in the aging population the number of alzheimer disease ( ad ) patients is expected to increase . however , the diagnostic accuracy of the clinical criteria is relatively low ( sensitivity 80% and specificity of 70% ) ) . with this in mind , biological markers in body fluids are urgently needed to sustain diagnosis , as they are an objective tool and reflect ongoing processes . biomarkers can aid not only in early diagnosis or in differential diagnosis but also in estimation of prognosis and , ideally , monitoring progression of this disease . the concentrations of amyloid - beta(1 - 42 ) ( a42 ) , total tau ( tau ) , and tau phosphorylated at position 181 ( ptau ) in cerebrospinal fluid ( csf ) of ad patients can be used as biomarkers . several laboratories measure these three biomarkers in csf , and a major challenge is to translate the technology from the lab to clinical practice . to reach this goal , the technique should be robust and laboratories should be adequately experienced . in addition , results obtained in different centres should be comparable to the highest possible degree . the comparability of results between different centres is crucially dependent on the performance of the biomarker tests in the various institutions , and this can be assessed with an external quality assessment scheme . no such scheme was available and that is why we took the initiative in 2004 to send samples to a number of laboratories with previous experience in performing these csf biomarker assays , with their own elisa assays . the results revealed large variation in the concentrations of the three biomarkers between the different laboratories and a difference in variation at each evaluated time point . overall variation for tau was slightly better in 2008 than in 2004 , since the mean interlaboratory cv was 21% in 2004 and 16% in 2008 . for ptau the mean between - laboratory cv increased slightly , that is , from 13% in 2004 to 15% in 2008 . the largest overall change was seen for a42 , where the between - laboratory variation increased from 31% to 37% . the introduction of other elisa methods appeared to be responsible for this overall increase in variation . laboratories that used the innotest assays improved the between - laboratory variation of a42 from 30% in 2004 to 22% in 2008 , suggesting that experience and standardization of assay procedure may contribute significantly to reduce between laboratory variation . with the aim to improve between - laboratory performance we set out to identify the specific differences in procedures between laboratories . for this , we organised a hands - on workshop at the end of 2009 . 26 participants from 17 different international centres with previous experience in performing the assays were divided in 14 groups , and every group performed the a42 , tau , and ptau assays . identical samples containing pooled anonymised csf samples , with concentrations of these biomarkers covering concentrations observed in controls , in ad patients and an intermediate value , were provided to the groups . standard curves were diluted by each group according to the protocol , starting with dissolving the solid powder . all groups analysed the same samples , and the assays were performed simultaneously in the same laboratory and used the protocol ( incubation procedures ) as provided by the manufacturer . one exception was the incubation temperature of the tau assay , which should be in an incubator at 25  2c , and was at room temperature due to practical reasons . during the performance of the assays , the two persons in every group discussed their usual laboratory practice in performing the assays and differences were recorded . the standard curves were calculated using a 5-parameter logistic ( 5pl ) curve fitting on the biorad microplate manager software . the mean and standard deviations of the concentrations in the pools were calculated per group . table 1 lists the items that were noticed to vary among the laboratories participating in the workshop . the items involved procedures for pipetting , incubation , washing , finishing , and sample handling . several items listed in table 1 were standardised during the workshop due to its setup . therefore , we were able to evaluate the inter - assay variation under circumstances that excluded several items that varied between the laboratories ( table 2 ) , providing an indication of the relative contribution of these sources of error . the cause was identified as lack of experience ( group 3 ) and an error in the calculation of the dilution of the standard to make the standard curve ( group 11 ) . for tau , no outliers were identified while , for ptau , the outcomes of the concentration of the highest pool were deviating in group 14 . the intra - assay variation based on the concentrations of the unknowns was high for pool 1 and 2 of abeta but on average was below 10% for tau and ptau . the principle aim of this study was to address the issue of procedural differences as a source of variation between outcomes of csf biomarker analysis . one of the results of workshop was a list with differences in procedures among the labs , containing 23 items . examples of these items are the use of polypropylene plate for sample predilution , the inclusion of 1500  pg / ml standard in the curve , the use of 50  l h2so4 in the a42 assay , and the use of 5pl curve fitting