nmo can be diagnosed based on a blood - derived biomarker , that is antibodies against aquaporin-4 , a channel protein present on astrocytes , extensively discussed in other contributions in this special issue . the presence of antibodies against aquaporin-4 has been proven as one of the most successful results of biomarker studies , and is supportive for the idea that central nervous system ( cns ) abnormalities are reflected in changes in body fluids . it also proofs the autoimmune component of this disorder and of pathologies that are related to the nmo spectrum disorders , such as longitudinally extensive transverse myelitis . determination of serum anti - aquaporin-4 antibody levels is a mainstay in the diagnosis of nmo , but the discovery of such disease - specific antibodies is relatively recent , and , therefore , further studies in body fluids are warranted . one case report suggested that nmo - immunoglobulin g ( igg ) , the nmo - associated antibodies that are reactive to cerebellar tissue , can be absent in serum , but present in csf . however , another study on a relative large cohort of patients showed that testing csf does not increase diagnostic sensitivity . another recently identified candidate biomarker for nmo is glial fibrillary acid protein ( gfap ) . takano and colleagues observed that the analysis of csf glial fibrillary acid protein is useful in the differential diagnosis between nmo and multiple sclerosis or acute demyelinating encephalomyelitis , and that its csf levels at disease onset correlated with expanded disability score scale ( edss ) in nmo . however , studies on larger cohorts are needed before drawing definite conclusions . taken together , no biomarkers are available yet for prognosis or therapy response in nmo and in nmo - related disorders . one important flaw in several previously performed biomarker studies in cns diseases has been the lack of large cohorts to sufficiently power the study . this is especially an issue for such a rare disease as nmo , where a single center will not be able to collect a large cohort within a reasonable time frame . the need for collaboration was the reason for biomarker researchers in multiple sclerosis to start a network ( bioms - eu , http://www.bioms.eu/ ) . the aim of this collaboration is to obtain well - proven , high - quality biomarkers , which will be achieved by sharing patient samples , standardization , and improvement of procedures important in the research area . one of the most urgent prerequisites for collaboration was felt to be standardization of biobanking protocols . therefore , a consensus - meeting was organised and the result was collection and biobanking guidelines , which the network developed and published in 2009 . there are currently major efforts worldwide to professionalize biobanks and the collection and biobanking guidelines established by consensus among 26 groups participating in bioms - eu ( http://www.bioms.eu/ ) is a major achievement in the cns biomarker field . one year after publication of the guidelines , over 90% of the bioms - eu laboratories had already adapted their procedures in agreement with the guidelines . a great use of the guidelines is the applicability for any neurological disease , including nmo , and that it provides guidelines for setting up a novel biobank . furthermore , it will greatly facilitate biomarker studies in the cns biomarker research area . in the concensus discussions , we have sought a balance between practicality and scientific rationale , and the background of each decision is provided . before the consensus , it was clear that large differences were present between collection protocols , highlighting the need to address these differences ( figure 1 and table 1 ) . in the current paper , we include only the items and their rationale from the original paper that are relevant for biobanking for nmo . other modifications from the original protocol is an adaptation of item 1 ( samples should be pooled if multiple collection tubes are used for one patient ) , and the inclusion of an item addressing transportation ( item 20 ) and information of some more physiological confounders ( item 25 ) . we would like to stress that researchers adhere to these protocols for optimal collaboration in the field of csf biomarker research . we suggest using tables 2 and 3 as a checklist for csf biomarker research and recommend that future studies of csf biomarker take these issues into account . in discovery - based biomarker research , all these items should be considered carefully before initiating a study . some procedures may not be possible in everyday clinical practice ( e.g. , processing within one hour ) , but less stringent requirements will suffice for specific research questions . therefore , careful documentation of these issues is crucial to facilitate retrieval of appropriate samples dictated by specific study aims . as indicated before , the procedures for withdrawal and storage of csf ( table 2 ) are broadly applicable for any neurological disease .   besides methodological issues the signed informed consent should include a statement that exchange of samples between ( international ) centers is allowed . furthermore , to bring a biomarker to clinical practice , one may need patents and the involvement of industrial partners could be needed , who have the infrastructure for large - scale production , quality control procedures , and to reach as many laboratories as possible . for large - scale validation studies , patient samples are of course also needed , and it will be wise to consider this possibility at the start of biobank formation and , if ethical laws permit and the option is perceived to be important , indicate the possibility for industrial cooperation in the patient information and consent . lastly , researchers should be willing to share their samples and information for the benefit of the whole , that is , obtaining reliable biomarkers that can be used for patient care and cure . ( a ) collection procedures : 
