multiple sclerosis ( ms ) is the most common reason of neurological disability among young adults . . different mechanisms of inflammation - demyelination , axonal damage - neurodegeneration , gliosis , and remyelination - repair combine together in various degrees ( influenced by idiosyncratic factors ) to create a unique clinical result for each patient . identifying those idiosyncratic factors , as well as understanding which mechanism is prominent in each case , is the first step towards a rational therapeutical choice . thus , guiding research towards distinguishing reliable biomarkers for every independent ms pathogenic factor is of primary importance . an adequate definition of the term  biomarker  would be as follows :  biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes , pathogenic processes or pharmacological responses to a therapeutical intervention  . a biomarker would ideally serve as a  surrogate endpoint  of a clinical outcome , only if it was fully capable to represent it . at this time , such a measure exists only in a theoretical basis , because none of the existing biomarkers can fully reflect the immensity of diverse ms pathogenic mechanisms creating a unique result . arguably , the applicability of a biomarker for ms should be judged by the use of certain criteria . the criteria that were used in the present paper , in accordance with previous efforts of systematization [ 2 , 3 ] , and are reflected in a critical way in table 1 , are the following : biological rationale : degree of the correlation of the biomarker with an identified pathogenic mechanism ; clinical rationale : accuracy in depicting clinical status;predictability of disease initiation , reactivation , or progression , or of disease differentiation of other demyelinating spectrum diseases , like neuromyelitis optica ( nmo);sensitivity and specificity : false negative or false positive results in depicting an event;reproducibility of a result;practicality of the method in use for the measurement;correlation with therapeutic outcome : reflecting the negative and positive effects of a therapy;correlation with prognosis and disability : the latter being objectively measured by instruments such as the expanded disability status scale ( edss ) . biological rationale : degree of the correlation of the biomarker with an identified pathogenic mechanism ;   clinical rationale : accuracy in depicting clinical status ; predictability of disease initiation , reactivation , or progression , or of disease differentiation of other demyelinating spectrum diseases , like neuromyelitis optica ( nmo ) ; sensitivity and specificity : false negative or false positive results in depicting an event ; reproducibility of a result ; practicality of the method in use for the measurement ; correlation with therapeutic outcome : reflecting the negative and positive effects of a therapy ; correlation with prognosis and disability : the latter being objectively measured by instruments such as the expanded disability status scale ( edss ) . its value as a means of providing biomarkers is undebatable , due to its natural proximity to the central nervous system ( cns ) . the levels of a csf biomarker can not be influenced by liver or kidney function . on the other hand , the invasiveness of the collecting method narrows the potential of multiple measurements . circadian fluctuation in csf 's production rate dictates the necessity of standardizing the time of performing a lumbar puncture . little is known about circadian fluctuation in the concentrations of csf biomarkers . experience acquired in other fields of neurological science suggests that this may be the case ( i.e. , circadian fluctuation in the levels of hypocretin-1 , dopamine , and tryptophan ) [ 58 ] . it is hypothesized , although not clarified in all papers , that csf collection via lumbar puncture is done in morning hours , after night fasting . . tumor necrosis factor ( tnf ) receptor-1 and receptor-2 , soluble intercellular adhesion molecule-1 ( sicam-1 ) , and soluble vascular cell adhesion molecule-1 ( svcam-1 ) also display diurnal variations . kidney function , liver function , and concomitant infections can influence the levels measured , as well as the time from collection to process . it is the easiest material to collect , even in a 24-hour basis , overcoming the obstacles of fluctuations previously mentioned . ms patients with bladder dysfunction may regulate the amount of the fluids taken in a daily basis , affecting the quantity of produced urine . there have been previous efforts in measuring oligoclonal bands ( ocbs ) in tears , with results comparable to those of csf . it has served as a means of specifying soluble human leucocyte antigens ( hlas ) type ii . magnetic resonance imaging ( mri ) although in depth analysis of all the different techniques providing biomarkers for ms is out of the scopes of this paper , a short comment in relevance to the most important of them is going to follow . an example biomarker will be provided in each case , extracted from the literature and references sited in this paper . the term refers to a solid - phase enzyme immunoassay which gives the ability to detect the presence of a substance , in a liquid or wet sample . performing an elisa technique requires the use of at least one specific antibody against the antigen under investigation . there are numerous different commercial kits available . by the use of standardization protocols , the assays are easy to reproduce by different laboratories . elisa techniques give also the opportunity to analyse samples that are properly stored ( i.e. , il-1 and il-6 measurements ; see section 6.1.8 ) . this is a highly sensitive technique which gives the opportunity to specify , in vivo , proteins of variable size , haptens , and antibodies . immunofluorescence techniques use the affinity of a certain antibody against its epitope , which is made visible through microscope by the use of proper fluorescent dyes . further categorization in direct and indirect