Gus a o Miguel da Rocha Rod igues
The ole o bioma ke s in he diagnosis o Mul iple scle osis
2011/2012
ma ço, 2012
Gus a o Miguel da Rocha Rod igues
The ole o bioma ke s in he diagnosis o Mul iple Scle osis
Mes ado In eg ado em Medicina
Á ea: Neu ologia
T abalho e e uado sob a O ien ação de:
D a. Joana da C uz Guima ães Fe ei a de Almeida
T abalho o ganizado de aco do com as no mas da e is a:
Mul iple Scle osis and ela ed Diso de s
ma ço, 2012
IIIPORTO
P oje o de Opção do 6° ano - DECLARAÇÃO DE INTEGRIDADE
!FMUP!
FACULDADE DE MEDICINA
UNIVERSIDADE DO PORTO
UNIDADE CURRICUlAR
PROJETO DE
o e c z o
DISSERTAÇÃO
MONOGRAFIA
Eu, Gus a o Miguel da Rocha Rod igues, abaixo assinado, nOmecanog á ico 060801225, es udan e do
6
0
ano do Mes ado In eg ado em Medicina, na Faculdade de Medicina da Uni e sidade do Po o,
decla o e a uado com absolu a in eg idade na elabo ação des e p oje o de opção.
Nes e sen ido, con i mo que NÃO inco i em plágio (a o pelo qual um indi íduo, mesmo po omissão,
assume a au o ia de um de e minado abalho in elec ual, ou pa es dele). Mais decla o que odas as
ases que e i ei de abalhos an e io es pe encen es a ou os au o es, o am e e enciadas, ou
edigidas com no as pala as, endo colocado, nes e caso, a ci ação da on e bibliog á ica.
Faculdade de Medicina da Uni e sidade do Po o,
1
b
/ó'? /
~o
1),
Assina u a:
DlpORTO
FMUP FACULDADE DE MEDICINA
UNIVERSIDADE DO PORTO
P ojec o de Opção do 6° ano - DECLARAÇÃODEREPRODUÇÃO
Nome: Gus a o Miguel da Rocha Rod igues
Ende eço elec ónico: gus a o [email p o ec ed] Tele one ou Telemó el: 912248477
Núme o do Bilhe e de Iden idade: 13213739
Tí ulo da Monog a ia:
The ole o bioma ke s in he diagnosis o Mul iple Scle osis
O ien ado :
D a. Joana da C uz Guima ães Fe ei a de Almeida
Ano de conclusão: 2012
Designação da á ea do p ojec o:
Neu ologia
É
au o izada a ep odução in eg al des a Monog a ia pa a e ei os de in es igação e de di ulgação
pedagógica, em p og amas e p ojec os coo denados pela FMUP.
Faculdade de Medicina da Uni e sidade do Po o,
0~
lo
11
Assina u a:
L~josJ{)
Dedica ó ia
Dedico es e abalho á minha amília pela disponibilidade e apoio incondicional
ao longo de odo o meu pe cu so académico.

MESTRADO INTEGRADO EM MEDICINA
Ano le i o: 2011/2012
Nome do(a) Es udan e: Gus a o Miguel da Rocha Rod igues
O ien ado (a): D a. Joana da C uz Guima ães Fe ei a de Almeida
Á ea do P oje o: Neu ologia
Tí ulo do P oje o: The ole o bioma ke s in he diagnosis o Mul iple Scle osis.
Resumo: Escle ose Múl ipla (EM) é uma doença neu ológica in lama ó ia desmielinizan e
impo an e de ido à sua incidência, c onicidade e maio p e alência em adul os jo ens. No
es udo inicial de uma doença desmielinizan e do Sis ema Ne oso Cen al exis e a necessidade
de encon a bioma cado es que possam melho disc imina a EM de ou as pa ologias.
A ualmen e, apenas as bandas oligoclonais (BOC) são u ilizadas. Es a e isão p e ende, a a és
de uma pesquisa na base de dados PubMed, examina a li e a u a publicada nos úl imos 5
anos, sob e o papel dos bioma cado es no diagnós ico da escle ose múl ipla. O uso de
ma cado es no líquido encé alo- aquidiano, aumen a a especi icidade e alo p edi i o
nega i o, diminuindo os possí eis alsos posi i os da, mui o sensí el, essonância magné ica.
As BOC con i ma am o seu alo , apoiando o diagnós ico quando p esen es e suge indo a
e isão do mesmo quando ausen es, mos ando con udo algumas limi ações: a iabilidade
in e populacional, labo a o ial e me odológica, con o é sia sob e o uso do índice IgG, a
in e p e ação de uma única BOC e a elação das BOC com a EM. Enquan o o í us Eps ein-Ba
e os an ico pos an imielina alha am quando es ados, as cadeias le es li es K demons a am
esul ados p omisso es, especialmen e, como odos os bioma cado es, quando combinadas
com essonância magné ica. Na p e isão de con e são do sínd ome clínico isolado pa a EM, as
BOC con i ma am o seu papel, enquan o CXC quimiocina mo i 13 e a eação MRZ mos a am
bons alo es p edi i os, e os ma cado es de lesão axonal e an ico pos con a a mielina
alha am. Além da de eção de an ico pos aquapo ina-4 e a eação MRZ no líquido
cé alo aquidiano a de eção de N-ace il aspa a o no sangue mos a am bons esul ados a
dis ingui EM de neu omieli e ó ica. Há uma p ocu a pa a desen ol e mé odos au oma izados
pa a es uda bioma cado es, e o desen ol imen o de écnicas p o eómicas é p omisso pa a a
iden i icação de no os al os molecula es pa a ensaios de diagnós ico e e apêu ica.
Concluindo, o p incipal con ibu o dos bioma cado es no diagós ico de EM, consis e no
aumen o de especi icidade e alo p edi i o. É necessá io co igi qualque possí el e o
me odológico, en ando es anda diza ao máximo de modo a ob e esul ados mais
consis en es. Apesa dos es o ços na pesquisa de no os bioma cado es é necessá io
comp eende melho a EM de modo a encon a um es e simpli icado.
Pala as-cha e: Escle ose Múl ipla, Sínd ome clínico isolado, bandas oligoclonais, líquido encé alo-
aquidiano, bioma cado es, diagnós ico.
The ole o bioma ke s in he diagnosis o Mul iple Scle osis
Rod igues, Gus a o Facul y o Medicine o Po o Uni e si y (FMUP)*
Neu ology Depa men , Facul y o Medicine, Uni e si y o Po o, Hospi al de São João,
Alameda P o esso He nâni Mon ei o, 4200, Po o, Po ugal Facul y o Medicine o Po o Uni e si y
Telephone and Fax: + 00351 225511200
*Co esponding au ho , P aça, 135 Vila No a, 4465-767 P aia da Vi ó ia, Aço es - Po ugal
E-mail: Gus a o_ od igues17@ho mail.com; Telephone: +00351 912248477
Abs ac
Mul iple Scle osis (MS) is an impo an in lamma o y demyelina i e neu ologic
disease, due o i s, incidence, ch onici y and highe p e alence among young adul s. In
he ini ial app oach o a demyelina i e disease o he Cen al Ne ous Sys em he e is a
u ge o bioma ke s o bes disc imina e MS om o he diseases. Despi e he b oad
ange o bioma ke s udies, only oligoclonal bands (OCB) a e used by ou ine in
di e en ial diagnosis. This e iew aims o access he published li e a u e in he las 5
yea s ( ough a sea ch in PubMed da abase), ega ding he ole o bioma ke s in he
diagnosis o MS. Rega ding MS di e en ial diagnosis, he use o ce eb ospinal luid
ma ke s (CSF) inc eases speci ici y and nega i e p edic i e, diminishing he possible
alse posi i es om he high sensible magne ic esonance imaging (MRI). OCB
con i med hey alue, suppo ing he MS diagnosis when p esen and sugges ing
e ision o his, bu some limi a ions appea ed ega ding in e -popula ion, in e -
labo a o ies and in e -me hods de ec ion a iabili y and he e’s con o e sy conce ning
he cu en use o IgGindex, in e p e a ion o a single weak OCB and he ela ion o
OCB wi h MS. While Eps ein Ba Vi us (EBV) and an i-myelin an ibodies ailed when
es ed, Kappa ee ligh chains (KFLC) pe o med in a p omising way, specially, as all
bioma ke s, when combined wi h MRI.