 item 1 volume of withdrawal of at least 12  mlthe csf volume taken can influence the concentration of biomarkers . most molecules and cell numbers have a rostrocaudal concentration gradient [ 6 , 7 ] . if a small volume is taken , the csf will reflect the composition of the lumbar dural sac , whereas large volumes may reflect the rostral spinal or even ventricular csf . therefore , if biomarker concentrations in a sample from a puncture of 2  ml are compared to that in a puncture of 15  ml , this can lead to erroneous results . also collecting different portions of the csf for biobanking ( e.g. , initial and final volumes of the puncture ) may introduce errors . thus , a standard volume of csf should be collected during lumbar puncture , the first 2  ml can be used for basic csf analysis ( item 26 ) , and the remainder of the sample should be pooled before spinning and aliquoting . at least , the procedure must be recorded . the volume of collected csf does not correlate with the risk of postlumbar puncture headache [ 8 , 9 ] . item 2 location of puncture : vertebral body l3l5usually , diagnostic csf is obtained by lumbar puncture . because of the increasing gradient in protein concentration from ventricular to lumbar csf , the site of csf withdrawal must be recorded . when csf is taken from other locations such as the cervical cisterns or from the lateral ventricles ( e.g. , ventricular drainage ) , item 3 removal of bloody csf samplesa traumatic tap causing blood contamination of csf occurs in about 1420% of standard lumbar punctures . for biomarkers that have high serum concentrations , such as coagulation factors , in addition , blood proteins lead to suppressed matrix - assisted laser desorption / ionization - mass spectrometry ( maldi - tof / ms ) proteomics patterns in csf . this suppression by blood proteins is , however , highly reduced after removal of the blood cells by centrifugation prior to initial freezing [ 12 , 13 ] . csf samples with an erythrocyte count above 500/l should not be used for biomarker studies . item 4 use of atraumatic needle ( sprotte or whitacre needle)there is no evidence that the type of lumbar puncture needle influences biomarker concentrations . however , atraumatic needles are best tolerated by patients , and are associated with a lower risk for postlumbar puncture headache , that is about 12% for a needle size of 2022  g compared to about 70% for a needle size of 1619  g [ 14 , 15 ] . 
 item 5 use of polypropylene collection tubesthere are several reports showing that the type of collection tube influences biomarker outcomes , for example , total tau proteins and amyloid  peptides . we propose to use polypropylene tubes , with their low protein binding potential , for collecting csf . when multiple tubes are used , the total volume should be mixed after centrifugation to avoid gradient effects . item 6 time of the day of withdrawalfor biomarkers that are influenced by circadian rhythm , time of withdrawal is important . since it is often difficult to accomplish standardization of withdrawal time in everyday clinical practice , documentation is necessary to select the appropriate samples to minimize the effect of this variable . items 7 and 8 serum , plasma , and dna linked to the csf sampleit is important to collect matched serum and/or plasma samples for evaluation of csf biomarkers because the concentration of the marker in blood often influences that in csf . further , serum / plasma pairs are essential to study the intrathecal origin of a biomarker and its cns specificity . furthermore , the presence of cns markers in serum / plasma may aid in disease monitoring . vacuum tubes that use edta ( in dried format ) are preferred over those that use citrate ( in solution ) because if tubes containing a standard volume of citrate are filled incompletely , the final biomarker concentration is diluted unequally compared to other samples . depending on the type of biomarkers and methods of study , we recommend collecting both serum and plasma ; for some methods , plasma is preferred over serum and vice versa . we advise to perform a blood draw using vacuum systems , since tourniquet use is related to additional confounding factors in the preanalytic phase include tourniquet time and posture . furthermore , instructions of the supplier should be followed , such as mixing.lastly , dna collection expands the possibilities for studying the phenotypes and genotypes within individuals . a protocol for storage and handling of dna can be found in the supplementary files ( e - appendix  1 ) . item 1 volume of withdrawal of at least 12  mlthe csf volume taken can influence the concentration of biomarkers . most molecules and cell numbers have a rostrocaudal concentration gradient [ 6 , 7 ] . if a small volume is taken , the csf will reflect the composition of the