção.

Neste sentido, confirmo que NÃO incorri em plágio (ato pelo qual um indivíduo, mesmo por omissão,

assume a autoria de um determinado trabalho intelectual, ou partes dele). Mais declaro que todas as

frases que retirei de trabalhos anteriores pertencentes a outros autores, foram referenciadas, ou

redigidas com novas palavras, tendo colocado, neste caso, a citação da fonte bibliográfica.

Faculdade de Medicina da Universidade do Porto, 1b /ó'? / ~o1),

Assinatura:

DlpORTOFMUP FACULDADE DE MEDICINA

UNIVERSIDADE DO PORTO

Projecto de Opção do 6° ano - DECLARAÇÃODEREPRODUÇÃO

Nome: Gustavo Miguel da Rocha Rodrigues

Endereço electrónico: gustavo rodrigues17@hotmail.com Telefone ou Telemóvel: 912248477

Número do Bilhete de Identidade: 13213739

Título da Monografia:

The role of biomarkers in the diagnosis of Multiple Sclerosis

Orientador:

Dra. Joana da Cruz Guimarães Ferreira de Almeida

Ano de conclusão: 2012

Designação da área do projecto:

Neurologia

É autorizada a reprodução integral desta Monografia para efeitos de investigação e de divulgação

pedagógica, em programas e projectos coordenados pela FMUP.

Faculdade de Medicina da Universidade do Porto, t0~ lo 11

Assinatura: L~josJ{)

Dedicatória

Dedico este trabalho á minha família pela disponibilidade e apoio incondicional

ao longo de todo o meu percurso académico.

MESTRADO INTEGRADO EM MEDICINA

Ano letivo: 2011/2012

Nome do(a) Estudante: Gustavo Miguel da Rocha Rodrigues

Orientador(a): Dra. Joana da Cruz Guimarães Ferreira de Almeida

Área do Projeto: Neurologia

Título do Projeto: The role of biomarkers in the diagnosis of Multiple Sclerosis.

Resumo: Esclerose Múltipla (EM) é uma doença neurológica inflamatória desmielinizante

importante devido à sua incidência, cronicidade e maior prevalência em adultos jovens. No

estudo inicial de uma doença desmielinizante do Sistema Nervoso Central existe a necessidade

de encontrar biomarcadores que possam melhor discriminar a EM de outras patologias.

Atualmente, apenas as bandas oligoclonais (BOC) são utilizadas. Esta revisão pretende, através

de uma pesquisa na base de dados PubMed, examinar a literatura publicada nos últimos 5

anos, sobre o papel dos biomarcadores no diagnóstico da esclerose múltipla. O uso de

marcadores no líquido encéfalo-raquidiano, aumenta a especificidade e valor preditivo

negativo, diminuindo os possíveis falsos positivos da, muito sensível, ressonância magnética.

As BOC confirmaram o seu valor, apoiando o diagnóstico quando presentes e sugerindo a

revisão do mesmo quando ausentes, mostrando contudo algumas limitações: variabilidade

interpopulacional, laboratorial e metodológica, controvérsia sobre o uso do índice IgG, a

interpretação de uma única BOC e a relação das BOC com a EM. Enquanto o vírus Epstein-Barr

e os anticorpos antimielina falharam quando testados, as cadeias leves livres K demonstraram

resultados promissores, especialmente, como todos os biomarcadores, quando combinadas

com ressonância magnética. Na previsão de conversão do síndrome clínico isolado para EM, as

BOC confirmaram o seu papel, enquanto CXC quimiocina motif 13 e a reação MRZ mostraram

bons valores preditivos, e os marcadores de lesão axonal e anticorpos contra a mielina

falharam. Além da deteção de anticorpos aquaporina-4 e a reação MRZ no líquido

céfaloraquidiano a deteção de N-acetil aspartato no sangue mostraram bons resultados a

distinguir EM de neuromielite ótica. Há uma procura para desenvolver métodos automatizados

para estudar biomarcadores, e o desenvolvimento de técnicas proteómicas é promissor para a

identificação de novos alvos moleculares para ensaios de diagnóstico e terapêutica.

Concluindo, o principal contributo dos biomarcadores no diagóstico de EM, consiste no

aumento de especificidade e valor preditivo. É necessário corrigir qualquer possível erro

metodológico, tentando estandardizar ao máximo de modo a obter resultados mais

consistentes. Apesar dos esforços na pesquisa de novos biomarcadores é necessário

compreender melhor a EM de modo a encontrar um teste simplificado.

Palavras-chave: Esclerose Múltipla, Síndrome clínico isolado, bandas oligoclonais, líquido encéfalo-

raquidiano, biomarcadores, diagnóstico.

The role of biomarkers in the diagnosis of Multiple Sclerosis

Rodrigues, Gustavo Faculty of Medicine of Porto University (FMUP)*

Neurology Department, Faculty of Medicine, University of Porto, Hospital de São João,

Alameda Professor Hernâni Monteiro, 4200, Porto, Portugal Faculty of Medicine of Porto University

Telephone and Fax: + 00351 225511200

*Corresponding author, Praça, 135 Vila Nova, 4465-767 Praia da Vitória, Açores - Portugal

E-mail: Gustavo_rodrigues17@hotmail.com; Telephone: +00351 912248477

Abstract

Multiple Sclerosis (MS) is an important inflammatory demyelinative neurologic

disease, due to its, incidence, chronicity and higher prevalence among young adults. In

the initial approach to a demyelinative disease of the Central Nervous System there is a

urge for biomarkers to best discriminate MS from other diseases. Despite the broad

range of biomarker studies, only oligoclonal bands (OCB) are used by routine in

differential diagnosis. This review aims to access the published literature in the last 5

years (trough a search in PubMed database), regarding the role of biomarkers in the

diagnosis of MS. Regarding MS differential diagnosis, the use of cerebrospinal fluid

markers (CSF) increases specificity and negative predictive, diminishing the possible

false positives from the high sensible magnetic resonance imaging (MRI). OCB

confirmed they value, supporting the MS diagnosis when present and suggesting

revision of this, but some limitations appeared regarding inter-population, inter-

laboratories and inter-methods detection variability and there’s controversy concerning

the current use of IgGindex, interpretation of a single weak OCB and the relation of

OCB with MS. While Epstein Barr Virus (EBV) and anti-myelin antibodies failed when

tested, Kappa free light chains (KFLC) performed in a promising way, specially, as all

biomarkers, when combined with MRI.

In this review, OCB confirmed they importance while C-X-C motif chemokine

13 (CXCL13) and MRZ reaction showed good predictive values results while axonal

damage markers and antibodies against myelin didn’t perform well, predicting clinically

isolated syndrome (CIS) conversion to MS. Besides aquaporin-4 antibody detection and

MRZ reaction in CSF, N-acetyl aspartate detection in serum showed good results

distinguishing MS from neuromyelitis optica (NMO). There has been a struggle to

develop standard easy automated methods to biomarker studying and the development

of more sensitive proteomic techniques is very promising for the identification of new

molecular targets for diagnostic assays and therapies preventing axonal damage.

Concluding, biomarkers major contribute is in terms of Specificity and

predictive value. Methodological standardization, correcting any possible error, is vital

for a more consistent research. Despite the efforts in this field, an accurate simple test is

only possible when the pathophysiology of MS is better understood.

Keywords: Multiple Sclerosis, Clinically isolated syndrome, oligoclonal bands,

cerebrospinal fluid, biomarker, diagnosis.