In his e iew, OCB con i med hey impo ance while C-X-C mo i chemokine
13 (CXCL13) and MRZ eac ion showed good p edic i e alues esul s while axonal
damage ma ke s and an ibodies agains myelin didn’ pe o m well, p edic ing clinically
isola ed synd ome (CIS) con e sion o MS. Besides aquapo in-4 an ibody de ec ion and
MRZ eac ion in CSF, N-ace yl aspa a e de ec ion in se um showed good esul s
dis inguishing MS om neu omyeli is op ica (NMO). The e has been a s uggle o
de elop s anda d easy au oma ed me hods o bioma ke s udying and he de elopmen
o mo e sensi i e p o eomic echniques is e y p omising o he iden i ica ion o new
molecula a ge s o diagnos ic assays and he apies p e en ing axonal damage.
Concluding, bioma ke s majo con ibu e is in e ms o Speci ici y and
p edic i e alue. Me hodological s anda diza ion, co ec ing any possible e o , is i al
o a mo e consis en esea ch. Despi e he e o s in his ield, an accu a e simple es is
only possible when he pa hophysiology o MS is be e unde s ood.
Keywo ds: Mul iple Scle osis, Clinically isola ed synd ome, oligoclonal bands,
ce eb ospinal luid, bioma ke , diagnosis.
1. In oducion
1.1 His o ic no e and s a e o a
Mul iple Scle osis (MS) is an impo an in lamma o y demyelina i e neu ologic
disease, due o i s incidence, ch onici y and endency o a ec young adul s.(1) MS
ea u es a b oad he e ogenei y o clinical, his opa hological and immunological
pheno ypes, which u ges a mo e accu a e de ining o pa ien s by biological ma ke s ha
e lec he unde lying disease p ocess and allow he p edic ion o disease cou se and
ea men esponse.(2, 3) Thus, se ious e o s a e made in he ield o bioma ke s o
imp o e he diagnos ic disc imina ion, o de e mine p ognos ic ac o s, and o iden i y
ma ke s o moni o he clinical cou se and esponse o disease-modi ying he apies.(3)
S a ing wi h Cha co (1868) and his iad (nys agmus, in en ion emo and
scanning speech), he diagnos ic c i e ia o MS ha e e ol ed o e lec he
b eak h oughs in ou unde s anding o he disease and he de elopmen o new
diagnos ic echniques, changing om pu ely clinical c i e ia o inc easing dependency
upon imaging o he cen al ne ous sys em (CNS). Only in 1954 he i s ue
diagnos ic scheme appea ed, published by Allison and Milla desc ibing h ee
ca ego ies: ea ly, p obable and possible dissemina ed scle osis.(1)
The e was a need o eliable and ep oducible diagnos ic c i e ia in o de o
p o ide accu a e epidemiological s udies and he apeu ic ials. A majo s ep in ha
di ec ion occu ed in 1965, when B oman e al. ou lined he p inciples ha we e he
basis o ollowing schemes.(4) They consis ed o age o onse , he empi ic signi icance
o symp oms o an MS diagnosis, he mul iplici y o lesions, he numbe o bou s, and
he amilial “ ecu ence” o MS. The esul s o he newly desc ibed elec opho e ic
examina ion o ce eb ospinal luid (CSF) p o ein, he p ecu so o oligoclonal bands
(OCB), we e also in oduced. Addi ionally, all o he possible diagnoses we e o be
conside ed and, i possible, excluded. Fo he i s ime was s a ed he win p inciples o
dissemina ion in ime and in space, ha o his day emain he undamen al c i e ia o
he diagnosis o MS.(4) The u he need o accu a e diagnosis c i e ia, lead o he
o ma ion o an expe panel and elabo a ion o he Schumache e al. c i e ia, used
wo ldwide.(5)
A adap a ion o he Schumache c i e ia eme ged om a wo ldwide
in es iga ion by Baue ha was he i s o include CSF OCB.(1)
syn hesis is di ec ed agains di e se an igens. An associa ion o hese an ibodies wi h
he cause o MS is s ill missing.(2) Eps ein-Ba i us (EBV) in ec ion has been
suspec ed as an ini ial igge o au oimmuni y in MS and, hus, e idence o EBV
in ec ion could ha e ele ance o MS diagnosis.(10)
A numbe o s udies ha e shown ha almos 100% o MS pa ien s ha e e idence
o p io EBV in ec ion, whe eas app oxima ely 90% o heal hy adul s also ha e
e idence o EBV in ec ion.(40) The e o e, he p esence o EBV an ibodies is no
conside ed o be diagnos ic ele an . Fu he mo e, CSF eac i i y o EBV an igens in
MS compa ed wi h heal hy con ols has no yielded any speci ic associa ions.(41)
Cu en in es iga ions a e ocused on de e mining whe he MS pa ien s ha e
an ibodies o unique de e minan s o EBV, o i i al-clea ance esponses o EBV
in ec ion in MS pa ien s a e abno mal.(42) I hese in es iga ions a e ui ul, hey could
ha e applica ion as diagnos ic ma ke s.(10)
Addi ionally, Ba os e al. (2007) epo ed no di e ence in an ibodies agains
ligh neu o ilamen s in all sub- ypes o MS and o he diseases.(43)
Nogo-A, an inhibi o o axonal egene a ion, is a pe ec example o he
inconsis en and con adic o y da a ga he ed in his e iew. Ju ewicz e al. (2007)
epo ed ha CSF Nogo-A may be speci ic (100%) o MS and i s p esence may p edic
ailu e o axonal egene a ion wi hin he CNS. Nogo-A was ound in CSF o 96% o
MS pa ien s and wasn’ p esen in meningo-encephalomyeli is, con ol subjec s wi he
neu ologic, and au oimmune diseases.(44) These indings lacked con i ma ion by
William e al. (2008) wi h he same me hodology.(45) Co espondence be ween au ho s,
showed high disco dance in his subjec .
Nume ous s udies ha e demons a ed ele a ed kappa ee ligh chains (KFCLs)
in CSF o MS pa ien s. Howe e , so a only small coho s ha e been examined, and
gene ally only h ough quali a i e KFLCs analysis. De e mining a single quan i a i e
diagnos ic FLC pa ame e (KFLC index) by nephelome y (highly au oma ed) and IEF,
P esslaue e al. (2008) showed highe sensi i i y (96% s 91%) bu lowe speci ici y
(86% s 92%) compa ed wi h OCB, bu was a mo e accu a e han IgG index.
Addi ionally, he e was no co ela ion be ween MS ype, EDSS sco e, disease du a ion,
o numbe /localiza ion o MRI lesions and he alue o KFLC o KFLC index.(14)
The combined use o h ee FLC indices accoun ing o monome ic FLC –k le el
and k/λ a io alues in he CSF and se um was ound o be o p omising diagnos ic
impo ance o di e en ia ion o MS om o he non-MS neu ological diseases

(speci ici y 93%, sensi i i y 100% s OCB 76% and 87% in he same coho ).(46) In
con as o he OCB es , whe e he esul is in e p e ed as “posi i e”, “nega i e” o
“ ace”, his me hod p o ides quan i a i e e alua ion o ela i e amoun s o FLCs, as a
measu e o he se e i y o in a hecal immunoglobulin syn hesis. This p ocedu e o
FLC analysis can se e as a basis o a new diagnos ic es o MS. The desc ibed
me hod is inexpensi e, i equi es no special equi emen , and may be applied in clinical
labo a o ies.(46) This p ocedu e also allows p epa a ion o samples in a d y s a e, ha is
help ul o p olonged s o age and anspo a ion o CSF and se um samples p io o
hei analysis.(46)
As i is measu ed by au oma ed, ou inely a ailable labo a o y me hods, KFLC
quan i ica ion can p o ide a apid and ep oducible indica ion o in a hecal
immunological p ocesses suppo ing cu en MS diagnos ic c i e ia.(14) These esul s
s ongly sugges ha he KFLC index should be pa o he ou ine MS diagnos ic
algo i hm.(14, 46) Addi ional LFLC index de e mina ion can suppo he exclusion o
al e na i e diagnoses. Al e na i e diagnoses should be conside ed i hese a e associa ed
wi h concu en signi ican ele a ion o LFLC le els.(14)
Ele a ed CSF an i-MBP an ibodies co ela e well wi h acu e myelin damage in
he CNS, bu a e no speci ic o MS. Rep oducible quan i ica ion o MBP has been
di icul since immunoassays a e no s anda dized and no comme cially a ailable.(3)
CSF MBP has lacked speci ici y in mos s udies. The es was ecen ly e-
e alua ed by Tian e al. (2009) who epo ed a diagnos ic sensi i i y o 83.7% and
speci ici y o 78.3%. These esul s need o be con i med in u he s udies.(16, 47)
P elimina y s udies, app oaching Gelsolin, leukemia inhibi o y ac o and 7-
ke ocholes e ol, showed good esul , needing u he e alua ion.(48-50)
In able 1, a e displayed o he bioma ke s e ised in his sea ch.