lumbar dural sac , whereas large volumes may reflect the rostral spinal or even ventricular csf . therefore , if biomarker concentrations in a sample from a puncture of 2  ml are compared to that in a puncture of 15  ml , this can lead to erroneous results . also collecting different portions of the csf for biobanking ( e.g. , initial and final volumes of the puncture ) may introduce errors . thus , a standard volume of csf should be collected during lumbar puncture , the first 2  ml can be used for basic csf analysis ( item 26 ) , and the remainder of the sample should be pooled before spinning and aliquoting . at least , the procedure must be recorded . the volume of collected csf does not correlate with the risk of postlumbar puncture headache [ 8 , 9 ] . item 2 location of puncture : vertebral body l3l5usually , diagnostic csf is obtained by lumbar puncture . because of the increasing gradient in protein concentration from ventricular to lumbar csf , the site of csf withdrawal must be recorded . when csf is taken from other locations such as the cervical cisterns or from the lateral ventricles ( e.g. , ventricular drainage ) , this should be documented . item 3 removal of bloody csf samplesa traumatic tap causing blood contamination of csf occurs in about 1420% of standard lumbar punctures . for biomarkers that have high serum concentrations , such as coagulation factors , in addition , blood proteins lead to suppressed matrix - assisted laser desorption / ionization - mass spectrometry ( maldi - tof / ms ) proteomics patterns in csf . this suppression by blood proteins is , however , highly reduced after removal of the blood cells by centrifugation prior to initial freezing [ 12 , 13 ] . csf samples with an erythrocyte count above 500/l should not be used for biomarker studies . item 4 use of atraumatic needle ( sprotte or whitacre needle)there is no evidence that the type of lumbar puncture needle influences biomarker concentrations . however , atraumatic needles are best tolerated by patients , and are associated with a lower risk for postlumbar puncture headache , that is about 12% for a needle size of 2022  g compared to about 70% for a needle size of 1619  g [ 14 , 15 ] . item 5 use of polypropylene collection tubesthere are several reports showing that the type of collection tube influences biomarker outcomes , for example , total tau proteins and amyloid  peptides . therefore , standardization is important . we propose to use polypropylene tubes , with their low protein binding potential , for collecting csf . when multiple tubes are used , the total volume should be mixed after centrifugation to avoid gradient effects . item 6 time of the day of withdrawalfor biomarkers that are influenced by circadian rhythm , time of withdrawal is important . since it is often difficult to accomplish standardization of withdrawal time in everyday clinical practice , documentation is necessary to select the appropriate samples to minimize the effect of this variable . items 7 and 8 serum , plasma , and dna linked to the csf sampleit is important to collect matched serum and/or plasma samples for evaluation of csf biomarkers because the concentration of the marker in blood often influences that in csf . further , serum / plasma pairs are essential to study the intrathecal origin of a biomarker and its cns specificity . furthermore , the presence of cns markers in serum / plasma may aid in disease monitoring . vacuum tubes that use edta ( in dried format ) are preferred over those that use citrate ( in solution ) because if tubes containing a standard volume of citrate are filled incompletely , the final biomarker concentration is diluted unequally compared to other samples . depending on the type of biomarkers and methods of study , we recommend collecting both serum and plasma ; for some methods , plasma is preferred over serum and vice versa . we advise to perform a blood draw using vacuum systems , since tourniquet use is related to additional confounding factors in the preanalytic phase include tourniquet time and posture . furthermore , instructions of the supplier should be followed , such as mixing.lastly , dna collection expands the possibilities for studying the phenotypes and genotypes within individuals . a protocol for storage and handling of dna can be found in the supplementary files ( e - appendix  1 ) . most molecules and cell numbers have a rostrocaudal concentration gradient [ 6 , 7 ] . if a small volume is taken , the csf will reflect the composition of the lumbar dural sac , whereas large volumes may reflect the rostral spinal or even ventricular csf . therefore , if biomarker concentrations in a sample from a puncture of 2  ml are compared to that in a puncture of 15  ml , this can lead to erroneous results . also collecting different portions of the csf for biobanking ( e.g. , initial and final volumes of the puncture ) may introduce errors . thus , a standard volume of csf should be collected during lumbar puncture , the first 2  ml can be used for basic csf analysis ( item 26 ) , and the remainder of the sample should be pooled before spinning and aliquoting . at least , the procedure must be recorded . the volume of collected csf does not correlate with the risk of postlumbar puncture headache [ 8 , 9 ] . usually , diagnostic csf is obtained by lumbar puncture . because of the increasing gradient in protein concentration from ventricular to lumbar csf , the