1. Introducion

1.1 Historic note and state of art

Multiple Sclerosis (MS) is an important inflammatory demyelinative neurologic

disease, due to its incidence, chronicity and tendency to affect young adults.(1) MS

features a broad heterogeneity of clinical, histopathological and immunological

phenotypes, which urges a more accurate defining of patients by biological markers that

reflect the underlying disease process and allow the prediction of disease course and

treatment response.(2, 3) Thus, serious efforts are made in the field of biomarkers to

improve the diagnostic discrimination, to determine prognostic factors, and to identify

markers to monitor the clinical course and response to disease-modifying therapies.(3)

Starting with Charcot (1868) and his triad (nystagmus, intention tremor and

scanning speech), the diagnostic criteria for MS have evolved to reflect the

breakthroughs in our understanding of the disease and the development of new

diagnostic techniques, changing from purely clinical criteria to increasing dependency

upon imaging of the central nervous system (CNS). Only in 1954 the first true

diagnostic scheme appeared, published by Allison and Millar describing three

categories: early, probable and possible disseminated sclerosis.(1)

There was a need for reliable and reproducible diagnostic criteria in order to

provide accurate epidemiological studies and therapeutic trials. A major step in that

direction occurred in 1965, when Broman et al. outlined the principles that were the

basis for following schemes.(4) They consisted of age of onset, the empiric significance

of symptoms for an MS diagnosis, the multiplicity of lesions, the number of bouts, and

the familial “recurrence” of MS. The results of the newly described electrophoretic

examination of cerebrospinal fluid (CSF) protein, the precursor of oligoclonal bands

(OCB), were also introduced. Additionally, all other possible diagnoses were to be

considered and, if possible, excluded. For the first time was stated the twin principles of

dissemination in time and in space, that to this day remain the fundamental criteria for

the diagnosis of MS.(4) The further need for accurate diagnosis criteria, lead to the

formation of an expert panel and elaboration of the Schumacher et al. criteria, used

worldwide.(5)

A adaptation of the Schumacher criteria emerged from a worldwide

investigation by Bauer that was the first to include CSF OCB.(1)

One problem with the diverse published diagnostic classifications is their

different terminology and the subjective judgment required. This problem cannot be

completely overcome but can be diminished in the absence of a totally specific

laboratory test for MS by adding to the clinical evaluation, the results of laboratory,

neuroimaging, and neurophysiological procedures.(1)

New criteria (Poser et al. criteria) were published in 1983 and consisted of two

main groups, definite and probable, each with two subgroups, clinical and laboratory

supported.(6) Furthermore, for the first time definitions of the terms used were agreed.

An attack/exacerbation could consist of symptoms alone lasting at least 24 h. The term

“paraclinical” evidence was introduced to comprise the results of evoked response

studies and neuroimaging. One problem of this scheme was the failure to incorporate

consideration of primary progressive MS (PPMS). These criteria promptly replaced

previous ones for epidemiological studies and selection of patients for therapeutic drug

trials as well in clinical practice.(6) The 2001 McDonald et al. diagnostic criteria

resulted in two major changes: MRI criteria were incorporated into the scheme, and

long-needed guidelines for the diagnosis of primary progressive MS were defined.(7)

OCBs, best detected by isoelectric focusing followed by immunoblotting, is the

only biomarker present on the latest 2005 Revisions to the Mcdonald Criteria for MS.

According to these criteria, “elevated immunoglobulin G (IgG) index or ≥2 OCB in

CSF can be important to support the inflammatory demyelinating nature of the

underlying condition, to evaluate differential diagnosis and to predict clinically

definitive MS”.(8)

1.2 Biomarkers

Biomarkers are measurable indicators of a physiologic or pathologic process or

the effects of a therapeutic intervention. An ideal biomarker would thus be binary,

missing in a healthy individual, only present in the disease state and increasing with the

severity of the latter.(9) Characteristics that define biomarkers are usually limited to

detection of specific proteins in body fluids that become altered as a consequence of a

biologic or pathologic process.(2, 9, 10)

Since MS lesions are rarely biopsied, CSF analysis remains an important and

practical tool to understand the underlying immunopathogenesis in patients presenting

with a first demyelinating event or with early MS. It is possible to detect and measure a

various number of particles by several techniques.(3, 11) Differential therapeutic

approaches requires the development of biological markers that objectively: 1)reflect

the targeted immunopathological process, 2)select patients in which the pathogenic

process predominates, 3)show responses to treatment, 4)provide a simple and economic

monitoring tool in clinical trials and routine patient management.(2) In a complex

disease like MS with different pathogenetic mechanisms, a single biomarker is likely to

reflect only one of many ongoing pathogenic processes. Thus, the stratification of

patients might only be possible by using a defined set of biomarkers. Each biomarker

will have to be validated across diverse patient cohorts in prospective, multicentre

studies for the following goals:

1) clinical relevance: the biomarker has the capability to reflect the change of a

pathologic process and/or therapeutic intervention in a relatively short period;

2) sensitivity: the ability to be measured with adequate precision and sufficient

magnitude of change to reflect a meaningful change in important clinical endpoints;

3) specificity: the power to identify patients by the occurrence of pathologic

processes or the response to an intervention in terms of changes in a clinical endpoint;

4) probability of false positive or false negatives: defined by the situations in

which an estimated change of the biomarker is not reflected by the respective change in

clinical endpoint;

5) accuracy, precision, reproducibility and variability of the laboratory assay or

test measurement of the biomarker.(2, 9)

Some challenges to biomarker discovery and validation are: highly variable

clinical course, heterogeneous mechanisms of disease, nonspecificity of disease

markers, intra- and inter-individual variability in markers, variable responses to

treatment, diagnosis of exclusion without “gold standard”, frequent subclinical disease

activity, heterogeneous treatments with diverse mechanism of action.(12)

The latest promise in biomarker discovery is the proteomic studies, which

revealed a broad range of new possible biomarkers for MS. The discovery of new

biomarkers would be crucial for accurate and earlier diagnosis.(3)

1.3 Objective

The aim of this review is to assess the literature published in the last 5 years

concerning the role of biomarkers in the diagnosis of MS and conversion of CIS to MS.

The biomarkers concerning the MS evolution were excluded.

2. Material and Methods

The bibliography used for this article was obtained on 15/11/2011 by a double

research in the pubmed database. The first was made with the following query:

(("cerebrospinal fluid" [Subheading]) AND ( "Multiple Sclerosis/cerebrospinal

fluid"[Mesh] OR "Multiple Sclerosis/diagnosis"[Mesh] )) AND ( "Biological

Markers/analysis"[Mesh] OR "Biological Markers/cerebrospinal fluid"[Mesh] OR

"Biological Markers/diagnostic use"[Mesh] ), and the limits used were language

(Portuguese, Spanish and English) and publication date (<5 years). After reading the

abstract and/or full article, 68 articles were selected from the 158 that resulted from the

search. A second query (“multiple sclerosis AND diagnosis AND biomarkers”) without

Mesh terms was used, with the same limits for language and with publication data

limited to 6 months. 13 were selected from the 62 obtained and most of them were

included in the previous research. From all the articles, only 4 of them couldn’t be

acquired.

The bibliography of the studies above and additional literature considered

relevant, was included in this review. Microsoft word from Microsoft office 2007,

EndNote X5 and My NCBI were the tools used.

3. Results and discussion

3.1 MS diagnosis

3.1.1 Role of OCB in MS diagnosis

While MRI is now very sensitive, specificity is a problem. The MRI lesions are

compatible with many pathophysiological mechanisms and thus different diseases,

creating “false positives”.(13) MRI criteria for MS show limitations in equivocal MS

cases. Therefore, it is imperative that other investigative procedures, such as CSF

examinations, are established for MS diagnosis.(14)

The major diagnostic contribution of OCB was in terms of Specificity and

negative predictive value. Improved specificity is vital in differential diagnosis to rule

out non-MS cases. Moreover the combination of MRI and CSF analysis proved to be

more accurate than MRI alone.(15) The only marker used in the diagnostic procedure of

MS is the qualitative and quantitative assessment of immunoglobulin G in CSF.(2, 12,

16) Using optimized, standardized methodology, preferentially protein separation by

isoelectric focusing (IEF) followed by immunoblotting, more than 95% of patients with

MS have CSF OCB not detectable in serum, thereby providing powerful evidence for

the diagnosis of MS. Because of the high sensitivity of CSF OB in MS as well as its

high specificity in the appropriate clinical setting, examination of CSF for OB of IgG

class can be strongly recommended to obtain support for the diagnosis of MS.(17)

The IgG index equal to CSF/serum IgG:CSF/serum albumin is elevated in about

70% of MS patients, but infrequently in CSF OB-negative MS. Because of lower

diagnostic sensitivity, IgG index cannot be recommended as surrogate of CSF OB in the

diagnosis of MS but, when elevated, as additional evidence for an augmented B-cell

response within the CNS compatible with MS.(17, 18)

A drawback with determination of the IgG index is that it may become falsely

elevated in patients with moderate severe blood–CSF barrier damage. Detection of a

moderately–strongly elevated CSF/serum albumin ratio should lead to reevaluation of

an MS diagnosis. Still, the IgG index is more or less obtained for free in the context of

practically every routine CSF examination.(17)

Although the clinical picture as well as findings from magnetic resonance

imaging of the brain and spinal cord is essential for an MS diagnosis, this should be

reevaluated in CSF OB-negative patients, remembering the possible diseases imitating

MS. Conversely, OCB are not specific and are found less frequently in other conditions

that can clinically mimic MS, though usually OCB are only transient in these

conditions, whereas they are present continuously in MS patients, regardless of disease

activity or therapy, indicating a stable humoral immune response in CNS of MS

patients.(2, 17, 19) Consequently, a positive laboratory test for OCBs has a high

negative predictive value and a low positive predictive value, making it a sensitive

screening tool but not a diagnostic one.(16)