methods refers to the use of one or two antibodies . time - resolved immunofluorescence assays display high sensibility , tracking down proteins in very small concentrations . major drawback is the danger of photobleaching , as a result of prolonged light exposure ( i.e. , sicam-1 and svcam-1 ; see section 6.1.9 ) it is a laser - based method applied in the study of cellular subpopulations and biomarkers by the form of surface antigens , dna and rna variations , protein expression , enzymes , and intracellular antigens . several techniques , like fluorescein dyes and intracellular cytokine dying , allow the collection of functional data for specific cellular populations . the major drawback of these assays is that sample handling may influence the results . because of that , comparisons between different biomarker measurements are at risk of being inaccurate ( i.e. , 4 integrin ; see section 6.3.1 ) pcr provides the researcher with the ability to detect trivial amount of genetic information . basic components of the method are primers ( short dna fragments , sequences complementary to the target area ) , as well as a dna polymerase , which enhances the replication . subsequently , the results may not be fully compatible with the functional state , in vivo . cost and sample handling limitations should also be considered ( i.e. , hla - drb1 * polymorphisms ; see section 5.1 ) the term nephelometry refers to a technique of estimating protein concentrations in different bodily fluids . the liquid sample is beamed by light by a certain angle , and afterwards the degree of light scatter is estimated . nephelometry techniques are widely performed , reliably reproducible by many different laboratories ( i.e. , csf albumin ; see section 6.4 ) the term refers to a protein detection method , which uses gel electrophoresis of a sample and subsequent dying of the protein target by the use of a specific antibody , on a membranic surface . several final detection techniques have been developed , namely , colorimetric and fluorescent ( i.e. , b - crystalline measurements ; see section 6.5.2 ) . this is another electrophoresis technique , which takes advantage of the phenomenon of the different isoelectric point between different molecules , in order to separate them . for this purpose , acrylamide gels with ph gradient are used ( i.e. , detection of csf oligoclonal bands - kappa and lambda free chains ; see sections 6.1.1 , 6.1.2 and 6.1.3 ) . refers to a group of rapidly emerging novel technologies that give the opportunity of large - scale analysis and identification of candidate biomarkers in multiple levels of cell biology ( dna , rna , proteins , lipids , metabolites , and epigenetic modifications ) . subsequently , the  -omics  technologies are further categorized in : genomics : large - scale studies of the whole dna sequence ( i.e. , vitamin d receptor element recognition ; see section 6.2.2);transcriptomics : genome - wide studies of rna sequences . two main types of transcriptomics technologies are in common use , microarrays and next generation sequencing ( i.e. , tob-1 gene downregulation ; see section 5.3 ) ; proteomics : large - scale studies of protein distribution ( i.e. , ninjurin-1 ; see section 6.4.2);lipidomics : recognition studies of important cellular lipid pathways . recent knowledge implicates specific cns lipid epitopes in the generation of anti - lipid antibodies in ms ( see also igm against myelin lipids section 6.1.2 ) ; metabolomics : studies of important metabolic pathways , as a result of ms specific pathogenic mechanisms ( i.e. , n - acetyloaspartate csf measurements ; see section 6.8.6);epigenomics : large - scale studies of epigenetic modifications . they explore the potential influence that alterations in chromatin architecture may have in ms susceptibility . genomics : large - scale studies of the whole dna sequence ( i.e. , vitamin d receptor element recognition ; see section 6.2.2 ) ; transcriptomics : genome - wide studies of rna sequences . two main types of transcriptomics technologies are in common use , microarrays and next generation sequencing ( i.e. , tob-1 gene downregulation ; see section 5.3 ) ; proteomics : large - scale studies of protein distribution ( i.e. , ninjurin-1 ; see section 6.4.2 ) ; lipidomics : recognition studies of important cellular lipid pathways . recent knowledge implicates specific cns lipid epitopes in the generation of anti - lipid antibodies in ms ( see also igm against myelin lipids section 6.1.2 ) ; metabolomics : studies of important metabolic pathways , as a result of ms specific pathogenic mechanisms ( i.e. , n - acetyloaspartate csf measurements ; see section 6.8.6 ) ; epigenomics : large - scale studies of epigenetic modifications . they explore the potential influence that alterations in chromatin architecture may have in ms susceptibility . in this paper biomarkers are further categorized in three subgroups for reasons of systematic approach , and according to their pathophysiological implication in ms pathogenesis : genetic / immunogenetic : biomarkers specified via genomics and immunogenetic techniques;laboratorial : all other biomarkers that can be measured in body fluids;imaging : biomarkers provided by imaging techniques . genetic / immunogenetic : biomarkers specified via genomics and immunogenetic techniques ; laboratorial : all other biomarkers that can be measured in body fluids ; imaging : biomarkers provided by imaging techniques . the fact that genetic factors may influence ms was already known by epidemiological studies of previous decades . recent research , using modern techniques previously mentioned , led to the implication of multiple genetic loci , with polymorphisms of major histocompatibility complex ( mhc ) antigens having the primary role . polymorphisms in hla class ii antigens seem to be decisive in attributing genetic burden for ms . initial studies found positive correlation between drb1 * 1501-drb5 * 0101-dqa1 * 0102-dqb1 * 0602 haplotype and disease frequency . multiple