3.1.3 Con e sion o Clinically Isola ed Synd ome o Clinically De ini i e Mul iple
Scle osis
In a signi ican pe cen age o pa ien s who la e de elop MS, he disease ini ially
p esen s wi h an acu e o sub-acu e episode o neu ological symp oms due o a single
demyelina ing lesion, known as clinically isola ed synd ome (CIS). P edic he isk o
u u e e en s, con i ming he MS diagnosis is c ucial o begin an impo an ea ly
ea men . In he las ew yea s, he main ocus has been on diagnos ic sensi i i y in
o de o ea ly de ec he pa ien s in need o ea ly he apeu ics, esul ing he e o e in
possible o e diagnoses.(51) In his con ex , he mos ele an s a is ical pa ame e s o
p edic con e sion om CIS o MS a e posi i e p edic i e alue (PPV) and nega i e
p edic i e alue (NPV).(51) MRI is he main exam used o his p edic ion bu he e is
an ongoing sea ch o accu a e bioma ke s o his con e sion.(52, 53)
Tin o é e al. (2008) showed ha de ec ion o OCB in CSF app oxima ely
doubles he isk o ha ing a second a ack, au onomously o MRI, bu does no seem o
in luence he de elopmen o disabili y.(54) This highe isk is mo e signi ican when
MRI is no mal o when he e is no MRI c i e ia o dissemina ion in space (DIS) (4%
o pa ien s wi h no mal MRI and nega i e OCB con e ed and 23% o hose wi h
no mal MRI and posi i e OCB con e ed).(54)
The associa ion o OCB o MRI maximizes he isk; he e o e hey can be used
oge he o be e p edic con e sion o CDMS. OCB associa ed wi h numbe o
Ba kho c i e ia and numbe o lesions bu he absence o OCB should sugges
ex ending he diagnosis wo kup.(15, 17)
Simila esul s we e ound by o he displayed on able 2.(15, 55-58)
Addi ionally, wo o hese s udies sugges ed ha pa ien s wi h OCB con e s as e o
CDMS (55, 57), especially when IgM is associa ed.(55)
B e sschneide e al. (2010) in a p ospec i e pilo s udy wi h 91 pa ien s and 2
yea s o ollow-up, showed ha he p esence o C-X-C mo i chemokine 13 (CXCL13)
in CSF has a be e PPV (70%, augmen ed o 80% wi h Ba kho c i e ia) han OCB,
Measles, Rubella and Va icella zos e eac ion (MRZR, in a- hecal syn hesis o speci ic
an i i al IgG an ibodies ha suppo s he diagnosis) o Ba kho c i e ia.(59)
A la ge clinical case-con ol coho , including he whole spec um o MS o ms,
as well as la ge numbe s o in lamma o y and non-in lamma o y subjec s wi h o he
neu ologic diseases con i med he po en ial ole o CXCL13 as a MS disease ac i i y
ma ke . CXCL13 co ela ed wi h elapse a e, Expanded Disabili y S a us Scale
(EDSS), numbe o lesion on MRI and esponse o ea men .(60) Howe e , his
bioma ke is no a disease-speci ic bioma ke , bu showed a close ela ionship wi h
OCB and CSF cell coun s.(59-61)
Rega ding an ibodies agains myelin, myelin oligodend ocy e glycop o ein
(MOG), and myelin basic p o ein (MBP), disco dan unexplained esul s ha e
appea ed.(62) Co ela ion anged om highly signi ican (63), signi ican (64, 65), o
no signi ican a all (62, 66). I is impo an o no e ha hese con o e sial esul s we e
ob ained wi h he same ype o analysis o an imyelin an ibodies, immunoblo ing.
Thus, hese di e en esul s may p ima ily e lec di e ences in s udy coho s a he
han me hodological p oblems.(2) Cu en ly he e is no p o en alue in he diagnos ic
and p ognos ic u ili y o CSF an i-myelin an ibodies in pa ien s wi h CIS.(16)
CSF o pa ien s wi h MS is cha ac e ized by a polyspeci ic, in a hecal B-cell
esponse wi h a p ominen an ibody p oduc ion agains neu o opic i uses like measles,
ubella and a icella zos e .(51) A p ospec i e s udy wi h 89 CIS pa ien s compa ing
MRZR wi h OCB and MRI lesion load showed ha MRZR was signi ican ly mo e
equen in CIS ha de eloped MS du ing ollow-up. Thei da a showed MRZR o ha e
a high PPV. MRZ sco e, p oduced acco ding o he di e en p ognos ic ele ance o
he h ee an ibody included in MRZR, had he bes single PPV (79%), augmen ed when
added o MRI (≥2 lesions, 91%).(51)
An impo an d awback o MRZR is ha he e’s no wo kable combined assay o
de e mine hese an ibodies. The e o e, ano he s udy om he same au ho s, ollowing
121 indi iduals o 2 yea s alues mo e CXCL13 de e mina ion (PPV o 70%).(59)
Axonal damage ma ke s ( au and N HSMI35) we e measu ed by B e schneide
e al. (2006) in o de o access hei powe o p edic con e sion om CIS o MS. CSF
au and N HSMI35 we e signi ican ly ele a ed in CIS compa ed o con ols, and
co ela ed wi h disease ac i i y.(67) The sensi i i y p edic ing he con e sion om CIS
o MS was highe o he combina ion o CSF ma ke s (ei he au o N HSMI35
ele a ed) han o MRI (40 e sus 34%), bu could be u he inc eased o 60% i CSF
and MRI c i e ia we e combined. Simila ly, he combina ion o au and N HSMI35
showed highe speci ici y (94%) han MRI (82%). Tau and N HSMI35, acco ding o
his s udy, a e aluable bioma ke s o axonal damage in he CIS pa ien s.(67)
O he molecules showed esul s ha need u he e alua ion: inc eased CSF
cys a in C (in con as wi h o me s udies), dissocia ion 14-3-3/ au p o ein in CSF,
blood and CSF whi e blood cell coun and neu o ilamen ligh .(68-71)
In o he hand, IgM and human leukocy e an igen G ailed o be ele an in he
e alua ion o CIS.(72, 73)
3.1.4 Mul iple Scle osis s Neu omyeli is op ica (NMO)
An example o success in esea ching bioma ke s o di e en ial diagnosis o
MS is he aquapo in-4 an ibody. Recen ly, highly speci ic se um IgG an ibodies we e
de ec ed by immuno luo escence s aining o mouse b ain issue in a majo i y (73%) o
pa ien s wi h de ini e NMO and in 46% o pa ien s a high isk o NMO.(74) NMO-IgG
had 91% speci ici y o NMO and we e sugges ed o be a use ul diagnos ic bioma ke in
pa ien s wi h hei i s episode o myeli is.(75, 76) Mos ecen ly a numbe o
con i ma o y s udies om di e en labo a o ies using di e se assays and di e en
coho s o pa ien s we e published.(77-80)
A posi i e MRZR, de ined by a combina ion o a leas wo an ibody indices
was ound in 37/42 MS pa ien s, while only in 1/20 NMO pa ien s by Ja ius e al.
(2008) allowing he di e en ia ion be ween MS and NMO.(81)
To o ella e al. (2011) demons a ed ha MS pa ien s show highe se um and
CSF N-ace yl aspa a e (NAA) le els han NMO pa ien s, and highe se um NAA le els
han heal hy con ols. High se um NAA alues, exceeding he 95 h pe cen ile o se um
NAA alue in heal hy con ols, we e ound in all MS pa ien s and in none wi h NMO.