site of csf withdrawal must be recorded . when csf is taken from other locations such as the cervical cisterns or from the lateral ventricles ( e.g. , ventricular drainage ) , a traumatic tap causing blood contamination of csf occurs in about 1420% of standard lumbar punctures . for biomarkers that have high serum concentrations , such as coagulation factors , in addition , blood proteins lead to suppressed matrix - assisted laser desorption / ionization - mass spectrometry ( maldi - tof / ms ) proteomics patterns in csf . this suppression by blood proteins is , however , highly reduced after removal of the blood cells by centrifugation prior to initial freezing [ 12 , 13 ] . csf samples with an erythrocyte count above 500/l should not be used for biomarker studies . however , atraumatic needles are best tolerated by patients , and are associated with a lower risk for postlumbar puncture headache , that is about 12% for a needle size of 2022  g compared to about 70% for a needle size of 1619  g [ 14 , 15 ] . there are several reports showing that the type of collection tube influences biomarker outcomes , for example , total tau proteins and amyloid  peptides . therefore , standardization is important . we propose to use polypropylene tubes , with their low protein binding potential , for collecting csf . when multiple tubes are used , the total volume should be mixed after centrifugation to avoid gradient effects . for biomarkers that are influenced by circadian rhythm , time of withdrawal since it is often difficult to accomplish standardization of withdrawal time in everyday clinical practice , documentation is necessary to select the appropriate samples to minimize the effect of this variable . it is important to collect matched serum and/or plasma samples for evaluation of csf biomarkers because the concentration of the marker in blood often influences that in csf . further , serum / plasma pairs are essential to study the intrathecal origin of a biomarker and its cns specificity . furthermore , the presence of cns markers in serum / plasma may aid in disease monitoring . vacuum tubes that use edta ( in dried format ) are preferred over those that use citrate ( in solution ) because if tubes containing a standard volume of citrate are filled incompletely , the final biomarker concentration is diluted unequally compared to other samples . depending on the type of biomarkers and methods of study , we recommend collecting both serum and plasma ; for some methods , plasma is preferred over serum and vice versa . we advise to perform a blood draw using vacuum systems , since tourniquet use is related to additional confounding factors in the preanalytic phase include tourniquet time and posture . lastly , dna collection expands the possibilities for studying the phenotypes and genotypes within individuals . a protocol for storage and handling of dna can be found in the supplementary files ( e - appendix  1 ) . ( b ) processing for storage : 
 item 9 storage at room temperature until spinning and aliquotingfor csf , there are no data available yet that support a preference for leaving the samples at room temperature or at 4c until processing . for serum / plasma preprocessing temperature therefore , processing at room temperature for both serum / plasma and csf , including during and after spinning , is suitable for most studies . we would recommend exploratory studies to define the effect of temperature on specific biomarkers . item 10 standardized spinning conditionswe propose to adhere to a standardized spinning protocol of 400  g for 10 minutes at room temperature when fragile cells need to be preserved for rna of cell isolation , and otherwise at 2,000  g. for serum / plasma , we propose to spin at 2,000  g for 10  min at room temperature . standardization of spinning temperature and speed may be important for some biomarkers , although no studies have addressed these specific preanalytical variables for csf . for plasma and serum , temperature of processing item 11 standardization of time - delay between withdrawal , spinning , and freezingstudies of the effects of preanalytical variables by maldi - tof / ms proteomics ( proteins / peptides < 20  kd ) have shown that the time between sampling and storage is more crucial for specific serum proteins or peptides than for csf , [ 12 , 13 , 23 ] . for csf , it was observed that processing within two hours does not lead to artefactual results [ 12 , 13 ] . for serum , it was observed that small differences in processing time ( ~1030  min ) can result in changes in the protein profile . some biomarkers , such as antibodies or specific cytokines , are not very sensitive to sampling and storage conditions . for practical reasons , and in view of the standard of 3060  min clotting time for serum , we recommend a time delay of 1.5 hours ( 30  min ) for both matrices . when csf cells are to be preserved , processing as soon as possible is to be advised as cell numbers decrease quickly . however , in most of the centers , processing of the body fluid samples within one hour is not common practice . therefore , documentation of time of withdrawal and storage is required in order to select uniform samples . for newly discovered biomarkers item 12 use of small polypropylene tubes for aliquotingdue to the same rationale as for csf withdrawal ( item 5 ) , we recommend that polypropylene tubes should be used for aliquoting and storage . . 