recent researches , conducted in many ms cohorts , made clear that hla - drb1 * 1501 is the mainly responsible allele for attributing genetic risk in ms population [ 1721 ] . moreover , hla - drb1 * 1501 expression is partially regulated by vitamin d through interaction in a genomic level , thus explaining the long known connection between latitude and risk for ms ( see also section 6.2.2 ) . additionally , coexistence of certain alleles probably leads to augmentation of the overall risk , via epistatic mechanisms ( i.e. , drb1 * 1501 and dq1 * 0102 ) . nevertheless , there have been reports of association with other hla loci in some studies , like dr3 and dr4 haplotypes in sardinia   and drb1 * 04 in hutterite families , as a mere reflection of the disease 's genetic complexity . a positive association of the haplotype drb1 * 1303-dqa1 * 05-dqb1 * 030 with ms was found in the non - ashkenazi jewish population . finally , hla - dq  hla - dp polymorphisms were found to correlate with opticospinal ms frequency in japanese cohorts . positive correlation of hla - drb1 * 1501 and negative correlation of hla - drb1 * 0405 alleles with ocb in the csf of ms patients were established by observations in a japanese cohort   and further confirmed by subsequent studies . its correlation with early progression from relapsing - remitting ms ( rrms ) to secondary progressive ms ( spms ) remains controversial [ 28 , 29 ] . hla - drb1 * 01 allele is considered to protect against malignant disease form . zivadinov et al . observed the following in a study of ms patients : drb1 * 1501 positive had worst brain atrophy scores and bigger t1 lesions ' burden in mri;dqb1 * 0301 positive had worst brain atrophy scores and bigger t2 lesions ' burden;dqb1 * 0602 positive had worst grey matter atrophy scores . drb1 * 1501 positive had worst brain atrophy scores and bigger t1 lesions ' burden in mri ; dqb1 * 0301 positive had worst brain atrophy scores and bigger t2 lesions ' burden ; dqb1 * 0602 positive had worst grey matter atrophy scores .   in another study , drb1 * 1501 positive patients had the following : lower n - acetyl - aspartate ( naa ) levels within normal appearing white matter ( nawm ) ; bigger white matter lesions ; worst brain atrophy scores ; impaired cognitive function . lower n - acetyl - aspartate ( naa ) levels within normal appearing white matter ( nawm ) ;   bigger white matter lesions ;   worst brain atrophy scores ;   impaired cognitive function . hla - drb1 * 0401 , 0408 , and 1601 alleles correlate with greater risk of developing neutralizing antibodies against interferon beta ( ifn- ) , resulting in poor therapeutical outcome . various genome - wide studies revealed many non - mhc single nucleotide polymorphisms as candidates for genetic burden augmentation in ms . polymorphisms of the il2ra and il7ra regions seem as the most promising at the moment . increasing evidence also implicates other loci , like evi5 , cd58 , kiaa0350 , and rpl5 genes . another recent genome - wide association study identified 57 non - mhc genes associated with ms . tob-1 gene has a role against t - cell multiplication , keeping autoreactive cells in a dormant state . its degreased expression leads towards a more intense immune response ( higher percentage of th1 and th17 cells and lower percentage of t - regulatory cells ) . tob-1 polymorphisms represent an independent factor influencing the progression from clinically isolated syndrome ( cis ) to clinically definite multiple sclerosis ( cdms ) . carrying 4 allele of apoe seems to attribute greater risk of developing mental disorders in ms patients . positive ocb igg in the csf of patients with cis was found to duplicate the risk of progression in cdms in a 4-year observation period . additional studies provide more evidence for ocb igg being a relevant factor for conversion to cdms . their diagnostic sensitivity is high ( > 90% ) , but they lack in specificity ( ~35% ) among inflammatory disorders of the cns . ocb igg could not be connected with known protein targets inside cns , like myelin basic protein ( mbp ) , proteolipid protein , and myelin oligodendrocyte glycoprotein ( mog ) . some researchers consider them as a bad prognostic biomarker , correlating with disability progression both qualitatively and quantitatively ( igm index ) . kflc high csf levels have been repeatedly reported in ms . in comparison to ocb igg , slightly higher sensitivity with slightly lower specificity has been found . mrz igg reaction in csf displays , compared to ocb igg , a higher specificity for ms diagnosis and higher prognostic value of progression from cis to cdms . moreover , mrz reaction indicates a primarily b - cell mediated immune response , guiding therapeutical choice towards a relevant immunomodulating agent . reported a high percentage of igg antibodies against protein epitopes brrf2 and ebna-1 of the virus , in the serum and csf samples from ms patients . . isolated from ms patients in 2006   highly specific t - cells for the epitope ebna-1 . recently , infection of blood - brain barrier ( bbb ) endothelial cells has been reported , initiating disruption mechanisms , as well as proinflammatory cytokines production . ebv antibodies are considered as indicative of higher inflammatory activity and early disease onset . specifically , it has been made clear that the anti - mog antibodies recognize only conformational epitopes of mog and not their linear counterparts displayed in soluble phase [ 56 , 57 ] . likewise , denaturation of mbp leads to a marked decrease in igm antibody induced in vitro deposition . nevertheless , occurrence of anti - myelin antibodies alone could not support a diagnosis of increased risk for future ms , in the preclinical stage of the disease . considerably higher levels of serum autoantibodies against the fragments 4870 and 85170 of mbp were found in ms patients compared to healthy controls . reactivity against fragments 4368 and 146170 was also found significantly different between ms patients and donors suffering from