The significance of IgG migrating as one sharp (rare finding), monoclonal band

in CSF while not detectable in corresponding serum is debated. If the IgG index is

simultaneously elevated, then there is evidence for a local B-cell response, and this

should be reported. Usually, however, the IgG index is normal. The monoclonal CSF

IgG band may reflect a technical problem, and the examination for CSF OCB should be

repeatedly performed on the same CSF and serum specimens. The significance of a

single band in CSF remains unclear.(14, 17, 20)

There are trans-ethnic and/or trans-population differences in OCB sensitivity in

MS. Lower frequencies of CSF OCB have been reported from Asian and Southern

countries: China (63.3%), Italy (73.2%), Turkey (85.7%) and Brazil (54.4%).(17, 21-

25) Different methods adopted for CSF OB detection, various disease sub-types, diverse

genetic background, methodology or patient selection issues, can play a role in this

differences.(17, 21, 22) Due to these differences, OCB are of less diagnostic value for

MS in these areas (there was no significant difference between MS and other

inflammatory neurological diseases in a Chinese study).(21)

Different methods adopted for CSF detection can play a significant role in this

matter. Despite the awareness of the advantages inherent to IEF + immunoblotting,

several laboratories do not this method.(26, 27) It was argued that OCB detection

usefulness is limited by high inter-laboratory variability.(13) A Spanish study

demonstrated a high inter-laboratory reproducibility in high sensitivity techniques like

IEF, while an Italian group identified an unacceptable large inter-laboratory variation

not only in OCB numbering, but also in qualitative reporting of the OCB pattern and in

differentiating OCB+ from OCB-.(23, 28) Nevertheless, results of CSF OCB testing

should be considered in the context of the method used.(17, 28, 29) Recently OCB have

been detected in the tears of patients with CIS with 100% specificity.(30)

External quality control schemes are fundamental steps in standardization

processes, particularly in the field of IEF, the recommended technique for OCB

detection, because many IEF steps may be difficult to standardize and new centers lack

specialized sections for CSF analysis. Misinterpretation of art factual bands, insufficient

IEF skills or poor blotting and staining skills contribute to the inter-laboratory

variability. Educational support and external quality control schemes, scientific

associations involved in CSF analysis play essential roles in promoting quality.(29)

A various number of studies comparing OCB positive MS patients with OCB

negative, sometimes with contradictories results, regarding the OCB pattern, the

fulfillment of MRI criteria, location of the lesions, disease course, CSF pattern

demonstrated once more the heterogeneity of MS.(10, 31-33) Even the correlation

between both parameters of intra-thecal synthesis is not yet clear.(34)

In a patient where CSF OCB are expected but not detectable, the pair of

CSF+serum might be re-examined, but this only very rarely leads to a different result. It

is recommended that a complete routine CSF examination including test for IgG OCB

with appropriate methodology is done before a MS patient is included in clinical trials

with e.g. disease modulating drugs. The intent was that the Criteria could “be used by

the practicing neurologist to better and more reliably diagnose MS, balancing earlier

diagnosis with the need to avoid false-positive diagnosis”.(17)

The diagnosis of PPMS is particularly difficult. The single large study referred

to in the 2005 Revisions consists of 943 patients with a diagnosis of PPMS based on

clinical, MRI and evoked potential data. “Supportive CSF findings” were obtained in

about 80% of the patients, while the remaining 20% were ineligible because they did

not fulfill the supportive CSF findings.(35) These consisted of CSF OCB, elevated IgG

index or elevated IgG synthesis rate. The sensitivity of these variables differs

significantly in the context of a diagnosis of MS (95%, 70%, and 70%, respectively if

optimal tests are performed). Unfortunately, the three variables are equalized in the

study.(35) This is not noticed in the 2005 Revisions. Further, the study gives no

information regarding the procedures used for protein separation. A well-performed

study published in 2004, showed that 58 of 60 patients with PPMS (97%) have IgG OB

in CSF when examined by IEF+immunoblotting.(36) By adopting the 2005 Revisions,

there could be a risk that patients not suffering from MS might be included, thereby

jeopardizing the outcome of the trial.(17)

Other OCB isotypes, like IgA and IgM appear to be of limited additional value

in the diagnosis of MS.(16) IgA presence in MS is uncommon, suggesting other

diagnosis.(37, 38) IgM OCB are identified up to 60% of MS patients (especially in

childhood MS) and are more common in early phases of MS, being useful to select

patients to receive early treatment, because may influence disease prognosis.(16, 39)

Fifty years after their discovery, the OCB therefore continue to puzzle neuro-

immunologists.(19) It is highly recommended that the diagnostic MS criteria are further

revised to incorporate the minimum of CSF tests recommended for use in order to

support a diagnosis of MS and reduce the likelihood of another disease that mimics MS.

Many questions regarding CSF OCB remain to be solved. Some of them are minor like

the significance of a monoclonal band in CSF, the optimal way to proceed with CSF

examinations in a patient who according to clinical and MRI criteria has MS but has no

CSF OCB, the diagnostic role of detection of other Immunoglobulins or free light

chains migrating as OCB in CSF, improved and simplified methods for detection etc.

Some remaining questions are major like elucidating the role of CSF OCB in the

pathogenesis of MS.(2, 17)

3.1.2 Additional markers.

Despite the numerous studies, there has been no definite association of OCB

with a specific antigen in patients with MS and it is clear that the intra-thecal antibody

synthesis is directed against diverse antigens. An association of these antibodies with

the cause of MS is still missing.(2) Epstein-Bar virus (EBV) infection has been

suspected as an initial trigger of autoimmunity in MS and, thus, evidence of EBV

infection could have relevance for MS diagnosis.(10)

A number of studies have shown that almost 100% of MS patients have evidence

of prior EBV infection, whereas approximately 90% of healthy adults also have

evidence of EBV infection.(40) Therefore, the presence of EBV antibodies is not

considered to be diagnostic relevant. Furthermore, CSF reactivity to EBV antigens in

MS compared with healthy controls has not yielded any specific associations.(41)

Current investigations are focused on determining whether MS patients have

antibodies to unique determinants of EBV, or if viral-clearance responses to EBV

infection in MS patients are abnormal.(42) If these investigations are fruitful, they could

have application as diagnostic markers.(10)

Additionally, Bartos et al. (2007) reported no difference in antibodies against

light neurofilaments in all sub-types of MS and other diseases.(43)

Nogo-A, an inhibitor of axonal regeneration, is a perfect example of the

inconsistent and contradictory data gathered in this review. Jurewicz et al. (2007)

reported that CSF Nogo-A may be specific (100%) for MS and its presence may predict

failure of axonal regeneration within the CNS. Nogo-A was found in CSF of 96% of

MS patients and wasn’t present in meningo-encephalomyelitis, control subjects wither

neurologic, and autoimmune diseases.(44) These findings lacked confirmation by

William et al. (2008) with the same methodology.(45) Correspondence between authors,

showed high discordance in this subject.

Numerous studies have demonstrated elevated kappa free light chains (KFCLs)

in CSF of MS patients. However, so far only small cohorts have been examined, and

generally only through qualitative KFLCs analysis. Determining a single quantitative

diagnostic FLC parameter (KFLC index) by nephelometry (highly automated) and IEF,

Presslauer et al. (2008) showed higher sensitivity (96% vs 91%) but lower specificity

(86% vs 92%) compared with OCB, but was far more accurate than IgG index.

Additionally, there was no correlation between MS type, EDSS score, disease duration,

or number/localization of MRI lesions and the value of KFLC or KFLC index.(14)

The combined use of three FLC indices accounting for monomeric FLC –k level

and k/λ ratio values in the CSF and serum was found to be of promising diagnostic

importance for differentiation of MS from other non-MS neurological diseases

(specificity 93%, sensitivity 100% vs OCB 76% and 87% in the same cohort).(46) In

contrast to the OCB test, where the result is interpreted as “positive”, “negative” or

“trace”, this method provides quantitative evaluation of relative amounts of FLCs, as a

measure of the severity of intrathecal immunoglobulin synthesis. This procedure for

FLC analysis can serve as a basis of a new diagnostic test for MS. The described

method is inexpensive, it requires no special requirement, and may be applied in clinical

laboratories.(46) This procedure also allows preparation of samples in a dry state, that is

helpful for prolonged storage and transportation of CSF and serum samples prior to

their analysis.(46)

As it is measured by automated, routinely available laboratory methods, KFLC

quantification can provide a rapid and reproducible indication of intrathecal

immunological processes supporting current MS diagnostic criteria.(14) These results

strongly suggest that the KFLC index should be part of the routine MS diagnostic

algorithm.(14, 46) Additional LFLC index determination can support the exclusion of

alternative diagnoses. Alternative diagnoses should be considered if these are associated

with concurrent significant elevation of LFLC levels.(14)

Elevated CSF anti-MBP antibodies correlate well with acute myelin damage in

the CNS, but are not specific to MS. Reproducible quantification of MBP has been

difficult since immunoassays are not standardized and not commercially available.(3)

CSF MBP has lacked specificity in most studies. The test was recently re-

evaluated by Tian et al. (2009) who reported a diagnostic sensitivity of 83.7% and

specificity of 78.3%. These results need to be confirmed in further studies.(16, 47)

Preliminary studies, approaching Gelsolin, leukemia inhibitory factor and 7-

ketocholesterol, showed good result, needing further evaluation.(48-50)

In table 1, are displayed other biomarkers revised in this search.