 item 13 aliquotingfreeze / thaw cycles can influence biomarker concentrations . for example , one - time freezing of csf samples can lead to a highly significant loss of amyloid (1 - 42 ) which is decreased a further 20% after three more thawing cycles [ 26 , 27 ] . by contrast , no effects on csf proteome profiles obtained by maldi - tof / ms have been observed after up to four freeze / thaw cycles .in principle , repeated freeze / thawing of samples should be avoided , as data addressing this topic are available for only a few biomarkers and the response to freeze / thaw cycles of new biomarkers is not known . thus , splitting the pooled sample in multiple small aliquots is optimal , and possible freeze / thaw cycles should be recorded . item 14 volumes of aliquots of 0.2 , 0.5 , and 1  mlsmall aliquot volumes are optimal to avoid freeze / thawing and to avoid waste of csf . tubes should be filled up to 75% to prevent freeze - drying within the tube , which will affect the concentration of biomarkers , although it may only be a problem if the seal of the cryogenic tubes are not airtight . this issue has not been formally studied and is not referred to in related standard operating procedures . item 15 coding and use of freezing - proof labelsunique codes are necessary to track samples and pair with clinical data . ideally barcodes should be used to facilitate searching , to aid in blinding the analysis , and to protect the privacy of patients . item 9 storage at room temperature until spinning and aliquotingfor csf , there are no data available yet that support a preference for leaving the samples at room temperature or at 4c until processing . for serum / plasma preprocessing temperature is more crucial . to avoid platelet activation , therefore , processing at room temperature for both serum / plasma and csf , including during and after spinning , is suitable for most studies . we would recommend exploratory studies to define the effect of temperature on specific biomarkers . item 10 standardized spinning conditionswe propose to adhere to a standardized spinning protocol of 400  g for 10 minutes at room temperature when fragile cells need to be preserved for rna of cell isolation , and otherwise at 2,000  g. for serum / plasma , we propose to spin at 2,000  g for 10  min at room temperature . standardization of spinning temperature and speed may be important for some biomarkers , although no studies have addressed these specific preanalytical variables for csf . for plasma and serum , temperature of processing item 11 standardization of time - delay between withdrawal , spinning , and freezingstudies of the effects of preanalytical variables by maldi - tof / ms proteomics ( proteins / peptides < 20  kd ) have shown that the time between sampling and storage is more crucial for specific serum proteins or peptides than for csf , [ 12 , 13 , 23 ] . for csf , it was observed that processing within two hours does not lead to artefactual results [ 12 , 13 ] . for serum , it was observed that small differences in processing time ( ~1030  min ) can result in changes in the protein profile . some biomarkers , such as antibodies or specific cytokines , are not very sensitive to sampling and storage conditions . for practical reasons , and in view of the standard of 3060  min clotting time for serum , we recommend a time delay of 1.5 hours ( 30  min ) for both matrices . when csf cells are to be preserved , processing as soon as possible is to be advised as cell numbers decrease quickly . however , in most of the centers , processing of the body fluid samples within one hour is not common practice . therefore , documentation of time of withdrawal and storage is required in order to select uniform samples . for newly discovered biomarkers item 12 use of small polypropylene tubes for aliquotingdue to the same rationale as for csf withdrawal ( item 5 ) , we recommend that polypropylene tubes should be used for aliquoting and storage . . 
 item 13 aliquotingfreeze / thaw cycles can influence biomarker concentrations . for example , one - time freezing of csf samples can lead to a highly significant loss of amyloid (1 - 42 ) which is decreased a further 20% after three more thawing cycles [ 26 , 27 ] . by contrast , no effects on csf proteome profiles obtained by maldi - tof / ms have been observed after up to four freeze / thaw cycles .in principle , repeated freeze / thawing of samples should be avoided , as data addressing this topic are available for only a few biomarkers and the response to freeze / thaw cycles of new biomarkers is not known . thus , splitting the pooled sample in multiple small aliquots is optimal , and possible freeze / thaw cycles should be recorded . item 14 volumes of aliquots of 0.2 , 0.5 , and 1  mlsmall aliquot volumes are optimal to avoid freeze / thawing and to avoid waste of csf . tubes should be filled up to 75% to prevent freeze - drying within the tube , which will affect the concentration of biomarkers , although it may only be a problem if the seal of the cryogenic tubes are not airtight . this issue has not been formally studied and is not referred to in related standard operating procedures . item 15 coding and use of freezing - proof labelsunique codes are necessary to track samples and pair with clinical data . ideally barcodes should be used to facilitate searching , to aid in blinding the analysis , and to protect the privacy of patients . labels must be water and frost ( 80c ) resistant . 