No di e ences in se um NAA le els we e ound be ween NMO and heal hy
con ols.(82) The e o e, se um and CSF NAA may be aluable in he diagnos ic wo kup
o di e en ia e MS om NMO.(82)
3.1.5 P o eomics
Mul iple p o eomic p elimina y s udies ca ied ou in he pas ew yea s ha e
e ealed po en ial bioma ke s candida es ha may no o he wise ha e been disco e ed
h ough unc ion-based app oaches.(10, 53, 83-88) Fo mos o hese candida e p o eins,
he pa hophysiological ele ance in MS emains o be cla i ied. Gene ally he disease
speci ici y o hese p o eins is a he low, possibly due o common pa hological
mechanisms unde lying di e en diseases.(53, 89)
A p oblem o CSF p o eome analysis is ha high-abundance p o eins, such as
albumin and immunoglobulin, may o e lay b ain-speci ic low-abundance CSF p o eins
in he elec opho esis. The e o e, CSF has o be p e-p ocessed o ex ac hose ex a-
p o eins, possibly esul ing in was ing p o eins ha a e bounded do albumin.(53, 85)
The ad an ages o mass spec ome y-based p o ein quan i ica ion a e p ecision,
sensi i i y, h oughpu and con enience.(83) An impo an aspec in u u e CSF
p o eome s udies is he s anda diza ion o sampling p o ocols, expe imen al design and
da a analysis, in o de o each consensus.(53)
Fu he s udies a e needed o be e cha ac e ize he co ela ion o hese new
candida e ma ke s wi h MS.(88-90)
4. Conclusion
Diagnosis o MS is cu en ly based on clinical p esen a ion, MRI indings, and
CSF examina ion (OCB and IgG index) and equi es he exclusion o al e na i e
diagnosis.(45) CSF is especially impo an in no making a diagnosis o MS; ha is, in
excluding o he condi ions ha mimic MS and as a p edic o o con e sion o CDMS.
As long as we do no know wha causes MS we will no ha e a simple diagnos ic es
.(91) To unde s and he ela ion o ma ke concen a ions in body luids and
neu opa hological p ocesses, he unde lying p ocess need u he in es iga ion.(92)
Wi h inc easing sensi i i y and esolu ion o analy ical me hods, he demands o
igo ous con ol o sample handling and s o age also inc ease.(93) O en, ea ly s udies
show g ea po en ial, bu subsequen epo s on he same ma ke yield inconsis en o
e en con adic o y esul s. The easons o his a e mainly me hodological al hough he
he e ogenei y and he lack o solid knowledge abou MS also con ibu e.(10, 12, 65, 72,
94, 95)
The use o OCB has some limi a ions and in esul o MS he e ogenei y, one
ma ke possibly isn’ enough. In his espec , de elopmen o no el labo a o y
echniques and sea ch o new eliable MS-speci ic ma ke s emains o conside able
diagnos ic impo ance o MS.(46, 96)
O he han OCB, MRZR and CXCL13 appea ed like aluable ma ke s o
con e sion o CIS o MS, and an i-myelin an ibodies he opposi e. P edic ing
con e sion om CIS o MS can be imp o ed combining MRI wi h CSF ma ke s, bu
MRI emains as he majo p ognos ic ac o .(16, 54, 59, 67)
CSF OCB u ili y was demons a ed in diagnosing and p edic ing MS, wi h he
awa eness o he equency a iabili y in he di e en communi ies. When p esen , is a
s ong suppo o MS bu he diagnosis should be e iewed in i absence.(21, 33) The
equi alen use o IgG index compa ing wi h OCB is inadequa e acco ding wi h some
au ho s.(17)
Quan i a i e KFCL’s p esen ed as a help ul ma ke o MS diagnosis, and
should be u he in es iga ed.(46)
Besides aquapo in-4 an ibody, he p esence o NAA in he se um can be
impo an o dis inguish MS om neu omyeli is.(82)
E en hough OCB p o ide impo an in o ma ion ega ding MS diagnosis, he e
a e cu en ly no CSF bioma ke s ha can eliably and de ini i ely diagnose MS a he

ime o ini ial symp oms.(10) The de elopmen o mo e sensi i e p o eomic echniques
is e y p omising o he iden i ica ion o new molecula a ge s o diagnos ic assays
and he apies p e en ing axonal damage.(92)
5. Acknowledgemen s
The help and a ailabili y o D Joana Guima ães we e ex emely help ul and
welcome du ing his pape elabo a ion, eason why I am since ely g a e ul.
6. Con lic s o in e es
The e a e no po en ial con lic s o in e es om each au ho ha ela e o he
esea ch co e ed in he a icle submi ed. The au ho s ha e no inancial disclosu es o
no hing o disclose.
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AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 2
EDITORIAL BOARD
.
Chie Edi o s:
B. Banwell, Resea ch Ins i u e, The Hospi al o Sick Child en, 555 Uni e si y A enue, To on o, ON, Canada
M5G 1X8
G. Gio annoni, Neu oimmunology Uni , Neu oscience Cen e; Ins i u e o Cell and Molecula Science, Ba s and
The London School o Medicine and Den is y; 4 Newa k S ee ; London E1 2AT, Uni ed Kingdom
C. H. Hawkes, Neu oimmunology Uni , Neu oscience Cen e; Ins i u e o Cell and Molecula Science Ba s and
The London School o Medicine and Den is y, 4 Newa k S ee , London E1 2AT, Uni ed Kingdom
F. D. Lublin, The Co inne Goldsmi h Dickinson Cen e o Mul iple Scle osis, Moun Sinai School o Medicine, 5
Eas 98 h S ee , New Yo k, NY 10029-6574, USA
Sec ion Edi o s:
Expe imen al Models
S. Amo , Ams e dam, The Ne he lands
Epidemiology
A. Asche io, Bos on, MA, USA
Neu o-op halmology
L. Balce , Philadelphia, PA, USA
In ec ion
J. Be ge , Lexing on, KY, USA
Neu oe hics
J. Be na , Lebanon, NH, USA
Pedia ic MS
R. Dale, Sydney, Aus alia
Bioma ke s
F. Deisenhamme , Innsb uck, Aus ia
Gene ics / ansc ip omics
R. Hin zen, Ro e dam, The Ne he lands
Measu emen / quali y o li e
J. Hoba , Plymou h, Uni ed Kingdom
Teaching
M. Keegan, Roches e , MN, USA
Rehabili a ion
J. Kessel ing, Valens, Swi ze land
Clinical Neu ology
J.I. Ki a, Fukuaka, Japan
Psychology and Fa igue
D. Langdon, Egham Su ey, Uni ed Kingdom
S em Cell Resea ch
G. Ma ino, Milan, I aly
The apy
A. Mille , New Yo k, NY, USA
Immunology
F. Sellebje g, Copenhagen, Denma k
Imaging
J. Wolinsky, Hous on, TX, USA
Edi o ial Boa d:
H. Bu zkue en, Melbou ne, Aus alia
V. B ina , Zag eb, C oa ia

AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 3
J. Co eale, Buenos Ai es, A gen ina
O. Fe nandez, Malaga, Spain
E. Ha do a, P ague, Czech Republic
H.P. Ha ung, Dusseldo , Ge many
L. Kappos, Basel, Swi ze land
N. Koch-Hen iksen, Aa hus, Denma k
P. Li, Hong Kong, China
J. Losy, Poznan, Poland
C. Lube zki, Pa is, F ance
A.H. Maghzi, Is ahan, I an
A. Mille , Hai a, Is ael
N. P ayoonwiwa , Bangkok, Thailand
T. Saida, Kyo o, Japan
B. Singhal, Mumbai, India
S. S einbjo nsdo i , Reykja ik, Iceland
J. To o, Bogo a, Columbia
C.P. Tsai, Taipei, Taiwan
L. Vécsei, Szeged, Hunga y
E. Willoughby, Auckland, New Zealand
B. Yamou , Bei ou , Lebanon
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 4
GUIDE FOR AUTHORS
.
INTRODUCTION
Types o a icle
O iginal Resea ch A icles
Full leng h esea ch pape s will no no mally be mo e han 3500 wo ds in leng h om he Ino duc ion
h ough he Discussion sec ion and will p e e ably be sho e . Submission o a pape o Mul iple
Scle osis and Rela ed Diso de s will be held o imply ha i ep esen s o iginal esea ch no p e iously
published (excep in he o m o an abs ac o p elimina y epo ), ha i is no being conside ed o
publica ion elsewhe e, and ha i accep ed by Mul iple Scle osis and Rela ed Diso de s i will no be
published elsewhe e in he same o m in any language wi hou he consen o he Publishe . Majo
pape s o opical con en will be gi en p io i y in publica ion.