3.1.3 Conversion of Clinically Isolated Syndrome to Clinically Definitive Multiple

Sclerosis

In a significant percentage of patients who later develop MS, the disease initially

presents with an acute or sub-acute episode of neurological symptoms due to a single

demyelinating lesion, known as clinically isolated syndrome (CIS). Predict the risk of

future events, confirming the MS diagnosis is crucial to begin an important early

treatment. In the last few years, the main focus has been on diagnostic sensitivity in

order to early detect the patients in need for early therapeutics, resulting therefore in

possible over diagnoses.(51) In this context, the most relevant statistical parameters to

predict conversion from CIS to MS are positive predictive value (PPV) and negative

predictive value (NPV).(51) MRI is the main exam used to this prediction but there is

an ongoing search for accurate biomarkers for this conversion.(52, 53)

Tintoré et al. (2008) showed that detection of OCB in CSF approximately

doubles the risk of having a second attack, autonomously of MRI, but does not seem to

influence the development of disability.(54) This higher risk is more significant when

MRI is normal or when there is not MRI criteria for dissemination in space (DIS) (4%

of patients with normal MRI and negative OCB converted and 23% of those with

normal MRI and positive OCB converted).(54)

The association of OCB to MRI maximizes the risk; therefore they can be used

together to better predict conversion to CDMS. OCB associated with number of

Barkhof criteria and number of lesions but the absence of OCB should suggest

extending the diagnosis workup.(15, 17)

Similar results were found by other displayed on table 2.(15, 55-58)

Additionally, two of these studies suggested that patients with OCB converts faster to

CDMS (55, 57), especially when IgM is associated.(55)

Brettsschneider et al. (2010) in a prospective pilot study with 91 patients and 2

years of follow-up, showed that the presence of C-X-C motif chemokine 13 (CXCL13)

in CSF has a better PPV (70%, augmented to 80% with Barkhof criteria) than OCB,

Measles, Rubella and Varicella zoster reaction (MRZR, intra-thecal synthesis of specific

antiviral IgG antibodies that supports the diagnosis) or Barkhof criteria.(59)

A large clinical case-control cohort, including the whole spectrum of MS forms,

as well as large numbers of inflammatory and non-inflammatory subjects with other

neurologic diseases confirmed the potential role of CXCL13 as a MS disease activity

marker. CXCL13 correlated with relapse rate, Expanded Disability Status Scale

(EDSS), number of lesion on MRI and response to treatment.(60) However, this

biomarker is not a disease-specific biomarker, but showed a close relationship with

OCB and CSF cell counts.(59-61)

Regarding antibodies against myelin, myelin oligodendrocyte glycoprotein

(MOG), and myelin basic protein (MBP), discordant unexplained results have

appeared.(62) Correlation ranged from highly significant (63), significant (64, 65), to

not significant at all (62, 66). It is important to note that these controversial results were

obtained with the same type of analysis of antimyelin antibodies, immunoblotting.

Thus, these different results may primarily reflect differences in study cohorts rather

than methodological problems.(2) Currently there is no proven value in the diagnostic

and prognostic utility of CSF anti-myelin antibodies in patients with CIS.(16)

CSF of patients with MS is characterized by a polyspecific, intrathecal B-cell

response with a prominent antibody production against neurotropic viruses like measles,

rubella and varicella zoster.(51) A prospective study with 89 CIS patients comparing

MRZR with OCB and MRI lesion load showed that MRZR was significantly more

frequent in CIS that developed MS during follow-up. Their data showed MRZR to have

a high PPV. MRZ score, produced according to the different prognostic relevance of

the three antibody included in MRZR, had the best single PPV (79%), augmented when

added to MRI (≥2 lesions, 91%).(51)

An important drawback of MRZR is that there’s no workable combined assay to

determine these antibodies. Therefore, another study from the same authors, following

121 individuals for 2 years values more CXCL13 determination (PPV of 70%).(59)

Axonal damage markers (tau and NfHSMI35) were measured by Brettschneider

et al. (2006) in order to access their power to predict conversion from CIS to MS. CSF

tau and NfHSMI35 were significantly elevated in CIS compared to controls, and

correlated with disease activity.(67) The sensitivity predicting the conversion from CIS

to MS was higher for the combination of CSF markers (either tau or NfHSMI35

elevated) than for MRI (40 versus 34%), but could be further increased to 60% if CSF

and MRI criteria were combined. Similarly, the combination of tau and NfHSMI35

showed higher specificity (94%) than MRI (82%). Tau and NfHSMI35, according to

this study, are valuable biomarkers for axonal damage in the CIS patients.(67)

Other molecules showed results that need further evaluation: increased CSF

cystatin C (in contrast with former studies), dissociation 14-3-3/tau protein in CSF,

blood and CSF white blood cell count and neurofilament light.(68-71)

In other hand, IgM and human leukocyte antigen G failed to be relevant in the

evaluation of CIS.(72, 73)

3.1.4 Multiple Sclerosis vs Neuromyelitis optica (NMO)

An example of success in researching biomarkers for differential diagnosis of

MS is the aquaporin-4 antibody. Recently, highly specific serum IgG antibodies were

detected by immunofluorescence staining of mouse brain tissue in a majority (73%) of

patients with definite NMO and in 46% of patients at high risk for NMO.(74) NMO-IgG

had 91% specificity for NMO and were suggested to be a useful diagnostic biomarker in

patients with their first episode of myelitis.(75, 76) Most recently a number of

confirmatory studies from different laboratories using diverse assays and different

cohorts of patients were published.(77-80)

A positive MRZR, defined by a combination of at least two antibody indices

was found in 37/42 MS patients, while only in 1/20 NMO patients by Jarius et al.

(2008) allowing the differentiation between MS and NMO.(81)

Tortorella et al. (2011) demonstrated that MS patients show higher serum and

CSF N-acetyl aspartate (NAA) levels than NMO patients, and higher serum NAA levels

than healthy controls. High serum NAA values, exceeding the 95th

percentile of serum

NAA value in healthy controls, were found in all MS patients and in none with NMO.

No differences in serum NAA levels were found between NMO and healthy

controls.(82) Therefore, serum and CSF NAA may be valuable in the diagnostic workup

to differentiate MS from NMO.(82)

3.1.5 Proteomics

Multiple proteomic preliminary studies carried out in the past few years have

revealed potential biomarkers candidates that may not otherwise have been discovered

through function-based approaches.(10, 53, 83-88) For most of these candidate proteins,

the pathophysiological relevance in MS remains to be clarified. Generally the disease

specificity of these proteins is rather low, possibly due to common pathological

mechanisms underlying different diseases.(53, 89)

A problem of CSF proteome analysis is that high-abundance proteins, such as

albumin and immunoglobulin, may overlay brain-specific low-abundance CSF proteins

in the electrophoresis. Therefore, CSF has to be pre-processed to extract those extra-

proteins, possibly resulting in wasting proteins that are bounded do albumin.(53, 85)

The advantages of mass spectrometry-based protein quantification are precision,

sensitivity, throughput and convenience.(83) An important aspect in future CSF

proteome studies is the standardization of sampling protocols, experimental design and

data analysis, in order to reach consensus.(53)

Further studies are needed to better characterize the correlation of these new

candidate markers with MS.(88-90)

4. Conclusion

Diagnosis of MS is currently based on clinical presentation, MRI findings, and

CSF examination (OCB and IgG index) and requires the exclusion of alternative

diagnosis.(45) CSF is especially important in not making a diagnosis of MS; that is, in

excluding other conditions that mimic MS and as a predictor of conversion to CDMS.