  for csf , there are no data available yet that support a preference for leaving the samples at room temperature or at 4c until processing . for serum / plasma preprocessing temperature therefore , processing at room temperature for both serum / plasma and csf , including during and after spinning , is suitable for most studies . we propose to adhere to a standardized spinning protocol of 400  g for 10 minutes at room temperature when fragile cells need to be preserved for rna of cell isolation , and otherwise at 2,000  g. for serum / plasma , we propose to spin at 2,000  g for 10  min at room temperature . standardization of spinning temperature and speed may be important for some biomarkers , although no studies have addressed these specific preanalytical variables for csf . for plasma and serum , temperature of processing studies of the effects of preanalytical variables by maldi - tof / ms proteomics ( proteins / peptides < 20  kd ) have shown that the time between sampling and storage is more crucial for specific serum proteins or peptides than for csf , [ 12 , 13 , 23 ] . for csf , it was observed that processing within two hours does not lead to artefactual results [ 12 , 13 ] . for serum , it was observed that small differences in processing time ( ~1030  min ) can result in changes in the protein profile . some biomarkers , such as antibodies or specific cytokines , are not very sensitive to sampling and storage conditions . for practical reasons , and in view of the standard of 3060  min clotting time for serum , we recommend a time delay of 1.5 hours ( 30  min ) for both matrices . when csf cells are to be preserved , processing as soon as possible is to be advised as cell numbers decrease quickly . however , in most of the centers , processing of the body fluid samples within one hour is not common practice . therefore , documentation of time of withdrawal and storage is required in order to select uniform samples . for newly discovered biomarkers , these preanalytical variables should be evaluated . due to the same rationale as for csf withdrawal ( item 5 ) , we recommend that polypropylene tubes should be used for aliquoting and storage . for example , one - time freezing of csf samples can lead to a highly significant loss of amyloid (1 - 42 ) which is decreased a further 20% after three more thawing cycles [ 26 , 27 ] . by contrast , no effects on csf proteome profiles obtained by maldi - tof / ms have been observed after up to four freeze / thaw cycles . in principle , repeated freeze / thawing of samples should be avoided , as data addressing this topic are available for only a few biomarkers and the response to freeze / thaw cycles of new biomarkers is not known . thus , splitting the pooled sample in multiple small aliquots is optimal , and possible freeze / thaw cycles should be recorded . small aliquot volumes are optimal to avoid freeze / thawing and to avoid waste of csf . tubes should be filled up to 75% to prevent freeze - drying within the tube , which will affect the concentration of biomarkers , although it may only be a problem if the seal of the cryogenic tubes are not airtight . this issue has not been formally studied and is not referred to in related standard operating procedures . unique codes are necessary to track samples and pair with clinical data . ideally barcodes should be used to facilitate searching , to aid in blinding the analysis , and to protect the privacy of patients . ( c ) storage and administration of samples ( table 2 , lower part ) : 
 item 16 freezing temperature of 80cproteins may not be stable at 20c for years . in one study , the effect of storing csf at 20c and 80c on cystatin c , an abundant csf protein , was investigated . cleavage of this protein occurred in all samples stored at 20c but not in samples stored at 80c . apart from the cystatin c truncation , changes in the low molecular weight polypeptide profile due to csf sample storage at 20c for three months appeared to be minimal [ 12 , 13 ] . oligoclonal bands in csf may be recovered after several years of storage at 20c indicating a high stability of immunoglobulins . no data are available showing the benefit of storage of csf or serum in liquid nitrogen . as this is expensive and not practical for csf biobanking , there is no basis yet to recommend storage in liquid nitrogen.taken together , we recommend that samples are stored at 80c to ensure long - term stability of biomarkers . item 17 location of samplesto enable easy tracking and fast relocation of samples , storage information should include freezer location , freezer identification , and sample location within freezer . items 18 and 19 : surveillance of freezers and splitting of samplesfreezers should be alarm controlled and a sample rescue plan established and documented . aliquots of samples should be distributed among different freezers , although not absolutely needed if good surveillance is in place . item 20 : transport conditions and thawing before usetransport of frozen samples should always be performed on dry - ice , and the volume should be sufficient for transport for minimal 3 days . preferably , transports are initiated on monday for the samples to arrive within the same week . once the samples have arrived and are ready for experiments , excessive thawing temperatures ( such as 37c ) are to be avoided to prevent protein degradation . furthermore , inadequate agitation can cause salt and protein gradients to form in thawed samples . item 16 freezing temperature of 80cproteins may not be stable at 20c for years . in one study , the effect of storing csf at 20c and 80c on cystatin c , an abundant csf protein , was investigated . cleavage of this protein occurred in all samples stored at 20c but not in samples stored at 80c . apart from the cystatin c truncation , changes in the low molecular weight polypeptide profile due to csf sample storage at 20c for three months appeared to be minimal [ 12 , 13 ] . oligoclonal bands in csf may be recovered after several years of storage at 20c indicating a high stability of immunoglobulins . no data are available showing the benefit of storage of csf or serum in liquid nitrogen . as this is expensive and not practical for csf biobanking , there is no basis yet to recommend storage in liquid nitrogen.taken together , we recommend that samples are stored at 80c to ensure long - term stability of biomarkers . item 17 location of samplesto enable easy tracking and fast relocation of samples , storage information should include freezer location , freezer identification , and sample location within freezer . 