Book Re iews
These a e no mally submi ed by he Book Re iew Edi o s, bu hey welcome sugges ions o books
o e iew.
Case Repo s
Case epo s should de ail he clinical, labo a o y and neu oimaging ea u es o in o ma i e pa ien s.
In o ma i e pa ien s should p o ide insigh s ha in o m on gene ic con ibu ions o disease, a e
clinical mani es a ions, no el labo a o y o imaging ea u es, o highligh impo an concep s in he
di e en ial o MS and ela ed diso de s. Case epo s should be app oxima ely 1200 wo ds, and should
ha e no mo e han i e key e e ences
Commen
Commen s should ocus on speci ic issues ele an o MS and ela ed diso de s, o should discuss
ecen publica ions. Commen s should be less han 800 wo ds and should e e ence he a icle(s)
upon which he commen a y is based.
Clinical T ial pape s
Manusc ip s de ailing he esul s o clinical ials in MS and ela ed diso de s a e encou age. The ial
me hodology should accoun o all sc eened pa icipan s, and analyses should obse e an in en ion-
o - ea model whe e app op ia e. All sou ces o unding o he s udy mus be disclosed, and he
in ol emen o he s udy sponso mus be de ailed. Clinical ial manusc ip s should be a maximum
o 3500 wo ds.
Edi o ials
The Edi o s welcome sugges ions o edi o ials which gi e pe sonal and opical iews on subjec s
wi hin he Jou nal's a ea o in e es . They should no no mally exceed 1500 wo ds in o al, including
e e ences.
Le e s o he Edi o s
These no mally e e o a icles p e iously published in he Jou nal. The Edi o s a e also willing o
conside le e s on subjec s o di ec ele ance o he Jou nal's in e es . Le e s should no exceed
1000 wo ds in o al and, whe e app op ia e, mus begin wi h he e e ence o he published a icle
abou which he au ho is commen ing. Resea ch le e s should be submi ed as 'le e o he Edi o s'
Re iew A icles
Re iew pape s a e no mally 4000-5000 wo ds in o al. Au ho s a e ad ised o consul one o he
Edi o s wi h an ou line be o e submi ing a e iew.
Con ac de ails o submission
Au ho s may send que ies conce ning he submission p ocess, manusc ip s a us, o jou nal
p ocedu es o he Edi o ial O ice a :
MSARD, Edi o ial O ice, ELSEVIER.
E-mail: msa d@else ie .com
BEFORE YOU BEGIN
E hics in publishing
Fo in o ma ion on E hics in publishing and E hical guidelines o jou nal publica ion see
h p://www.else ie .com/publishinge hics and h p://www.else ie .com/e hicalguidelines.
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 5
Policy and e hics
The wo k desc ibed in you a icle mus ha e been ca ied ou in acco dance wi h The Code
o E hics o he Wo ld Medical Associa ion (Decla a ion o Helsinki) o expe imen s in ol ing
humans h p://www.wma.ne /en/30publica ions/10policies/b3/index.h ml; EU Di ec i e 2010/63/EU
o animal expe imen s h p://ec.eu opa.eu/en i onmen /chemicals/lab_animals/legisla ion_en.h m;
Uni o m Requi emen s o manusc ip s submi ed o Biomedical jou nals h p://www.icmje.o g. This
mus be s a ed a an app op ia e poin in he a icle.
Con lic o in e es
All au ho s a e eques ed o disclose any ac ual o po en ial con lic o in e es including any inancial,
pe sonal o o he ela ionships wi h o he people o o ganiza ions wi hin h ee yea s o beginning he
submi ed wo k ha could inapp op ia ely in luence, o be pe cei ed o in luence, hei wo k. See
also h p://www.else ie .com/con lic so in e es .
Submission decla a ion
Submission o an a icle implies ha he wo k desc ibed has no been published p e iously (excep
in he o m o an abs ac o as pa o a published lec u e o academic hesis), ha i is no unde
conside a ion o publica ion elsewhe e, ha i s publica ion is app o ed by all au ho s and aci ly o
explici ly by he esponsible au ho i ies whe e he wo k was ca ied ou , and ha , i accep ed, i
will no be published elsewhe e including elec onically in he same o m, in English o in any o he
language, wi hou he w i en consen o he copy igh -holde .
Changes o au ho ship
This policy conce ns he addi ion, dele ion, o ea angemen o au ho names in he au ho ship o
accep ed manusc ip s:
Be o e he accep ed manusc ip is published in an online issue: Reques s o add o emo e an au ho ,
o o ea ange he au ho names, mus be sen o he Jou nal Manage om he co esponding au ho
o he accep ed manusc ip and mus include: (a) he eason he name should be added o emo ed,
o he au ho names ea anged and (b) w i en con i ma ion (e-mail, ax, le e ) om all au ho s ha
hey ag ee wi h he addi ion, emo al o ea angemen . In he case o addi ion o emo al o au ho s,
his includes con i ma ion om he au ho being added o emo ed. Reques s ha a e no sen by
he co esponding au ho will be o wa ded by he Jou nal Manage o he co esponding au ho , who
mus ollow he p ocedu e as desc ibed abo e. No e ha : (1) Jou nal Manage s will in o m he Jou nal
Edi o s o any such eques s and (2) publica ion o he accep ed manusc ip in an online issue is
suspended un il au ho ship has been ag eed.
A e he accep ed manusc ip is published in an online issue: Any eques s o add, dele e, o ea ange
au ho names in an a icle published in an online issue will ollow he same policies as no ed abo e
and esul in a co igendum.
Clinical ial esul s
In line wi h he posi ion o he In e na ional Commi ee o Medical Jou nal Edi o s, he jou nal will no
conside esul s pos ed in he same clinical ials egis y in which p ima y egis a ion esides o be
p io publica ion i he esul s pos ed a e p esen ed in he o m o a b ie s uc u ed (less han 500
wo ds) abs ac o able. Howe e , di ulging esul s in o he ci cums ances (e.g., in es o s' mee ings)
is discou aged and may jeopa dise conside a ion o he manusc ip . Au ho s should ully disclose all
pos ing in egis ies o esul s o he same o closely ela ed wo k.
Repo ing clinical ials
All andomised con olled ials submi ed o publica ion should include a comple ed Consolida ed
S anda ds o Repo ing T ials (CONSORT) low cha . Please e e o he CONSORT s a emen websi e
a h p://www.conso -s a emen .o g o mo e in o ma ion. This jou nal has adop ed he p oposal
om he In e na ional Commi ee o Medical Jou nal Edi o s (ICMJE) which equi e, as a condi ion
o conside a ion o publica ion o clinical ials, egis a ion in a public ials egis y. T ials mus
egis e a o be o e he onse o pa ien en olmen . The clinical ial egis a ion numbe should be
included a he end o he abs ac o he a icle. Fo his pu pose, a clinical ial is de ined as any
esea ch s udy ha p ospec i ely assigns human pa icipan s o g oups o humans o one o mo e
heal h- ela ed in e en ions o e alua e he e ec s o heal h ou comes. Heal h- ela ed in e en ions
include any in e en ion used o modi y a biomedical o heal h- ela ed ou come ( o example d ugs,
su gical p ocedu es, de ices, beha iou al ea men s, die a y in e en ions, and p ocess-o -ca e
changes). Heal h ou comes include any biomedical o heal h- ela ed measu es ob ained in pa ien s o
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 6
pa icipan s, including pha macokine ic measu es and ad e se e en s. Pu ely obse a ional s udies
( hose in which he assignmen o he medical in e en ion is no a he disc e ion o he in es iga o )
will no equi e egis a ion. Fu he in o ma ion can be ound a h p://www.icmje.o g.
Copy igh
Upon accep ance o an a icle, au ho s will be asked o comple e a 'Jou nal Publishing Ag eemen ' ( o
mo e in o ma ion on his and copy igh see h p://www.else ie .com/copy igh ). Accep ance o he
ag eemen will ensu e he wides possible dissemina ion o in o ma ion. An e-mail will be sen o
he co esponding au ho con i ming eceip o he manusc ip oge he wi h a 'Jou nal Publishing
Ag eemen ' o m o a link o he online e sion o his ag eemen .