As long as we do not know what causes MS we will not have a simple diagnostic test

.(91) To understand the relation of marker concentrations in body fluids and

neuropathological processes, the underlying process need further investigation.(92)

With increasing sensitivity and resolution of analytical methods, the demands of

rigorous control of sample handling and storage also increase.(93) Often, early studies

show great potential, but subsequent reports on the same marker yield inconsistent or

even contradictory results. The reasons for this are mainly methodological although the

heterogeneity and the lack of solid knowledge about MS also contribute.(10, 12, 65, 72,

94, 95)

The use of OCB has some limitations and in result of MS heterogeneity, one

marker possibly isn’t enough. In this respect, development of novel laboratory

techniques and search for new reliable MS-specific markers remains of considerable

diagnostic importance for MS.(46, 96)

Other than OCB, MRZR and CXCL13 appeared like valuable markers for

conversion of CIS to MS, and anti-myelin antibodies the opposite. Predicting

conversion from CIS to MS can be improved combining MRI with CSF markers, but

MRI remains as the major prognostic factor.(16, 54, 59, 67)

CSF OCB utility was demonstrated in diagnosing and predicting MS, with the

awareness of the frequency variability in the different communities. When present, is a

strong support for MS but the diagnosis should be reviewed in it absence.(21, 33) The

equivalent use of IgG index comparing with OCB is inadequate according with some

authors.(17)

Quantitative KFCL’s presented as a helpful marker for MS diagnosis, and

should be further investigated.(46)

Besides aquaporin-4 antibody, the presence of NAA in the serum can be

important to distinguish MS from neuromyelitis.(82)

Even though OCB provide important information regarding MS diagnosis, there

are currently no CSF biomarkers that can reliably and definitively diagnose MS at the

time of initial symptoms.(10) The development of more sensitive proteomic techniques

is very promising for the identification of new molecular targets for diagnostic assays

and therapies preventing axonal damage.(92)

5. Acknowledgements

The help and availability of Dr Joana Guimarães were extremely helpful and

welcome during this paper elaboration, reason why I am sincerely grateful.

6. Conflicts of interest

There are no potential conflicts of interest from each author that relate to the

research covered in the article submitted. The authors have no financial disclosures or

nothing to disclose.

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81. Jarius S, Franciotta D, Bergamaschi R, Rauer S, Wandinger KP, Petereit HF, et al. Polyspecific, antiviral immune response distinguishes multiple sclerosis and neuromyelitis optica. Journal of neurology, neurosurgery, and psychiatry. 2008;79(10):1134-6. Epub 2008/02/14. 82. Tortorella C, Ruggieri M, Di Monte E, Ceci E, Iaffaldano P, Direnzo V, et al. Serum and CSF N-acetyl aspartate levels differ in multiple sclerosis and neuromyelitis optica. Journal of neurology, neurosurgery, and psychiatry. 2011;82(12):1355-9. Epub 2011/05/31. 83. Comabella M, Fernandez M, Martin R, Rivera-Vallve S, Borras E, Chiva C, et al. Cerebrospinal fluid chitinase 3-like 1 levels are associated with conversion to multiple sclerosis. Brain : a journal of neurology. 2010;133(Pt 4):1082-93. Epub 2010/03/20. 84. Harris VK, Diamanduros A, Good P, Zakin E, Chalivendra V, Sadiq SA. Bri2-23 is a potential cerebrospinal fluid biomarker in multiple sclerosis. Neurobiology of disease. 2010;40(1):331-9. Epub 2010/07/06. 85. Li Y, Qin Z, Yang M, Qin Y, Lin C, Liu S. Differential expression of complement proteins in cerebrospinal fluid from active multiple sclerosis patients. Journal of cellular biochemistry. 2011;112(7):1930-7. Epub 2011/03/30. 86. Teunissen CE, Koel-Simmelink MJ, Pham TV, Knol JC, Khalil M, Trentini A, et al. Identification of biomarkers for diagnosis and progression of MS by MALDI-TOF mass spectrometry. Multiple sclerosis (Houndmills, Basingstoke, England). 2011;17(7):838-50. Epub 2011/04/21. 87. Rajalahti T, Kroksveen AC, Arneberg R, Berven FS, Vedeler CA, Myhr KM, et al. A multivariate approach to reveal biomarker signatures for disease classification: application to mass spectral profiles of cerebrospinal fluid from patients with multiple sclerosis. Journal of proteome research. 2010;9(7):3608-20. Epub 2010/05/27. 88. Chiasserini D, Di Filippo M, Candeliere A, Susta F, Orvietani PL, Calabresi P, et al. CSF proteome analysis in multiple sclerosis patients by two-dimensional electrophoresis. European journal of neurology : the official journal of the European Federation of Neurological Societies. 2008;15(9):998-1001. Epub 2008/07/22. 89. Lehmensiek V, Sussmuth SD, Tauscher G, Brettschneider J, Felk S, Gillardon F, et al. Cerebrospinal fluid proteome profile in multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England). 2007;13(7):840-9. Epub 2007/09/21. 90. Qin Z, Qin Y, Liu S. Alteration of DBP levels in CSF of patients with MS by proteomics analysis. Cellular and molecular neurobiology. 2009;29(2):203-10. Epub 2008/09/23. 91. Hintzen RQ, Giovannoni G. CSF analysis in suspected MS: do bands aid? Neurology. 2008;70(13 Pt 2):1059-60. Epub 2008/03/26. 92. Teunissen CE, Dijkstra C, Polman C. Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet neurology. 2005;4(1):32-41. Epub 2004/12/29. 93. Hansson SF, Simonsen AH, Zetterberg H, Andersen O, Haghighi S, Fagerberg I, et al. Cystatin C in cerebrospinal fluid and multiple sclerosis. Annals of neurology. 2007;62(2):193-6; discussion 205. Epub 2006/08/11. 94. Lim ET, Berger T, Reindl M, Dalton CM, Fernando K, Keir G, et al. Anti-myelin antibodies do not allow earlier diagnosis of multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England). 2005;11(4):492-4. Epub 2005/07/27. 95. Voumvourakis KI, Kitsos DK, Tsiodras S, Petrikkos G, Stamboulis E. Human herpesvirus 6 infection as a trigger of multiple sclerosis. Mayo Clinic proceedings Mayo Clinic. 2010;85(11):1023-30. Epub 2010/10/12. 96. Somers V, Govarts C, Somers K, Hupperts R, Medaer R, Stinissen P. Autoantibody profiling in multiple sclerosis reveals novel antigenic candidates. Journal of immunology (Baltimore, Md : 1950). 2008;180(6):3957-63. Epub 2008/03/07. 97. Franciotta D, Di Stefano AL, Jarius S, Zardini E, Tavazzi E, Ballerini C, et al. Cerebrospinal BAFF and Epstein-Barr virus-specific oligoclonal bands in multiple sclerosis and other

inflammatory demyelinating neurological diseases. Journal of neuroimmunology. 2011;230(1-2):160-3. Epub 2010/11/27. 98. Probst-Cousin S, Heuss D, Bergmann M. Annexin-1 is no useful surrogate marker of multiple sclerosis - an immunocytochemical study of the cerebrospinal fluid. Clinical neuropathology. 2011;30(1):18-24. Epub 2010/12/24. 99. Svarcova J, Fialova L, Bartos A, Steinbachova M, Malbohan I. Cerebrospinal fluid antibodies to tubulin are elevated in the patients with multiple sclerosis. European journal of neurology : the official journal of the European Federation of Neurological Societies. 2008;15(11):1173-9. Epub 2008/11/01. 100. Lazzarino G, Amorini AM, Eikelenboom MJ, Killestein J, Belli A, Di Pietro V, et al. Cerebrospinal fluid ATP metabolites in multiple sclerosis. Multiple sclerosis (Houndmills, Basingstoke, England). 2010;16(5):549-54. Epub 2010/03/03.

Appendices

Table 2. Association between OCB and/or imaging and conversion from CIS to

CDMS

N

Follow-

up

(years)

Sensibility Specificity PPV NPV RR1 RR2

Tintoré et al.

(2008) 415 4.2 83 48 50 84 1.7 8.9

Jai Perumal

et al.(2007) 58 5.2 100 61 53.1 100 53 -

Isabel Boscá

et al.(2010) 192 6.2 95 60 78 88 9.4

39.2

#

Juan Rojas

et al.(2010) 40 5 87 60 56 88 5 9

Valentina

Zipoli et

al.(2009)

90 3.8 76 37 60 56 1.4 -

Luisa Villar

et al.(2011)

*

58 6 94 96 97 91 - -

Table2. OCB: oligoclonal bands; CIS: Clinically isolated syndrome; CDMS: Clinically definitive

Multiple Sclerosis; *:All values concern OCB+2 MRI lesions. #:This value concerns the

combination of OCB and IgM. N: number of subjects in study; PPV: positive predictive value;

NPV: negative predictive value; RR1: OCB relative risk; RR2: OCB combined with imaging

relative risk.