other neurological disorders ( onds ) . naturally occurring mbp autoantibodies in healthy controls may have diminished capacity of facilitating interferon gamma ( ifn- ) and il-5 , than those occurring in ms . presence of anti - mbp antibodies in childhood ms increases the risk of an acute demyelinating encephalomyelitis-(adem- ) like onset of the disease . finally , children with cis were found to have high titres of anti - mog in a percentage of 30%40% . in general , anti - mog antibodies are considered to play significant role in childhood ms , anti - aquaporine 4 ( aqp4 ) negative nmo , adem , and isolated optic neuritis , but hardly in adult ms [ 63 , 64 ] . some markers of intrathecal anti - mog production though , like the rmog index , may have some use in adult ms , especially in progressive disease forms . chemokine cxcl13 mobilizes b - cells and t - helper cells towards active demyelinating lesions by interacting with cxcr5 receptor . consistent correlation of cxcl13 csf levels with csf b - cells , plasmablasts , and intrathecal ig synthesis has been reported . high levels of cxcl13 have been found in patients with cis and cdms . on the other hand , chemokine cxcl12 was shown to have a protective role against cns inflammation , in experimental autoimmune encephalomyelitis ( eae ) . ccl2 levels , normally induced by th2 immune response , were found diminished shortly after methylprednizolone treatment for ms relapse , but not one month later . inflammatory activity in active demyelinating lesions leads to the liberation of many different cytokines that can be used as biomarkers of disease activity . proinflammatory cytokines in the periphery primarily originate from t- and b - cells , whereas b - cells seem to be mainly responsible for their intrathecal production in rrms , with monocytes having a more immunoregulatory role inside csf . ifn- and tnf - a are the main products of th1 immune response . il-6 serves as linking arm between b - cell and t - cell immune response as well as a th-17 response triggering factor . il-6 serum levels were found to correlate significantly with the relapse frequency in female ms patients and age at onset for all ms patients . moreover , studying il-1 levels in mice led to the conclusion that any imbalance in the il-1 signalling ( increased or decreased ) may lead to cns demyelination . recent research implicates single nucleotide polymorphisms at the 592 position of the il-10 gene to the regulation of cns autoimmunity . flow cytometric analysis revealed that b - cells and monocytes from ms patients overexpress il-15 , and that stimulation of cd8(+ ) t - cells with the latter cytokine enhances their ability to kill glial cells and enter the bbb . il-15 was found elevated in the sera and csf of ms patients , in comparison with onds . finally , treating eae mice with anti - il-33 led to decreased levels of ifn- and il-17 and upregulation of proinflammatory il-10 and transforming growth factor- ( tgf- ) . higher csf levels of sicam-1 and svcam-1 were reported in nmo patients , in comparison with ms patients , suggesting that the bbb in nmo displays more severe alterations . finally , laminin 411 , which is situated within the vascular endothelium , interacts with adhesion molecule cd146 , allowing th17 cells to overcome the bbb . osteopontin is a macrophage derived phosphoprotein which enhances ifn- and il-12 levels and diminishes the levels of neuroprotective il-10 . serum and csf osteopontin levels are upregulated during an ms relapse , but this is also the case for many other inflammatory disorders . csf ccr2(+)ccr5(+ ) t - cells show a distinct ability to produce osteopontin during relapse . fetuin - a ( alpha2 hermans schmid glycoprotein ) is a calcium - regulating surface glycoprotein . protein 's coding m - rna is overexpressed in ms patients ' cns , resulting in its high concentrations in active demyelinating lesions . . diminished m - rna expression of vegf - a in serum monocytes of patients with spms compared to rrms patients has been reported . the vitamin 's potential pathogenic role in ms can be deducted by multiple previous epidemiological studies that showed correlation of latitude and sun exposure with relative risk for developing the disease . vitamin d suppresses th1 immune response in multiple levels and enables the production of many neurotrophic factors . recently , a vitamin d response element ( vdre ) was recognized close to the hla - drb1 * 1501 coding area , with the aid of genomics . vitamin d displays an inhibitory role in ms , also at a genetic level , by interacting with vdre . recently concluded that part of ifn- therapeutic effects during ms relapses may be attributed to greater production of vitamin d . evidence from animal model research also implicates vitamins other than vitamin d in ms pathogenesis . fat - soluble vitamins a and e are considered as modulators of disease activity . moreover , a small series of patients with ms found decreased csf and serum levels of the vitamin biotin , in comparison to healthy controls . mature b - cells and plasma - blasts were found to accumulate in the csf of rrms patients , correlating positively with higher disease activity in the mri . centroblasts , typically found in the germinal centre of lymphatic tissue , were also detected in the csf of ms patients , suggesting intrathecal production . transitional alterations of b - cells in early ms stages , like overexpression of cd80(+ ) , 4 , and 1 integrins , are associated with their ability to cross bbb . the expression of 4 integrin in b - cells was correlated with the number of gadolinium - enhanced lesions , during cis . cxcr3 has a low diagnostic specificity for ms , due to its high levels in many other inflammatory disorders . recent studies revealed expansion of cd4(+)cd28( ) t - cells in the sera of ms patients , t - cells phenotype with enhanced cytotoxic properties . cx(3)cr1 , the fractalkine receptor , may serve as biomarker for discriminating cd4(+)cd28( ) cells from their cd28(+ ) counterparts . csf ccr2(+)ccr5(+ ) t - cells show remarkable increase during ms relapse , which is not the case in onds , displaying reactivity against mbp . rrms patients in a remission phase display high levels of cd-56 surface antigen in their nk cells ( > 36% ) . trecs are intracellular by - products of t - cell receptor remodelling , gradually rejected via homeostatic mechanisms . thymic gland 's functional state can be estimated by the percentage of nave t - cells in the peripheral blood . a definite reduction of trecs in ms patients has been reported , indicative of thymic dysfunction in the disease . nave t - cells are found further diminished in patients with primary progressive multiple sclerosis ( ppms ) , compared to rrms . lipocalins are a family of proteins that transport small hydrophobic molecules , taking part in several processes of the immune system . the gene encoding for lipocalin 2 was found upregulated during relapses in the eae model of ms , mainly originating from neutrophils infiltrating the choroid plexus ( cp ) , as well as from astrocytes in affected regions . bbb disruption is an early feature of lesion formation , leading to edema , excitotoxicity , and entry of serum proteins and inflammatory cells inside cns . intercellular endothelial tight junctions breakdown possesses a primary role between events leading to bbb and blood - cerebrospinal fluid barrier ( bcb ) disruption [ 98 , 99 ] . tight junction proteins occluding and claudin-1 showed decrease after treatment of a bbb model with mbp .   apart from csf production , cp is actually considered as important regulator of cns autoimmunity , displaying properties of early bcb disruption site , allowing sentinel t - cells to enter noninflamed regions [ 101 , 102 ] . expression of various adhesion molecules in the vascular endothelium plays a vital role in facilitating inflammatory agents from the periphery to enter the barrier ( see also section 6.1.9 ) . various measures of bbb permeability have been proposed , like the csf  :  serum albumin ratio ( ar ) . ar levels are constantly higher in nmo in comparison to ms , and display correlation with clinical severity only in nmo . the degree of expression of the protein ninjurin-1 by endothelial cells of the bbb and myeloid antigen - presenting cells ( apcs ) plays an important role in the transmigration and localization of the latter inside cns , as it was made obvious by proteomic screen of human bbb cells . the csf sicam-1 levels from nmo patients were found to correlate adequately with other measures of bbb disruption , like the albumin quotient and the gadolinium - enhanced lesions in mri . the term refers to an endothelial proteinic system that plays role in the transmigration of monocytes through the bbb . major components of this system are the proteins endothelin-1 , endothelin type b receptor , and endothelin - converting enzyme-1 . mbp and its fragments are found in large quantities in the csf of most ms patients during a relapse ( 80% ) . a significant correlation of decrease in csf - mbp , contrast - enhancement in mri , and clinical disability in response to treatment with methylprednisolone suggests an association between inflammation and myelin breakdown in ms . immunohistochemical analysis of demyelinating lesions revealed increased expression of this protein , comparatively to healthy myelin . it is considered as primary target molecule for t - cells in ms , but it can also be found elevated in the csf of patients with onds . its mechanism of action encompasses activation of il-17 , il-10 , il-13 , tnf , and chemokines ccl5 and ccl1 . no and its metabolites can cause mitochondrial damage and tissue hypoxia leading to further damage in ms lesions . myelin cholesterol breaks down to 7-ketocholesterol , whose levels in the csf of ms patients have been reported to be elevated . extracellular levels of glutamate are normally regulated through its active reabsorption in oligodendrocytes . in active demyelinating lesions , active lesions seem to overexpress cystine / glutamate antiporter , aiming in intracellular accumulation of cystine for production of the antioxidant glutathione . neurofilaments are major axonal cytoskeleton proteins consisting of three subunits ( light chain / nf - l , intermediate chain / nf - m , and heavy chain / nf - h ) . nf - l csf levels in ms patients are considerably higher compared to healthy controls and onds patients , reaching their peak approximately three weeks after relapse onset . their correlation , however , with disability progression is poor . csf nf - l levels in natalizumab responders have been reported to return to normal . on the other hand , nf - h chains seem to correlate better with disease progression , with significant elevation recorded only in progressive disease forms . simultaneous elevation in tau and nf - h values in csf , in patients with cis , has a 70% predictive value of conversion to cdms , which is superior to the predictive value of mri . microtubules represent a major structural cytoskeleton component , consisting of two subunits , a- and b - tubulin . elevated csf tubulin and actin values have been reported in progressive disease forms , correlating well with disability measured by edss . in alzheimer 's disease , amyloid- ( 142 ) csf reduction can be also observed in ms patients , in correlation with greater risk for cognitive decline . apart from creutzfeldt - jacobs disease , elevated csf values have been reported in 10%30% of patients with rrms , but its potential utility as a biomarker for ms seems limited for the time being . naa is an aminoacid , highly expressed in neurons , which transfers actively water molecules extracellularly against concentration gradient . spectroscopy techniques revealed decreased naa values in ms lesions , but also in nawm , in conventional mri . serum and csf naa levels were significantly higher in rrms patients , in comparison to healthy donors and nmo patients . s100b is a calcium - binding protein , primarily expressed in astrocytes , whose csf