Subsc ibe s may ep oduce ables o con en s o p epa e lis s o a icles including abs ac s o in e nal
ci cula ion wi hin hei ins i u ions. Pe mission o he Publishe is equi ed o esale o dis ibu ion
ou side he ins i u ion and o all o he de i a i e wo ks, including compila ions and ansla ions
(please consul h p://www.else ie .com/pe missions). I exce p s om o he copy igh ed wo ks a e
included, he au ho (s) mus ob ain w i en pe mission om he copy igh owne s and c edi he
sou ce(s) in he a icle. Else ie has p ep in ed o ms o use by au ho s in hese cases: please consul
h p://www.else ie .com/pe missions.
Re ained au ho igh s
As an au ho you (o you employe o ins i u ion) e ain ce ain igh s; o de ails you a e e e ed
o: h p://www.else ie .com/au ho s igh s.
Role o he unding sou ce
You a e eques ed o iden i y who p o ided inancial suppo o he conduc o he esea ch and/o
p epa a ion o he a icle and o b ie ly desc ibe he ole o he sponso (s), i any, in s udy design; in
he collec ion, analysis and in e p e a ion o da a; in he w i ing o he epo ; and in he decision o
submi he a icle o publica ion. I he unding sou ce(s) had no such in ol emen hen his should
be s a ed. Please see h p://www.else ie .com/ unding.
Funding body ag eemen s and policies
Else ie has es ablished ag eemen s and de eloped policies o allow au ho s whose a icles appea in
jou nals published by Else ie , o comply wi h po en ial manusc ip a chi ing equi emen s as speci ied
as condi ions o hei g an awa ds. To lea n mo e abou exis ing ag eemen s and policies please isi
h p://www.else ie .com/ undingbodies.
Language and language se ices
Please w i e you ex in good English (Ame ican o B i ish usage is accep ed, bu no a mix u e o
hese). Au ho s who equi e in o ma ion abou language edi ing and copyedi ing se ices p e- and
pos -submission please isi h p://webshop.else ie .com/languagese ices o ou cus ome suppo
si e a h p://suppo .else ie .com o mo e in o ma ion.
Submission
Submission o his jou nal p oceeds o ally online and you will be guided s epwise h ough he c ea ion
and uploading o you iles. The sys em au oma ically con e s sou ce iles o a single PDF ile o he
a icle, which is used in he pee - e iew p ocess. Please no e ha e en hough manusc ip sou ce
iles a e con e ed o PDF iles a submission o he e iew p ocess, hese sou ce iles a e needed o
u he p ocessing a e accep ance. All co espondence, including no i ica ion o he Edi o 's decision
and eques s o e ision, akes place by e-mail emo ing he need o a pape ail.
Submi you a icle
Please submi you a icle ia h p://ees.else ie .com/msa d/
Re e ees
Please submi , wi h he manusc ip , he names, add esses and e-mail add esses o h ee po en ial
e e ees. No e ha he edi o e ains he sole igh o decide whe he o no he sugges ed e iewe s
a e used.
PREPARATION
Use o wo dp ocessing so wa e
I is impo an ha he ile be sa ed in he na i e o ma o he wo dp ocesso used. The ex should
be in single-column o ma . Keep he layou o he ex as simple as possible. Mos o ma ing codes
will be emo ed and eplaced on p ocessing he a icle. In pa icula , do no use he wo dp ocesso 's
op ions o jus i y ex o o hyphena e wo ds. Howe e , do use bold ace, i alics, subsc ip s,
supe sc ip s e c. When p epa ing ables, i you a e using a able g id, use only one g id o each
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 7
indi idual able and no a g id o each ow. I no g id is used, use abs, no spaces, o align columns.
The elec onic ex should be p epa ed in a way e y simila o ha o con en ional manusc ip s
(see also he Guide o Publishing wi h Else ie : h p://www.else ie .com/guidepublica ion). No e ha
sou ce iles o igu es, ables and ex g aphics will be equi ed whe he o no you embed you igu es
in he ex . See also he sec ion on Elec onic a wo k.
To a oid unnecessa y e o s you a e s ongly ad ised o use he 'spell-check' and 'g amma -check'
unc ions o you wo dp ocesso .
A icle s uc u e
Subdi ision - numbe ed sec ions
Di ide you a icle in o clea ly de ined and numbe ed sec ions. Subsec ions should be numbe ed
1.1 ( hen 1.1.1, 1.1.2, ...), 1.2, e c. ( he abs ac is no included in sec ion numbe ing). Use his
numbe ing also o in e nal c oss- e e encing: do no jus e e o ' he ex '. Any subsec ion may be
gi en a b ie heading. Each heading should appea on i s own sepa a e line.
In oduc ion
S a e he objec i es o he wo k and p o ide an adequa e backg ound, a oiding a de ailed li e a u e
su ey o a summa y o he esul s.
Ma e ial and me hods
P o ide su icien de ail o allow he wo k o be ep oduced. Me hods al eady published should be
indica ed by a e e ence: only ele an modi ica ions should be desc ibed.
Resul s
Resul s should be clea and concise.
Discussion
This should explo e he signi icance o he esul s o he wo k, no epea hem. A combined Resul s
and Discussion sec ion is o en app op ia e. A oid ex ensi e ci a ions and discussion o published
li e a u e.
Conclusions
The main conclusions o he s udy may be p esen ed in a sho Conclusions sec ion, which may s and
alone o o m a subsec ion o a Discussion o Resul s and Discussion sec ion.
Appendices
I he e is mo e han one appendix, hey should be iden i ied as A, B, e c. Fo mulae and equa ions in
appendices should be gi en sepa a e numbe ing: Eq. (A.1), Eq. (A.2), e c.; in a subsequen appendix,
Eq. (B.1) and so on. Simila ly o ables and igu es: Table A.1; Fig. A.1, e c.
Essen ial i le page in o ma ion
• Ti le. Concise and in o ma i e. Ti les a e o en used in in o ma ion- e ie al sys ems. A oid
abb e ia ions and o mulae whe e possible.
• Au ho names and a ilia ions. Whe e he amily name may be ambiguous (e.g., a double
name), please indica e his clea ly. P esen he au ho s' a ilia ion add esses (whe e he ac ual wo k
was done) below he names. Indica e all a ilia ions wi h a lowe -case supe sc ip le e immedia ely
a e he au ho 's name and in on o he app op ia e add ess. P o ide he ull pos al add ess o
each a ilia ion, including he coun y name and, i a ailable, he e-mail add ess o each au ho .
• Co esponding au ho . Clea ly indica e who will handle co espondence a all s ages o e e eeing
and publica ion, also pos -publica ion. Ensu e ha elephone and ax numbe s (wi h coun y
and a ea code) a e p o ided in addi ion o he e-mail add ess and he comple e pos al
add ess. Con ac de ails mus be kep up o da e by he co esponding au ho .
• P esen /pe manen add ess. I an au ho has mo ed since he wo k desc ibed in he a icle
was done, o was isi ing a he ime, a 'P esen add ess' (o 'Pe manen add ess') may be indica ed
as a oo no e o ha au ho 's name. The add ess a which he au ho ac ually did he wo k mus be
e ained as he main, a ilia ion add ess. Supe sc ip A abic nume als a e used o such oo no es.
Abs ac
A concise and ac ual abs ac is equi ed. The abs ac should s a e b ie ly he pu pose o he
esea ch, he p incipal esul s and majo conclusions. An abs ac is o en p esen ed sepa a ely om
he a icle, so i mus be able o s and alone. Fo his eason, Re e ences should be a oided, bu i
essen ial, hen ci e he au ho (s) and yea (s). Also, non-s anda d o uncommon abb e ia ions should
be a oided, bu i essen ial hey mus be de ined a hei i s men ion in he abs ac i sel .

AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 8
G aphical abs ac
A G aphical abs ac is op ional and should summa ize he con en s o he a icle in a concise, pic o ial
o m designed o cap u e he a en ion o a wide eade ship online. Au ho s mus p o ide images
ha clea ly ep esen he wo k desc ibed in he a icle. G aphical abs ac s should be submi ed as a
sepa a e ile in he online submission sys em. Image size: Please p o ide an image wi h a minimum
o 531 × 1328 pixels (h × w) o p opo ionally mo e. The image should be eadable a a size o 5 ×
13 cm using a egula sc een esolu ion o 96 dpi. P e e ed ile ypes: TIFF, EPS, PDF o MS O ice
iles. See h p://www.else ie .com/g aphicalabs ac s o examples.