 items 18 and 19 : surveillance of freezers and splitting of samplesfreezers should be alarm controlled and a sample rescue plan established and documented . aliquots of samples should be distributed among different freezers , although not absolutely needed if good surveillance is in place . item 20 : transport conditions and thawing before usetransport of frozen samples should always be performed on dry - ice , and the volume should be sufficient for transport for minimal 3 days . preferably , transports are initiated on monday for the samples to arrive within the same week . once the samples have arrived and are ready for experiments , excessive thawing temperatures ( such as 37c ) are to be avoided to prevent protein degradation . furthermore , inadequate agitation can cause salt and protein gradients to form in thawed samples . the effect of storing csf at 20c and 80c on cystatin c , an abundant csf protein , was investigated . cleavage of this protein occurred in all samples stored at 20c but not in samples stored at 80c . apart from the cystatin c truncation , changes in the low molecular weight polypeptide profile due to csf sample storage at 20c for three months appeared to be minimal [ 12 , 13 ] . oligoclonal bands in csf may be recovered after several years of storage at 20c indicating a high stability of immunoglobulins . no data are available showing the benefit of storage of csf or serum in liquid nitrogen . as this is expensive and not practical for csf biobanking , there is no basis yet to recommend storage in liquid nitrogen . taken together , we recommend that samples are stored at 80c to ensure long - term stability of biomarkers . to enable easy tracking and fast relocation of samples , storage information should include freezer location , freezer identification , and sample location within freezer . aliquots of samples should be distributed among different freezers , although not absolutely needed if good surveillance is in place . transport of frozen samples should always be performed on dry - ice , and the volume should be sufficient for transport for minimal 3 days . preferably , transports are initiated on monday for the samples to arrive within the same week . once the samples have arrived and are ready for experiments , excessive thawing temperatures ( such as 37c ) are to be avoided to prevent protein degradation . furthermore , inadequate agitation can cause salt and protein gradients to form in thawed samples . ( d ) patient information requirement in database : 
 items 21 - 22 basic demographics , such as age and genderinformation on the age at sampling is needed to allow comparability to age - matched reference values , since many proteins show age - dependent changes , for example , albumin or igg . ideally , date of birth and date of sampling are recorded . for example , a recent study observed a higher igg index in african americans than in caucasians , unrelated to socioeconomic status . criteria for race and ethnicity are available via the website of the national institutes of health . item 24 treatment at sampling and year before samplingit is well known that commonly used drugs for treatment of ms , including immunomodulatory agents and use of methylprednisolone for treatment or prevention of relapses , have an influence on expression of biomarkers [ 34 , 35 ] . therefore , type and duration of treatment should be documented in detail , preferably beginning at least one year before csf collection . item 25 fasting , infections and pregnanciesother relevant physiological variables that can influence csf and blood analyte levels should be recorded including fasting versus non fasting , pregnancy , and underlying nonneurological conditions such as infections . item 26 basic csf analysis ( protein , cell counts , erythrocytes , etc  )to enable stratification of patients according to their csf findings and to evaluate suitability of samples for further analysis , results of basic csf analysis should be recorded . in addition , quantitative changes of immunological markers are likely to occur depending on disease stage , relapse activity , and medication . inflammatory processes may influence the blood - csf barrier function and thereby biomarker concentrations .the presence of oligoclonal igg bands ( ogb ) in nmo is quite distinct from that in ms in that ogbs in ms are persistent , while they are transient in nmo [ 36 , 37 ] . for example , ogbs were detected in 399 of 411 ms patients ( 97% ) and never disappeared . in nmo , ogbs were detected in three of 11 patients ( 27% ) and always disappeared . we strongly recommend isoelectric focusing followed by immunoblotting and staining for igg [ 38 , 39 ] . preferably , the methods of all routine diagnostic procedures , including oligoclonal banding , should be documented . item 27 data in the csf database in englishthe mask on the database screen could be in the local language , but the underlying files will need to be in english . it is strongly suggested to use a commercially available program , if not a common database for networks like bioms - eu . items 21 - 22 basic demographics , such as age and genderinformation on the age at sampling is needed to allow comparability to age - matched reference values , since many proteins show age - dependent changes , for example , albumin or igg . for example , a recent study observed a higher igg index in african americans than in caucasians , unrelated to socioeconomic status . criteria for race and ethnicity are available via the website of the national institutes of health . item 24 treatment at sampling and year before samplingit is well known that commonly used drugs for treatment of ms , including immunomodulatory