elevated values have been previously correlated with cerebral injury . there are reports of csf elevation in rrms patients , but overall data remain inconclusive . gfap is a structural protein of the astrocytes whose csf levels increase in association with gliosis - astrocytosis . high csf values have been found in spms patients , but rarely in rrms patients , and seem to correlate well with disability progression [ 115 , 116 ] . csf - gfap levels are significantly higher during nmo relapse , in comparison with ms relapse [ 124126 ] , and show adequate correlation with clinical improvement and disability progression in nmo [ 125 , 126 ] . s100b possesses the same properties , but correlations tend to be weaker [ 125 , 126 ] . constant csf elevation of n - cam has been repeatedly reported immediately after ms relapse , in adequate correlation with clinical improvement . lower csf - bndf levels in spms patients comparatively to rrms patients have been reported . bdnf increased production was observed in glatiramer acetate responders , correlating well with clinical improvement . nogo - a is adequately specific for ms , as it could not be isolated in other autoimmune or infectious neurological disorders . ifn- converts peripheral immune response from the th1 towards the more anti - inflammatory th2 , influencing the expression of many genes . tnf - related inducing ligand ( trail ) and myxovirus resistance protein - a ( mxa ) serum levels may also reflect response to ifn- . finally , upregulation of svcam levels , following ifn- treatment , may represent a reliable predictor of therapeutical response . progress in understanding of ms pathophysiology shed light over the important role of th17 immune response . recently , researchers reached the conclusion that ms patients with prominent th17 response are probably more harmed than benefited by treatment with ifn- , because of il-17 exacerbation by the drug . nevertheless , il-17f levels could not be connected with poor therapeutic outcome during ifn- treatment by other researchers . expression levels of unblocked 4 integrin on peripheral mononuclear blood cells can serve as biomarker of natalizumab therapeutical efficacy , as well as biomarker of risk for progressive multifocal leucoencephalopathy ( pml ) . predictive role of fetuin - a is mentioned in section 6.1.11 , predictive role of lipocalin 2 is mentioned in section 6.3.5 , and predictive role of nf - l is mentioned in section 6.8.1 . csf levels reduction of sicam-1 and se - selectin may potentially serve as biomarkers of therapeutical efficacy after cladribine treatment . oct is a noninvasive technique using emission of infrared light through the pupil and detection of its reflection from the retina . rnfl thinning can be used as a reliable biomarker of axonal loss , correlating adequately with brain atrophy measures . rnfl thickness can serve as biomarker of disease progression and neuroprotection by a certain therapeutical agent . mri provides the clinical doctor with a substantial variety of neuroinflammation biomarkers . on the other hand , classical mri techniques lack in adequate correlation with neurodegeneration and disability progression . the most important mri biomarkers for ms although they are considered as the gold standard for bbb disruption imaging , recent research claims that the same diagnosis can be inferred in many cases by combination of t1 , t2 , and t2-weighted flair images characteristics alone ; hyperintense t2-weighted lesions : reflecting a combination of mechanisms like inflammation , demyelination , axonal damage and edema . their diagnostic value is high , but they correlate moderately with disability ; hypointense t1-weighted lesions ( black holes ) : considered as satisfactory biomarkers of axonal damage . their correlation with disability remains debatable ; whole brain atrophy biomarkers : the most widely used measure is the brain parenchymal fraction . brain atrophy worsening rates are higher in untreated ms patients ( 0.5%1% annualized decrease ) in comparison with healthy controls ( 0.1%0.3% ) . brain atrophy worsening rate at initial diagnosis has been proposed as prognostic biomarker of disability eight years afterwards ; gray matter atrophy biomarkers : recently acquired knowledge suggests gray matter demyelination , axonal damage , and atrophy in ms . double inversion recovery imaging techniques display gray matter atrophy in all ms stages and types , with higher worsening rates in spms patients . higher worsening rates of gray matter atrophy in cis patients correlate well with rapid conversion to rrms ; spinal cord atrophy biomarkers : upper cervical cord area ( ucca ) measuring techniques display atrophy most apparently in progressive ms forms , correlating well with disability progression . ucca atrophy presence in early disease stages in rrms patients is a bad prognostic biomarker of future disability . although they are considered as the gold standard for bbb disruption imaging , recent research claims that the same diagnosis can be inferred in many cases by combination of t1 , t2 , and t2-weighted flair images characteristics alone ; hyperintense t2-weighted lesions : reflecting a combination of mechanisms like inflammation , demyelination , axonal damage and edema . their diagnostic value is high , but they correlate moderately with disability ; hypointense t1-weighted lesions ( black holes ) : considered as satisfactory biomarkers of axonal damage . their correlation with disability remains debatable ; whole brain atrophy biomarkers : the most widely used measure is the brain parenchymal fraction . brain atrophy worsening rates are higher in untreated ms patients ( 0.5%1% annualized decrease ) in comparison with healthy controls ( 0.1%0.3% ) . brain atrophy worsening rate at initial diagnosis has been proposed as prognostic biomarker of disability eight years afterwards ;   gray matter atrophy biomarkers : recently acquired knowledge suggests gray matter demyelination , axonal damage , and atrophy in ms . double