Au ho s can make use o Else ie 's Illus a ion and Enhancemen se ice o ensu e he bes
p esen a ion o hei images also in acco dance wi h all echnical equi emen s: Illus a ion Se ice.
Highligh s
Highligh s a e a sho collec ion o bulle poin s ha con ey he co e indings o he a icle. Highligh s
a e op ional and should be submi ed in a sepa a e ile in he online submission sys em. Please use
'Highligh s' in he ile name and include 3 o 5 bulle poin s (maximum 85 cha ac e s, including spaces,
pe bulle poin ). See h p://www.else ie .com/highligh s o examples.
Keywo ds
Immedia ely a e he abs ac , p o ide a maximum o 6 keywo ds, using Ame ican spelling and
a oiding gene al and plu al e ms and mul iple concep s (a oid, o example, 'and', 'o '). Be spa ing
wi h abb e ia ions: only abb e ia ions i mly es ablished in he ield may be eligible. These keywo ds
will be used o indexing pu poses.
Abb e ia ions
De ine abb e ia ions ha a e no s anda d in his ield in a oo no e o be placed on he i s page
o he a icle. Such abb e ia ions ha a e una oidable in he abs ac mus be de ined a hei i s
men ion he e, as well as in he oo no e. Ensu e consis ency o abb e ia ions h oughou he a icle.
Acknowledgemen s
Colla e acknowledgemen s in a sepa a e sec ion a he end o he a icle be o e he e e ences and do
no , he e o e, include hem on he i le page, as a oo no e o he i le o o he wise. Lis he e hose
indi iduals who p o ided help du ing he esea ch (e.g., p o iding language help, w i ing assis ance
o p oo eading he a icle, e c.).
Ma h o mulae
P esen simple o mulae in he line o no mal ex whe e possible and use he solidus (/) ins ead o
a ho izon al line o small ac ional e ms, e.g., X/Y. In p inciple, a iables a e o be p esen ed in
i alics. Powe s o e a e o en mo e con enien ly deno ed by exp. Numbe consecu i ely any equa ions
ha ha e o be displayed sepa a ely om he ex (i e e ed o explici ly in he ex ).
Foo no es
Foo no es should be used spa ingly. Numbe hem consecu i ely h oughou he a icle, using
supe sc ip A abic numbe s. Many wo dp ocesso s build oo no es in o he ex , and his ea u e may
be used. Should his no be he case, indica e he posi ion o oo no es in he ex and p esen he
oo no es hemsel es sepa a ely a he end o he a icle. Do no include oo no es in he Re e ence
lis .
Table oo no es
Indica e each oo no e in a able wi h a supe sc ip lowe case le e .
A wo k
Elec onic a wo k
Gene al poin s
• Make su e you use uni o m le e ing and sizing o you o iginal a wo k.
• Sa e ex in illus a ions as 'g aphics' o enclose he on .
• Only use he ollowing on s in you illus a ions: A ial, Cou ie , Times, Symbol.
• Numbe he illus a ions acco ding o hei sequence in he ex .
• Use a logical naming con en ion o you a wo k iles.
• P o ide cap ions o illus a ions sepa a ely.
• P oduce images nea o he desi ed size o he p in ed e sion.
• Submi each igu e as a sepa a e ile.
A de ailed guide on elec onic a wo k is a ailable on ou websi e:
h p://www.else ie .com/a wo kins uc ions
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 9
You a e u ged o isi his si e; some exce p s om he de ailed in o ma ion a e gi en he e.
Fo ma s
Rega dless o he applica ion used, when you elec onic a wo k is inalised, please 'sa e as' o
con e he images o one o he ollowing o ma s (no e he esolu ion equi emen s o line d awings,
hal ones, and line/hal one combina ions gi en below):
EPS: Vec o d awings. Embed he on o sa e he ex as 'g aphics'.
TIFF: Colo o g ayscale pho og aphs (hal ones): always use a minimum o 300 dpi.
TIFF: Bi mapped line d awings: use a minimum o 1000 dpi.
TIFF: Combina ions bi mapped line/hal - one (colo o g ayscale): a minimum o 500 dpi is equi ed.
I you elec onic a wo k is c ea ed in a Mic oso O ice applica ion (Wo d, Powe Poin , Excel) hen
please supply 'as is'.
Please do no :
• Supply iles ha a e op imised o sc een use (e.g., GIF, BMP, PICT, WPG); he esolu ion is oo low;
• Supply iles ha a e oo low in esolu ion;
• Submi g aphics ha a e disp opo iona ely la ge o he con en .
Colo a wo k
Please make su e ha a wo k iles a e in an accep able o ma (TIFF, EPS o MS O ice iles) and wi h
he co ec esolu ion. I , oge he wi h you accep ed a icle, you submi usable colo igu es hen
Else ie will ensu e, a no addi ional cha ge, ha hese igu es will appea in colo on he Web (e.g.,
ScienceDi ec and o he si es) ega dless o whe he o no hese illus a ions a e ep oduced in colo
in he p in ed e sion. Fo colo ep oduc ion in p in , you will ecei e in o ma ion ega ding
he cos s om Else ie a e eceip o you accep ed a icle. Please indica e you p e e ence
o colo : in p in o on he Web only. Fo u he in o ma ion on he p epa a ion o elec onic a wo k,
please see h p://www.else ie .com/a wo kins uc ions.
Please no e: Because o echnical complica ions which can a ise by con e ing colo igu es o 'g ay
scale' ( o he p in ed e sion should you no op o colo in p in ) please submi in addi ion usable
black and whi e e sions o all he colo illus a ions.
Figu e cap ions
Ensu e ha each illus a ion has a cap ion. Supply cap ions sepa a ely, no a ached o he igu e. A
cap ion should comp ise a b ie i le (no on he igu e i sel ) and a desc ip ion o he illus a ion. Keep
ex in he illus a ions hemsel es o a minimum bu explain all symbols and abb e ia ions used.
Tables
Numbe ables consecu i ely in acco dance wi h hei appea ance in he ex . Place oo no es o ables
below he able body and indica e hem wi h supe sc ip lowe case le e s. A oid e ical ules. Be
spa ing in he use o ables and ensu e ha he da a p esen ed in ables do no duplica e esul s
desc ibed elsewhe e in he a icle.
Re e ences
Ci a ion in ex
Please ensu e ha e e y e e ence ci ed in he ex is also p esen in he e e ence lis (and ice
e sa). Any e e ences ci ed in he abs ac mus be gi en in ull. Unpublished esul s and pe sonal
communica ions a e no ecommended in he e e ence lis , bu may be men ioned in he ex . I hese
e e ences a e included in he e e ence lis hey should ollow he s anda d e e ence s yle o he
jou nal and should include a subs i u ion o he publica ion da e wi h ei he 'Unpublished esul s' o
'Pe sonal communica ion'. Ci a ion o a e e ence as 'in p ess' implies ha he i em has been accep ed
o publica ion.
Re e ence managemen so wa e
This jou nal has s anda d empla es a ailable in key e e ence managemen
packages EndNo e h p://www.endno e.com/suppo /ens yles.asp and Re e ence Manage
h p:// e man.com/suppo / ms yles.asp. Using plug-ins o wo dp ocessing packages, au ho s only
need o selec he app op ia e jou nal empla e when p epa ing hei a icle and he lis o e e ences
and ci a ions o hese will be o ma ed acco ding o he jou nal s yle which is desc ibed below.
Web e e ences
As a minimum, he ull URL should be gi en and he da e when he e e ence was las accessed. Any
u he in o ma ion, i known (DOI, au ho names, da es, e e ence o a sou ce publica ion, e c.),
should also be gi en. Web e e ences can be lis ed sepa a ely (e.g., a e he e e ence lis ) unde a
di e en heading i desi ed, o can be included in he e e ence lis .
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 10
Re e ences in a special issue
Please ensu e ha he wo ds ' his issue' a e added o any e e ences in he lis (and any ci a ions in
he ex ) o o he a icles in he same Special Issue.
Re e ence s yle
Tex : All ci a ions in he ex should e e o:
1. Single au ho : he au ho 's name (wi hou ini ials, unless he e is ambigui y) and he yea o
publica ion;
2. Two au ho s: bo h au ho s' names and he yea o publica ion;
3. Th ee o mo e au ho s: i s au ho 's name ollowed by 'e al.' and he yea o publica ion.
Ci a ions may be made di ec ly (o pa en he ically). G oups o e e ences should be lis ed i s
alphabe ically, hen ch onologically.