agents and use of methylprednisolone for treatment or prevention of relapses , have an influence on expression of biomarkers [ 34 , 35 ] . therefore , type and duration of treatment should be documented in detail , preferably beginning at least one year before csf collection . item 25 fasting , infections and pregnanciesother relevant physiological variables that can influence csf and blood analyte levels should be recorded including fasting versus non fasting , pregnancy , and underlying nonneurological conditions such as infections . item 26 basic csf analysis ( protein , cell counts , erythrocytes , etc  )to enable stratification of patients according to their csf findings and to evaluate suitability of samples for further analysis , results of basic csf analysis should be recorded . in addition , quantitative changes of immunological markers are likely to occur depending on disease stage , relapse activity , and medication . inflammatory processes may influence the blood - csf barrier function and thereby biomarker concentrations .the presence of oligoclonal igg bands ( ogb ) in nmo is quite distinct from that in ms in that ogbs in ms are persistent , while they are transient in nmo [ 36 , 37 ] . for example , ogbs were detected in 399 of 411 ms patients ( 97% ) and never disappeared . in nmo , we strongly recommend isoelectric focusing followed by immunoblotting and staining for igg [ 38 , 39 ] . preferably , the methods of all routine diagnostic procedures , including oligoclonal banding , should be documented . item 27 data in the csf database in englishthe mask on the database screen could be in the local language , but the underlying files will need to be in english . it is strongly suggested to use a commercially available program , if not a common database for networks like bioms - eu . information on the age at sampling is needed to allow comparability to age - matched reference values , since many proteins show age - dependent changes , for example , albumin or igg . for example , a recent study observed a higher igg index in african americans than in caucasians , unrelated to socioeconomic status . criteria for race and ethnicity are available via the website of the national institutes of health . it is well known that commonly used drugs for treatment of ms , including immunomodulatory agents and use of methylprednisolone for treatment or prevention of relapses , have an influence on expression of biomarkers [ 34 , 35 ] . therefore , type and duration of treatment should be documented in detail , preferably beginning at least one year before csf collection . other relevant physiological variables that can influence csf and blood analyte levels should be recorded including fasting versus non fasting , pregnancy , and underlying nonneurological conditions such as infections . to enable stratification of patients according to their csf findings and to evaluate suitability of samples for further analysis , results of basic csf analysis should be recorded . in addition , quantitative changes of immunological markers are likely to occur depending on disease stage , relapse activity , and medication . inflammatory processes may influence the blood - csf barrier function and thereby biomarker concentrations . the presence of oligoclonal igg bands ( ogb ) in nmo is quite distinct from that in ms in that ogbs in ms are persistent , while they are transient in nmo [ 36 , 37 ] . for example , ogbs were detected in 399 of 411 ms patients ( 97% ) and never disappeared . in nmo , we strongly recommend isoelectric focusing followed by immunoblotting and staining for igg [ 38 , 39 ] . preferably , the methods of all routine diagnostic procedures , including oligoclonal banding , should be documented . the mask on the database screen could be in the local language , but the underlying files will need to be in english . it is strongly suggested to use a commercially available program , if not a common database for networks like bioms - eu . the lists provided in table 2 can be used as an easy checklist for csf biobanking for any cns disease , applicable during setup of the procedures and also as a checklist for recording sample characteristics . it is expected that these standardizations will pave the way for large biomarker studies and fruitful collaborations . in the original paper , we present guidelines for outcome measures to be included for ms biomarker studies . for nmo , ultimately , these endeavors are to arrive at validated biomarker assays for diagnosis , prognosis , and treatment of cns diseases and a potential to elucidate relevant disease mechanisms .
there is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases . 
 however , only a few biomarkers in cerebrospinal fluid ( csf ) are being used in clinical practice . 
 anti - aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica ( nmo ) , but we could expect novel csf biomarkers that help define prognosis and response to treatment for this disease . 
 one of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers . 
 collaboration between investigators is needed to establish large biobanks of well - defined samples . 
 a key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of csf samples is not compromised by pre - analytical factors . here , consensus guidelines for csf collection and biobanking are presented , based on the guidelines that have been published by the bioms - eu network for csf biomarker research . 
 we focussed on csf collection procedures , pre - analytical factors and high quality clinical and paraclinical information . 
 importantly , the biobanking protocols are applicable for csf biobanks for research targeting any neurological disease .