inversion recovery imaging techniques display gray matter atrophy in all ms stages and types , with higher worsening rates in spms patients . higher worsening rates of gray matter atrophy in cis patients correlate well with rapid conversion to rrms ; spinal cord atrophy biomarkers : upper cervical cord area ( ucca ) measuring techniques display atrophy most apparently in progressive ms forms , correlating well with disability progression . ucca atrophy presence in early disease stages in rrms patients is a bad prognostic biomarker of future disability . it is a novel mri technique based on proton interaction between free water and macromolecules . in the absence of axonal loss , acute mri lesions that show recovery display optic nerve mtr decrease after optic neuritis shows good correlation with rnfl thickness in oct ( section 7.1 ) and with reduction of amplitude in visual evoked potentials , suggesting that mtr is primarily an axonal damage biomarker . dwi is based on mobility and spatial distribution of water molecules , while dti measures movement in several directions in space . dti technique provides two different measures , mean diffusivity ( md ) and fractional anisotropy ( fa ) . similar alterations can be recorded in nawm areas in conventional mri , as well as in normal appearing gray matter ( nagm ) areas , especially in progressive disease forms . corpus callosum dti abnormalities are present in early ms stages , even when lesions in conventional mri are still absent . md alterations precede visible in conventional mri bbb injury by at least 5 months , being thus a reliable predictive biomarker for ms relapse . corpus callosum dti abnormalities in spms patients constitute a bad prognostic biomarker of future disability . the following substances spectroscopic measurements are of particular value in ms : naa : biomarker of neuronal and axonal integrity . naa showed a progressive decline pattern in a two - year mrs followup of patients with rrms ; choline : biomarker of myelin loss;myoinositol and creatine : biomarkers of gliosis;glutamate : biomarker of acute inflammation . naa showed a progressive decline pattern in a two - year mrs followup of patients with rrms ; choline : biomarker of myelin loss ; myoinositol and creatine : biomarkers of gliosis ; glutamate : biomarker of acute inflammation . spectroscopic findings suggest that white matter abnormalities in rrms are more prominent than grey matter abnormalities where the injury is less diffuse . diffusion tensor spectroscopy ( dts ) , a technique combining properties of dti and mrs , seems promising in better distinguishing axonopathy , demyelination , inflammation , edema , and gliosis . modern pet tracers have the ability to bind in proteins that show upregulation in activated microglia , making possible an early visualization of nawm and nagm disorders , even before contrast enhancement in conventional mri . at present , the use of pet in ms remains experimental . eps estimate action potential conduction along somatosensory , motor , visual , and auditory pathways , providing a reliable means of demyelination and axonal loss assessment . mri techniques have diminished their spectrum of use , although visual and motor eps may have some utility as biomarkers of neurodegeneration . motor eps are of special value in patients with ms presentation as a spinal cord syndrome . sensory eps in combination with edss score could help in predicting short - term progression of disability in ms . vestibular evoked myogenic potentials ( vemps ) can offer complementary information in relevance with brainstem dysfunction .   combined ep data , like the ep score , for instance , could offer a reliable prognostic biomarker , especially for early recognition of benign ms forms . recent studies of ssr in ms patients report correlation of ssr abnormalities with higher edss scores and disease duration . pathophysiological complexity of ms leads inevitably to a great variety of potential biomarkers , as it was made obvious by the previous analysis . thus , for systematization reasons and after the completion of an elaborate quest for biomarkers in the international literature wesubgrouped all biomarkers in six broad categories , that is , diagnostic biomarkers , biomarkers of phenotypical expression , biomarkers of demyelination  neuroinflammation  relapse , biomarkers of axonal loss  neurodegeneration , prognostic biomarkers biomarkers of disability progression , and biomarkers of therapeutical response;gradually evaluated every biomarker with ( + + + ) to ( + / ) according to their implication in the category they referred to;commented on biomarker abilities to reflect the properties of the category in which they are presented . subgrouped all biomarkers in six broad categories , that is , diagnostic biomarkers , biomarkers of phenotypical expression , biomarkers of demyelination  neuroinflammation  relapse , biomarkers of axonal loss  neurodegeneration , prognostic biomarkers biomarkers of disability progression , and biomarkers of therapeutical response ; gradually evaluated every biomarker with ( + + + ) to ( + / ) according to their implication in the category they referred to ; commented on biomarker abilities to reflect the properties of the category in which they are presented . the previous three steps demanded as prerequisite hard , critical , and thorough data processing , in order to achieve the best and accurate results , since it is the first time that an attempt of such a systematization is done in a workable table . in this table , biomarkers such as hla - ii alleles and ocbs are for many years the  gold standard  for ms , while other well - described biomarkers are being implicated more and more every day , like conventional and nonconventional mri scans . additionally , many other laboratorial and imaging parameters are at the beginning of their characterization as biomarkers in ms . from every group of biomarkers , we collected those with characterization of ( + + + ) and presented them as an easy summary