Examples: 'as demons a ed in whea (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). K ame
e al. (2010) ha e ecen ly shown ....'
Lis : Re e ences should be a anged i s alphabe ically and hen u he so ed ch onologically i
necessa y. Mo e han one e e ence om he same au ho (s) in he same yea mus be iden i ied by
he le e s 'a', 'b', 'c', e c., placed a e he yea o publica ion.
Examples:
Re e ence o a jou nal publica ion:
Van de Gee J, Han aads JAJ, Lup on RA. The a o w i ing a scien i ic a icle. J Sci Commun
2010;163:51–9.
Re e ence o a book:
S unk J W, Whi e EB. The elemen s o s yle. 4 h ed. New Yo k: Longman; 1979.
Re e ence o a chap e in an edi ed book:
Me am GR, Adams LB. How o p epa e an elec onic e sion o you a icle. In: Jones BS, Smi h RZ,
edi o s. In oduc ion o he elec onic age. New Yo k: E-Publishing Inc; 2009. p. 281–304.
No e sho ened o m o las page numbe . e.g., 51–9, and ha o mo e han 6 au ho s he i s 6
should be lis ed ollowed by "e al." Fo u he de ails you a e e e ed o "Uni o m Requi emen s
o Manusc ip s submi ed o Biomedical Jou nals" (J Am Med Assoc 1997;277:927–34) (see also
h p://www.nlm.nih.go /bsd/uni o m_ equi emen s.h ml).
Jou nal abb e ia ions sou ce
Jou nal names should be abb e ia ed acco ding o
Index Medicus jou nal abb e ia ions: h p://www.nlm.nih.go / sd/se ials/lji.h ml;
Lis o i le wo d abb e ia ions: h p://www.issn.o g/2-22661-LTWA-online.php;
CAS (Chemical Abs ac s Se ice): h p://www.cas.o g/sen .h ml.
Video da a
Else ie accep s ideo ma e ial and anima ion sequences o suppo and enhance you scien i ic
esea ch. Au ho s who ha e ideo o anima ion iles ha hey wish o submi wi h hei a icle a e
s ongly encou aged o include hese wi hin he body o he a icle. This can be done in he same way
as a igu e o able by e e ing o he ideo o anima ion con en and no ing in he body ex whe e i
should be placed. All submi ed iles should be p ope ly labeled so ha hey di ec ly ela e o he ideo
ile's con en . In o de o ensu e ha you ideo o anima ion ma e ial is di ec ly usable, please p o ide
he iles in one o ou ecommended ile o ma s wi h a p e e ed maximum size o 50 MB. Video and
anima ion iles supplied will be published online in he elec onic e sion o you a icle in Else ie
Web p oduc s, including ScienceDi ec : h p://www.sciencedi ec .com. Please supply 's ills' wi h you
iles: you can choose any ame om he ideo o anima ion o make a sepa a e image. These will
be used ins ead o s anda d icons and will pe sonalize he link o you ideo da a. Fo mo e de ailed
ins uc ions please isi ou ideo ins uc ion pages a h p://www.else ie .com/a wo kins uc ions.
No e: since ideo and anima ion canno be embedded in he p in e sion o he jou nal, please
p o ide ex o bo h he elec onic and he p in e sion o he po ions o he a icle ha e e o
his con en .
Supplemen a y da a
Else ie accep s elec onic supplemen a y ma e ial o suppo and enhance you scien i ic esea ch.
Supplemen a y iles o e he au ho addi ional possibili ies o publish suppo ing applica ions, high-
esolu ion images, backg ound da ase s, sound clips and mo e. Supplemen a y iles supplied will be
published online alongside he elec onic e sion o you a icle in Else ie Web p oduc s, including
ScienceDi ec : h p://www.sciencedi ec .com. In o de o ensu e ha you submi ed ma e ial is
di ec ly usable, please p o ide he da a in one o ou ecommended ile o ma s. Au ho s should
AUTHOR INFORMATION PACK 8 Ma 2012 www.else ie .com/loca e/msa d 11
submi he ma e ial in elec onic o ma oge he wi h he a icle and supply a concise and desc ip i e
cap ion o each ile. Fo mo e de ailed ins uc ions please isi ou a wo k ins uc ion pages a
h p://www.else ie .com/a wo kins uc ions.
Submission checklis
The ollowing lis will be use ul du ing he inal checking o an a icle p io o sending i o he jou nal
o e iew. Please consul his Guide o Au ho s o u he de ails o any i em.
Ensu e ha he ollowing i ems a e p esen :
One au ho has been designa ed as he co esponding au ho wi h con ac de ails:
• E-mail add ess
• Full pos al add ess
• Telephone and ax numbe s
All necessa y iles ha e been uploaded, and con ain:
• Keywo ds
• All igu e cap ions
• All ables (including i le, desc ip ion, oo no es)
Fu he conside a ions
• Manusc ip has been 'spell-checked' and 'g amma -checked'
• Re e ences a e in he co ec o ma o his jou nal
• All e e ences men ioned in he Re e ence lis a e ci ed in he ex , and ice e sa
• Pe mission has been ob ained o use o copy igh ed ma e ial om o he sou ces (including he Web)
• Colo igu es a e clea ly ma ked as being in ended o colo ep oduc ion on he Web ( ee o cha ge)
and in p in , o o be ep oduced in colo on he Web ( ee o cha ge) and in black-and-whi e in p in
• I only colo on he Web is equi ed, black-and-whi e e sions o he igu es a e also supplied o
p in ing pu poses
Fo any u he in o ma ion please isi ou cus ome suppo si e a h p://suppo .else ie .com.
AFTER ACCEPTANCE
Use o he Digi al Objec Iden i ie
The Digi al Objec Iden i ie (DOI) may be used o ci e and link o elec onic documen s. The DOI
consis s o a unique alpha-nume ic cha ac e s ing which is assigned o a documen by he publishe
upon he ini ial elec onic publica ion. The assigned DOI ne e changes. The e o e, i is an ideal
medium o ci ing a documen , pa icula ly 'A icles in p ess' because hey ha e no ye ecei ed hei
ull bibliog aphic in o ma ion. The co ec o ma o ci ing a DOI is shown as ollows (example aken
om a documen in he jou nal Physics Le e s B):
doi:10.1016/j.physle b.2010.09.059
When you use he DOI o c ea e URL hype links o documen s on he web, he DOIs a e gua an eed
ne e o change.
P oo s
One se o page p oo s (as PDF iles) will be sen by e-mail o he co esponding au ho (i we do
no ha e an e-mail add ess hen pape p oo s will be sen by pos ) o , a link will be p o ided in
he e-mail so ha au ho s can download he iles hemsel es. Else ie now p o ides au ho s wi h
PDF p oo s which can be anno a ed; o his you will need o download Adobe Reade e sion 7 (o
highe ) a ailable ee om h p://ge .adobe.com/ eade . Ins uc ions on how o anno a e PDF iles
will accompany he p oo s (also gi en online). The exac sys em equi emen s a e gi en a he Adobe
si e: h p://www.adobe.com/p oduc s/ eade / ech-specs.h ml.
I you do no wish o use he PDF anno a ions unc ion, you may lis he co ec ions (including
eplies o he Que y Fo m) and e u n hem o Else ie in an e-mail. Please lis you co ec ions
quo ing line numbe . I , o any eason, his is no possible, hen ma k he co ec ions and any o he
commen s (including eplies o he Que y Fo m) on a p in ou o you p oo and e u n by ax, o scan
he pages and e-mail, o by pos . Please use his p oo only o checking he ypese ing, edi ing,
comple eness and co ec ness o he ex , ables and igu es. Signi ican changes o he a icle as
accep ed o publica ion will only be conside ed a his s age wi h pe mission om he Edi o . We will
do e e y hing possible o ge you a icle published quickly and accu a ely – please le us ha e all you
co ec ions wi hin 48 hou s. I is impo an o ensu e ha all co ec ions a e sen back o us in one
communica ion: please check ca e ully be o e eplying, as inclusion o any subsequen co ec ions
canno be gua an eed. P oo eading is solely you esponsibili y. No e ha Else ie may p oceed wi h
he publica ion o you a icle i no esponse is ecei ed.