Table 1. CSF biomarkers valuable or not for the diagnosis of MS

Not Valuable Valuable

12,5 kDa Cystatin C (93) Gelsolin (48)

BAAF (97) Leukemia inhibitory factor (49)

Annexin-1 (98) 7- ketocholesterol(50)

Anti-tubulin (99)

ATP metabolites (100)

Neurospecific enolase (92)

Anexos

AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 1

MULTIPLE SCLEROSIS AND RELATEDDISORDERS

AUTHOR INFORMATION PACK

TABLE OF CONTENTS.

XXX.

• Description• Audience• Abstracting and Indexing• Editorial Board• Guide for Authors

p.1p.1p.1p.2p.4

ISSN: 2211-0348

DESCRIPTION.

Multiple Sclerosis is an area of ever expanding research and escalating publications. Multiple Sclerosisand Related Disorders is a wide ranging international journal supported by key researchers from allneuroscience domains that focus on MS and associated disease of the central nervous system. Theprimary aim of this new journal is the rapid publication of high quality original research in the field.Important secondary aims will be timely updates and editorials on important scientific and clinicalcare advances, controversies in the field, and invited opinion articles from current thought leaders ontopical issues. One section of the journal will focus on teaching, written to enhance the practice ofcommunity and academic neurologists involved in the care of MS patients. Summaries of key articleswritten for a lay audience will be provided as an on-line resource.

A team of four chief editors is supported by leading section editors who will commissionand appraise original and review articles concerning: clinical neurology, neuroimaging,neuropathology, neuroepidemiology, therapeutics, genetics / transcriptomics, experimental models,neuroimmunology, biomarkers, neuropsychology, neurorehabilitation, measurement scales, teaching,neuroethics and lay communication.

The journal will publish the following types of articles: Reviews; Original Research Articles; Editorials;Comment; Clinical Trial papers; Letter to the Editors; ; Case Reports; Book reviews; News. Thesubmission of an on-line summary of selected papers of relevance for lay audience, Teaching Lessonsand supporting images and datasets is also encouraged.

AUDIENCE.

All branches of neuroscience: clinical neurologists, neurophysiologists, geneticists, psychologist,molecular biologists, MRI and allied imaging specialists, immunologists, major pharmaceuticalcompanies, ethical and legal specialists, MS specialist nurses, drug trial nurses.

ABSTRACTING AND INDEXING.

EMBASEGoogle Scholar®Scopus

AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 2

EDITORIAL BOARD.

Chief Editors:

B. Banwell, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, CanadaM5G 1X8G. Giovannoni, Neuroimmunology Unit, Neuroscience Centre; Institute of Cell and Molecular Science, Barts andThe London School of Medicine and Dentistry; 4 Newark Street; London E1 2AT, United KingdomC. H. Hawkes, Neuroimmunology Unit, Neuroscience Centre; Institute of Cell and Molecular Science Barts andThe London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, United KingdomF. D. Lublin, The Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Mount Sinai School of Medicine, 5East 98th Street, New York, NY 10029-6574, USA

Section Editors:

Experimental Models

S. Amor, Amsterdam, The Netherlands

Epidemiology

A. Ascherio, Boston, MA, USA

Neuro-opthalmology

L. Balcer, Philadelphia, PA, USA

Infection

J. Berger, Lexington, KY, USA

Neuroethics

J. Bernat, Lebanon, NH, USA

Pediatric MS

R. Dale, Sydney, Australia

Biomarkers

F. Deisenhammer, Innsbruck, Austria

Genetics / transcriptomics

R. Hintzen, Rotterdam, The Netherlands

Measurement / quality of life

J. Hobart, Plymouth, United Kingdom

Teaching

M. Keegan, Rochester, MN, USA

Rehabilitation

J. Kesselring, Valens, Switzerland

Clinical Neurology

J.I. Kira, Fukuaka, Japan

Psychology and Fatigue

D. Langdon, Egham Surrey, United Kingdom

Stem Cell Research

G. Martino, Milan, Italy

Therapy

A. Miller, New York, NY, USA

Immunology

F. Sellebjerg, Copenhagen, Denmark

Imaging

J. Wolinsky, Houston, TX, USA

Editorial Board:

H. Butzkueven, Melbourne, AustraliaV. Brinar, Zagreb, Croatia

AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 3

J. Correale, Buenos Aires, ArgentinaO. Fernandez, Malaga, SpainE. Havrdova, Prague, Czech RepublicH.P. Hartung, Dusseldorf, GermanyL. Kappos, Basel, SwitzerlandN. Koch-Henriksen, Aarhus, DenmarkP. Li, Hong Kong, ChinaJ. Losy, Poznan, PolandC. Lubetzki, Paris, FranceA.H. Maghzi, Isfahan, IranA. Miller, Haifa, IsraelN. Prayoonwiwat, Bangkok, ThailandT. Saida, Kyoto, JapanB. Singhal, Mumbai, IndiaS. Sveinbjornsdottir, Reykjavik, IcelandJ. Torro, Bogota, ColumbiaC.P. Tsai, Taipei, TaiwanL. Vécsei, Szeged, HungaryE. Willoughby, Auckland, New ZealandB. Yamout, Beirout, Lebanon

AUTHOR INFORMATION PACK 8 Mar 2012 www.elsevier.com/locate/msard 4

GUIDE FOR AUTHORS.

INTRODUCTIONTypes of articleOriginal Research ArticlesFull length research papers will not normally be more than 3500 words in length from the Inotrductionthrough the Discussion section and will preferably be shorter. Submission of a paper to MultipleSclerosis and Related Disorders will be held to imply that it represents original research not previouslypublished (except in the form of an abstract or preliminary report), that it is not being considered forpublication elsewhere, and that if accepted by Multiple Sclerosis and Related Disorders it will not bepublished elsewhere in the same form in any language without the consent of the Publisher. Majorpapers of topical content will be given priority in publication.

Book ReviewsThese are normally submitted by the Book Review Editors, but they welcome suggestions of booksfor review.

Case ReportsCase reports should detail the clinical, laboratory and neuroimaging features of informative patients.Informative patients should provide insights that inform on genetic contributions to disease, rareclinical manifestations, novel laboratory or imaging features, or highlight important concepts in thedifferential of MS and related disorders. Case reports should be approximately 1200 words, and shouldhave no more than five key references

CommentComments should focus on specific issues relevant to MS and related disorders, or should discussrecent publications. Comments should be less than 800 words and should reference the article(s)upon which the commentary is based.

Clinical Trial papersManuscripts detailing the results of clinical trials in MS and related disorders are encourage. The trialmethodology should account for all screened participants, and analyses should observe an intention-to -treat model where appropriate. All sources of funding for the study must be disclosed, and theinvolvement of the study sponsor must be detailed. Clinical trial manuscripts should be a maximumof 3500 words.

EditorialsThe Editors welcome suggestions for editorials which give personal and topical views on subjectswithin the Journal's area of interest. They should not normally exceed 1500 words in total, includingreferences.

Letters to the EditorsThese normally refer to articles previously published in the Journal. The Editors are also willing toconsider letters on subjects of direct relevance to the Journal's interest. Letters should not exceed1000 words in total and, where appropriate, must begin with the reference to the published articleabout which the author is commenting. Research letters should be submitted as 'letter to the Editors'

Review ArticlesReview papers are normally 4000-5000 words in total. Authors are advised to consult one of theEditors with an outline before submitting a review.

Contact details for submissionAuthors may send queries concerning the submission process, manuscript status, or journalprocedures to the Editorial Office at:MSARD, Editorial Office, ELSEVIER.E-mail: msard@elsevier.com

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Policy and ethicsThe work described in your article must have been carried out in accordance with The Codeof Ethics of the World Medical Association (Declaration of Helsinki) for experiments involvinghumans http://www.wma.net/en/30publications/10policies/b3/index.html; EU Directive 2010/63/EUfor animal experiments http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm;Uniform Requirements for manuscripts submitted to Biomedical journals http://www.icmje.org. Thismust be stated at an appropriate point in the article.

Conflict of interestAll authors are requested to disclose any actual or potential conflict of interest including any financial,personal or other relationships with other people or organizations within three years of beginning thesubmitted work that could inappropriately influence, or be perceived to influence, their work. Seealso http://www.elsevier.com/conflictsofinterest.