to the reader , as it follows . additionally , we present the biomarkers of differentiation between ms and nmo : ms diagnosis : the panel of potential biomarkers should definitely include hla - drb1 * characterization , csf ocb igg and/or kflc , csf mrz reaction , serum vitamin d levels , mri with contrast - enhancement , and eps ; ms phenotypical expression : the panel of potential biomarkers should primarily include hla - drb1 * characterization , antibodies against confrontational epitopes of mbp and mog , rmog index , and il-6 serum levels . dti abnormalities and ucca atrophy measures may also be helpful;demyelination - neuroinflammation - relapse : the panel of potential biomarkers should definitely include contrast - enhanced t1 mri lesions , csf mbp - glutamate - nf - l and ccr2(+)ccr5(+ ) t - cells , serum levels of ifn--tnf--il-6-vitamin d , and expression of tob-1-fetuin - a-b - crystalline . mrs offers reliable data in the study of demyelinating lesions;axonal loss - neurodegeneration : naa and nogo - a have the leading position as potential biomarkers . dts may help even more in the near future;prognosis - disability progression : ocb igg , kflc , mrz reaction , combination of nf - h with tau , tob-1 expression , and higher worsening rates of gray - matter atrophy are reliable prognostic biomarkers of cis conversion to cdms . naa , nf - h , and combined ep 's reflect adequately disability progression in ms , and gfap does the same in nmo . dti abnormalities offer prognostic data for relapse and disability progression;therapeutical response : hla - drb1 * polymorphisms and vitamin d levels should be considered as biomarkers for therapeutical outcome for ifn - b . fetuin - a , nf - l , and unblocked 4 integrin represent biomarkers for natalizumab treatment and bdnf for glatiramer acetate treatment . rnfl may be offered substantially in the near future;differentiation from nmo : mrs findings should help when differential diagnosis is required . csf naa , csf gfap , and ar can also help when there is a possibility for nmo ( apart from antiaqp4 of course ) . ms diagnosis : the panel of potential biomarkers should definitely include hla - drb1 * characterization , csf ocb igg and/or kflc , csf mrz reaction , serum vitamin d levels , mri with contrast - enhancement , and eps ;   ms phenotypical expression : the panel of potential biomarkers should primarily include hla - drb1 * characterization , antibodies against confrontational epitopes of mbp and mog , rmog index , and il-6 serum levels . dti abnormalities and ucca atrophy measures may also be helpful ; demyelination - neuroinflammation - relapse : the panel of potential biomarkers should definitely include contrast - enhanced t1 mri lesions , csf mbp - glutamate - nf - l and ccr2(+)ccr5(+ ) t - cells , serum levels of ifn--tnf--il-6-vitamin d , and expression of tob-1-fetuin - a-b - crystalline . mrs offers reliable data in the study of demyelinating lesions ; axonal loss - neurodegeneration : naa and nogo - a have the leading position as potential biomarkers . dts may help even more in the near future ; prognosis - disability progression : ocb igg , kflc , mrz reaction , combination of nf - h with tau , tob-1 expression , and higher worsening rates of gray - matter atrophy are reliable prognostic biomarkers of cis conversion to cdms . naa , nf - h , and combined ep 's reflect adequately disability progression in ms , and gfap does the same in nmo . dti abnormalities offer prognostic data for relapse and disability progression ; therapeutical response : hla - drb1 * polymorphisms and vitamin d levels should be considered as biomarkers for therapeutical outcome for ifn - b . fetuin - a , nf - l , and unblocked 4 integrin represent biomarkers for natalizumab treatment and bdnf for glatiramer acetate treatment . rnfl may be offered substantially in the near future ; differentiation from nmo : mrs findings should help when differential diagnosis is required . csf naa , csf gfap , and ar can also help when there is a possibility for nmo ( apart from antiaqp4 of course ) . even after decades of research , ms still remains at a significant proportion an unsolved mystery . this is mainly the reason why finding a biomarker with absolute surrogacy abilities remains elusive . further research in the field of ms biomarkers must be directed towards an earlier and accurate diagnosis and a more prompt , targeted , and individualized therapeutical approach , with the minimum intervention and economical cost .
during the last decades , the effort of establishing satisfactory biomarkers for multiple sclerosis has been proven to be very difficult , due to the clinical and pathophysiological complexities of the disease . 
 recent knowledge acquired in the domains of genomics - immunogenetics and neuroimmunology , as well as the evolution in neuroimaging , has provided a whole new list of biomarkers . 
 this variety , though , leads inevitably to confusion in the effort of decision making concerning strategic and individualized therapeutics . in this paper , 
 our primary goal is to provide the reader with a list of the most important characteristics that a biomarker must possess in order to be considered as reliable . 
 additionally , up - to - date biomarkers are further divided into three subgroups , genetic - immunogenetic , laboratorial , and imaging . 
 the most important representatives of each category are presented in the text and for the first time in a summarizing workable table , in a critical way , estimating their diagnostic potential and their efficacy to correlate with phenotypical expression , neuroinflammation , neurodegeneration , disability , and therapeutical response . 
 special attention is given to the  gold standards  of each category , like hla - drb1 polymorphisms , oligoclonal bands , vitamin d , and conventional and nonconventional imaging techniques . 
 moreover , not adequately established but quite promising , recently characterized biomarkers , like tob-1 polymorphisms , are further discussed .