Submission declarationSubmission of an article implies that the work described has not been published previously (exceptin the form of an abstract or as part of a published lecture or academic thesis), that it is not underconsideration for publication elsewhere, that its publication is approved by all authors and tacitly orexplicitly by the responsible authorities where the work was carried out, and that, if accepted, itwill not be published elsewhere including electronically in the same form, in English or in any otherlanguage, without the written consent of the copyright-holder.

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Submit your articlePlease submit your article via http://ees.elsevier.com/msard/

RefereesPlease submit, with the manuscript, the names, addresses and e-mail addresses of three potentialreferees. Note that the editor retains the sole right to decide whether or not the suggested reviewersare used.

PREPARATIONUse of wordprocessing softwareIt is important that the file be saved in the native format of the wordprocessor used. The text shouldbe in single-column format. Keep the layout of the text as simple as possible. Most formatting codeswill be removed and replaced on processing the article. In particular, do not use the wordprocessor'soptions to justify text or to hyphenate words. However, do use bold face, italics, subscripts,superscripts etc. When preparing tables, if you are using a table grid, use only one grid for each

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individual table and not a grid for each row. If no grid is used, use tabs, not spaces, to align columns.The electronic text should be prepared in a way very similar to that of conventional manuscripts(see also the Guide to Publishing with Elsevier: http://www.elsevier.com/guidepublication). Note thatsource files of figures, tables and text graphics will be required whether or not you embed your figuresin the text. See also the section on Electronic artwork.To avoid unnecessary errors you are strongly advised to use the 'spell-check' and 'grammar-check'functions of your wordprocessor.

Article structureSubdivision - numbered sectionsDivide your article into clearly defined and numbered sections. Subsections should be numbered1.1 (then 1.1.1, 1.1.2, ...), 1.2, etc. (the abstract is not included in section numbering). Use thisnumbering also for internal cross-referencing: do not just refer to 'the text'. Any subsection may begiven a brief heading. Each heading should appear on its own separate line.

IntroductionState the objectives of the work and provide an adequate background, avoiding a detailed literaturesurvey or a summary of the results.

Material and methodsProvide sufficient detail to allow the work to be reproduced. Methods already published should beindicated by a reference: only relevant modifications should be described.

ResultsResults should be clear and concise.

DiscussionThis should explore the significance of the results of the work, not repeat them. A combined Resultsand Discussion section is often appropriate. Avoid extensive citations and discussion of publishedliterature.

ConclusionsThe main conclusions of the study may be presented in a short Conclusions section, which may standalone or form a subsection of a Discussion or Results and Discussion section.

AppendicesIf there is more than one appendix, they should be identified as A, B, etc. Formulae and equations inappendices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent appendix,Eq. (B.1) and so on. Similarly for tables and figures: Table A.1; Fig. A.1, etc.

Essential title page information• Title. Concise and informative. Titles are often used in information-retrieval systems. Avoidabbreviations and formulae where possible.• Author names and affiliations. Where the family name may be ambiguous (e.g., a doublename), please indicate this clearly. Present the authors' affiliation addresses (where the actual workwas done) below the names. Indicate all affiliations with a lower-case superscript letter immediatelyafter the author's name and in front of the appropriate address. Provide the full postal address ofeach affiliation, including the country name and, if available, the e-mail address of each author.• Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeingand publication, also post-publication. Ensure that telephone and fax numbers (with countryand area code) are provided in addition to the e-mail address and the complete postaladdress. Contact details must be kept up to date by the corresponding author.• Present/permanent address. If an author has moved since the work described in the articlewas done, or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicatedas a footnote to that author's name. The address at which the author actually did the work must beretained as the main, affiliation address. Superscript Arabic numerals are used for such footnotes.

AbstractA concise and factual abstract is required. The abstract should state briefly the purpose of theresearch, the principal results and major conclusions. An abstract is often presented separately fromthe article, so it must be able to stand alone. For this reason, References should be avoided, but ifessential, then cite the author(s) and year(s). Also, non-standard or uncommon abbreviations shouldbe avoided, but if essential they must be defined at their first mention in the abstract itself.

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Graphical abstractA Graphical abstract is optional and should summarize the contents of the article in a concise, pictorialform designed to capture the attention of a wide readership online. Authors must provide imagesthat clearly represent the work described in the article. Graphical abstracts should be submitted as aseparate file in the online submission system. Image size: Please provide an image with a minimumof 531 × 1328 pixels (h × w) or proportionally more. The image should be readable at a size of 5 ×13 cm using a regular screen resolution of 96 dpi. Preferred file types: TIFF, EPS, PDF or MS Officefiles. See http://www.elsevier.com/graphicalabstracts for examples.Authors can make use of Elsevier's Illustration and Enhancement service to ensure the bestpresentation of their images also in accordance with all technical requirements: Illustration Service.

HighlightsHighlights are a short collection of bullet points that convey the core findings of the article. Highlightsare optional and should be submitted in a separate file in the online submission system. Please use'Highlights' in the file name and include 3 to 5 bullet points (maximum 85 characters, including spaces,per bullet point). See http://www.elsevier.com/highlights for examples.

KeywordsImmediately after the abstract, provide a maximum of 6 keywords, using American spelling andavoiding general and plural terms and multiple concepts (avoid, for example, 'and', 'of'). Be sparingwith abbreviations: only abbreviations firmly established in the field may be eligible. These keywordswill be used for indexing purposes.

AbbreviationsDefine abbreviations that are not standard in this field in a footnote to be placed on the first pageof the article. Such abbreviations that are unavoidable in the abstract must be defined at their firstmention there, as well as in the footnote. Ensure consistency of abbreviations throughout the article.

AcknowledgementsCollate acknowledgements in a separate section at the end of the article before the references and donot, therefore, include them on the title page, as a footnote to the title or otherwise. List here thoseindividuals who provided help during the research (e.g., providing language help, writing assistanceor proof reading the article, etc.).

Math formulaePresent simple formulae in the line of normal text where possible and use the solidus (/) instead ofa horizontal line for small fractional terms, e.g., X/Y. In principle, variables are to be presented initalics. Powers of e are often more conveniently denoted by exp. Number consecutively any equationsthat have to be displayed separately from the text (if referred to explicitly in the text).

FootnotesFootnotes should be used sparingly. Number them consecutively throughout the article, usingsuperscript Arabic numbers. Many wordprocessors build footnotes into the text, and this feature maybe used. Should this not be the case, indicate the position of footnotes in the text and present thefootnotes themselves separately at the end of the article. Do not include footnotes in the Referencelist.Table footnotesIndicate each footnote in a table with a superscript lowercase letter.

ArtworkElectronic artworkGeneral points• Make sure you use uniform lettering and sizing of your original artwork.• Save text in illustrations as 'graphics' or enclose the font.• Only use the following fonts in your illustrations: Arial, Courier, Times, Symbol.• Number the illustrations according to their sequence in the text.• Use a logical naming convention for your artwork files.• Provide captions to illustrations separately.• Produce images near to the desired size of the printed version.• Submit each figure as a separate file.

A detailed guide on electronic artwork is available on our website:http://www.elsevier.com/artworkinstructions

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You are urged to visit this site; some excerpts from the detailed information are given here.FormatsRegardless of the application used, when your electronic artwork is finalised, please 'save as' orconvert the images to one of the following formats (note the resolution requirements for line drawings,halftones, and line/halftone combinations given below):EPS: Vector drawings. Embed the font or save the text as 'graphics'.TIFF: Color or grayscale photographs (halftones): always use a minimum of 300 dpi.TIFF: Bitmapped line drawings: use a minimum of 1000 dpi.TIFF: Combinations bitmapped line/half-tone (color or grayscale): a minimum of 500 dpi is required.If your electronic artwork is created in a Microsoft Office application (Word, PowerPoint, Excel) thenplease supply 'as is'.Please do not:• Supply files that are optimised for screen use (e.g., GIF, BMP, PICT, WPG); the resolution is too low;• Supply files that are too low in resolution;• Submit graphics that are disproportionately large for the content.

Color artworkPlease make sure that artwork files are in an acceptable format (TIFF, EPS or MS Office files) and withthe correct resolution. If, together with your accepted article, you submit usable color figures thenElsevier will ensure, at no additional charge, that these figures will appear in color on the Web (e.g.,ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in colorin the printed version. For color reproduction in print, you will receive information regardingthe costs from Elsevier after receipt of your accepted article. Please indicate your preferencefor color: in print or on the Web only. For further information on the preparation of electronic artwork,please see http://www.elsevier.com/artworkinstructions.Please note: Because of technical complications which can arise by converting color figures to 'grayscale' (for the printed version should you not opt for color in print) please submit in addition usableblack and white versions of all the color illustrations.

Figure captionsEnsure that each illustration has a caption. Supply captions separately, not attached to the figure. Acaption should comprise a brief title (not on the figure itself) and a description of the illustration. Keeptext in the illustrations themselves to a minimum but explain all symbols and abbreviations used.

TablesNumber tables consecutively in accordance with their appearance in the text. Place footnotes to tablesbelow the table body and indicate them with superscript lowercase letters. Avoid vertical rules. Besparing in the use of tables and ensure that the data presented in tables do not duplicate resultsdescribed elsewhere in the article.

ReferencesCitation in textPlease ensure that every reference cited in the text is also present in the reference list (and viceversa). Any references cited in the abstract must be given in full. Unpublished results and personalcommunications are not recommended in the reference list, but may be mentioned in the text. If thesereferences are included in the reference list they should follow the standard reference style of thejournal and should include a substitution of the publication date with either 'Unpublished results' or'Personal communication'. Citation of a reference as 'in press' implies that the item has been acceptedfor publication.

Reference management softwareThis journal has standard templates available in key reference managementpackages EndNote http://www.endnote.com/support/enstyles.asp and Reference Managerhttp://refman.com/support/rmstyles.asp. Using plug-ins to wordprocessing packages, authors onlyneed to select the appropriate journal template when preparing their article and the list of referencesand citations to these will be formatted according to the journal style which is described below.

Web referencesAs a minimum, the full URL should be given and the date when the reference was last accessed. Anyfurther information, if known (DOI, author names, dates, reference to a source publication, etc.),should also be given. Web references can be listed separately (e.g., after the reference list) under adifferent heading if desired, or can be included in the reference list.

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References in a special issuePlease ensure that the words 'this issue' are added to any references in the list (and any citations inthe text) to other articles in the same Special Issue.

Reference styleText: All citations in the text should refer to:1. Single author: the author's name (without initials, unless there is ambiguity) and the year ofpublication;2. Two authors: both authors' names and the year of publication;3. Three or more authors: first author's name followed by 'et al.' and the year of publication.Citations may be made directly (or parenthetically). Groups of references should be listed firstalphabetically, then chronologically.Examples: 'as demonstrated in wheat (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Krameret al. (2010) have recently shown ....'List: References should be arranged first alphabetically and then further sorted chronologically ifnecessary. More than one reference from the same author(s) in the same year must be identified bythe letters 'a', 'b', 'c', etc., placed after the year of publication.Examples:Reference to a journal publication:Van der Geer J, Hanraads JAJ, Lupton RA. The art of writing a scientific article. J Sci Commun2010;163:51–9.Reference to a book:Strunk Jr W, White EB. The elements of style. 4th ed. New York: Longman; 1979.Reference to a chapter in an edited book:Mettam GR, Adams LB. How to prepare an electronic version of your article. In: Jones BS, Smith RZ,editors. Introduction to the electronic age. New York: E-Publishing Inc; 2009. p. 281–304.Note shortened form for last page number. e.g., 51–9, and that for more than 6 authors the first 6should be listed followed by "et al." For further details you are referred to "Uniform Requirementsfor Manuscripts submitted to Biomedical Journals" (J Am Med Assoc 1997;277:927–34) (see alsohttp://www.nlm.nih.gov/bsd/uniform_requirements.html).

Journal abbreviations sourceJournal names should be abbreviated according toIndex Medicus journal abbreviations: http://www.nlm.nih.gov/tsd/serials/lji.html;List of title word abbreviations: http://www.issn.org/2-22661-LTWA-online.php;CAS (Chemical Abstracts Service): http://www.cas.org/sent.html.

Video dataElsevier accepts video material and animation sequences to support and enhance your scientificresearch. Authors who have video or animation files that they wish to submit with their article arestrongly encouraged to include these within the body of the article. This can be done in the same wayas a figure or table by referring to the video or animation content and noting in the body text where itshould be placed. All submitted files should be properly labeled so that they directly relate to the videofile's content. In order to ensure that your video or animation material is directly usable, please providethe files in one of our recommended file formats with a preferred maximum size of 50 MB. Video andanimation files supplied will be published online in the electronic version of your article in ElsevierWeb products, including ScienceDirect: http://www.sciencedirect.com. Please supply 'stills' with yourfiles: you can choose any frame from the video or animation or make a separate image. These willbe used instead of standard icons and will personalize the link to your video data. For more detailedinstructions please visit our video instruction pages at http://www.elsevier.com/artworkinstructions.Note: since video and animation cannot be embedded in the print version of the journal, pleaseprovide text for both the electronic and the print version for the portions of the article that refer tothis content.

Supplementary dataElsevier accepts electronic supplementary material to support and enhance your scientific research.Supplementary files offer the author additional possibilities to publish supporting applications, high-resolution images, background datasets, sound clips and more. Supplementary files supplied will bepublished online alongside the electronic version of your article in Elsevier Web products, includingScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material isdirectly usable, please provide the data in one of our recommended file formats. Authors should

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submit the material in electronic format together with the article and supply a concise and descriptivecaption for each file. For more detailed instructions please visit our artwork instruction pages athttp://www.elsevier.com/artworkinstructions.

Submission checklistThe following list will be useful during the final checking of an article prior to sending it to the journalfor review. Please consult this Guide for Authors for further details of any item.Ensure that the following items are present:One author has been designated as the corresponding author with contact details:• E-mail address• Full postal address• Telephone and fax numbersAll necessary files have been uploaded, and contain:• Keywords• All figure captions• All tables (including title, description, footnotes)Further considerations• Manuscript has been 'spell-checked' and 'grammar-checked'• References are in the correct format for this journal• All references mentioned in the Reference list are cited in the text, and vice versa• Permission has been obtained for use of copyrighted material from other sources (including the Web)• Color figures are clearly marked as being intended for color reproduction on the Web (free of charge)and in print, or to be reproduced in color on the Web (free of charge) and in black-and-white in print• If only color on the Web is required, black-and-white versions of the figures are also supplied forprinting purposesFor any further information please visit our customer support site at http://support.elsevier.com.

AFTER ACCEPTANCEUse of the Digital Object IdentifierThe Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOIconsists of a unique alpha-numeric character string which is assigned to a document by the publisherupon the initial electronic publication. The assigned DOI never changes. Therefore, it is an idealmedium for citing a document, particularly 'Articles in press' because they have not yet received theirfull bibliographic information. The correct format for citing a DOI is shown as follows (example takenfrom a document in the journal Physics Letters B):doi:10.1016/j.physletb.2010.09.059When you use the DOI to create URL hyperlinks to documents on the web, the DOIs are guaranteednever to change.

ProofsOne set of page proofs (as PDF files) will be sent by e-mail to the corresponding author (if we donot have an e-mail address then paper proofs will be sent by post) or, a link will be provided inthe e-mail so that authors can download the files themselves. Elsevier now provides authors withPDF proofs which can be annotated; for this you will need to download Adobe Reader version 7 (orhigher) available free from http://get.adobe.com/reader. Instructions on how to annotate PDF fileswill accompany the proofs (also given online). The exact system requirements are given at the Adobesite: http://www.adobe.com/products/reader/tech-specs.html.If you do not wish to use the PDF annotations function, you may list the corrections (includingreplies to the Query Form) and return them to Elsevier in an e-mail. Please list your correctionsquoting line number. If, for any reason, this is not possible, then mark the corrections and any othercomments (including replies to the Query Form) on a printout of your proof and return by fax, or scanthe pages and e-mail, or by post. Please use this proof only for checking the typesetting, editing,completeness and correctness of the text, tables and figures. Significant changes to the article asaccepted for publication will only be considered at this stage with permission from the Editor. We willdo everything possible to get your article published quickly and accurately – please let us have all yourcorrections within 48 hours. It is important to ensure that all corrections are sent back to us in onecommunication: please check carefully before replying, as inclusion of any subsequent correctionscannot be guaranteed. Proofreading is solely your responsibility. Note that Elsevier may proceed withthe publication of your article if no response is received.

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OffprintsThe corresponding author, at no cost, will be provided with a PDF file of the article via e-mail. For anextra charge, paper offprints can be ordered via the offprint order form which is sent once the articleis accepted for publication. The PDF file is a watermarked version of the published article and includesa cover sheet with the journal cover image and a disclaimer outlining the terms and conditions of use.

AUTHOR INQUIRIESFor inquiries relating to the submission of articles (including electronic submission) pleasevisit this journal's homepage. Contact details for questions arising after acceptance ofan article, especially those relating to proofs, will be provided by the publisher. Youcan track accepted articles at http://www.elsevier.com/trackarticle. You can also checkour Author FAQs (http://www.elsevier.com/authorFAQ) and/or contact Customer Support viahttp://support.elsevier.com.

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