and incubation temperature of 25  2c for total tau . the type of pipette is not prescribed by the manufacturer , neither was the mode of pipetting . we do not expect wiping off the tips , which was historically done with specific types of tissues ; inverse pipetting and the pipette brand are factors inducing much variation , as long as one mode of pipetting of the samples is consistently used during the assays . the use of a single pipette versus using a multichannel pipette may influence the time needed to fill an entire plate and can be important when incubation time is short , such as for a42 . for preparation of standards a new tip for each concentration is required . during pipetting in the plate , however , the magnitude of this effect , if any , should be tested , to provide a better basis for recommendation . incubation temperatures , incubation in the dark , and shaking were other items that varied between the laboratories . whether this variation in incubation conditions is relevant for the current tests is not known but likely . this should be tested , and explicit information regarding influence of shaking and temperature requirements should be provided in assay protocols . regarding exclusion of the 1500  pg / ml standard in the curve , we did a recalculation of the pools for one of the groups on a curve fitting without 1500  pg / ml standard and the outcomes differed by 7.6% , 9.4% , and 7.2% for pool 1 , 2 and 3 from the original data . washing procedures ( number of washing steps , volume , purity of washing solutions ) are important issues influencing variation and background . this can lead to removing antibody - antigen complexes from the plate when washing too much , as well as insufficient removal of unbound complexes , thereby causing high background . furthermore , incomplete dissolution of crystals from the washing buffer may lead to aberrant buffer concentration when only part of the buffer is used .   the difference in volume of the stop - solution h2so4 probably does not influence the outcomes , as long as it is performed for all samples in one test similarly , as the ph of the reaction mixture is only marginally influenced by variation in the volume of the stop solution . sample handling ( storage at 4c or repeated freezing ) can be very important as well , specifically for instable proteins . for the current proteins , a42 , tau , and ptau , a previous study has shown that repeated freezing and storage at 4c do not have a significant effect on tau concentrations while a42 concentrations may be reduced due to storage for a few days at 4c . results from another study , however , reported no effect of storage temperatures on a42 concentrations ( blennow , personal communication ) . closing the vial is important to avoid losing the contents upon accidentally falling , and to avoid contamination . for scientific purposes , ideally , the influence of each of the procedures listed in table 1 on the intra- and interassay variation is systematically tested . the current study excluded several potential sources of error , indicated in the last column of table 1 , such as differences in incubation temperatures , washing , variation in standardised curve fitting , and lot - to - lot variation . the inter - assay variation in results obtained during the workshop was below the limits for intra - assay variation for all assays , with a few exceptions ( table 2 ) , suggesting that standardisation indeed leads to reduced inter - assay variation . this may be caused by increased experience as a42 assay was started with during the workshop and the participants may have needed some time to get used to the laboratory . alternatively , the observed variation may be the normal variation for these assays , as these differences were similar to what has been reported before in   and what we observe in our own laboratory . the importance of lot - to - lot variation is stressed by the results in table 3 , showing the reduction of inter - assay variation in our laboratory when we started using multiple assays from the same batch , purchased at once . the variation could be reduced if for instance clear quality control criteria and who - approved standards would be available . the issue of quality control is currently addressed in a large multicenter study , and our initiative has been adopted by the alzheimers association . standardisation of csf collection and biobanking procedures would be another strategy to tackle pre - analytical variation . we recently published such guidelines for standardised csf collection and biobanking protocols , that was based on a broad consensus between multiple centers . adherence to these guidelines will reduce variation induced by pre - analytical factors as well and increase the quality of studies aimed at discovery and validation of novel biomarkers .   in conclusion , the evaluation of the workshop showed that even under standardised conditions as in this workshop , with the same protocol and laboratory circumstances , inter - assay variation is comparable to intra - assay variation and is acceptable for the large majority of groups . the influence of several items that varied between the labs should be studied and protocols should be adapted accordingly . provision of information on the influence of specific items in the data sheets might be needed to convince the users of this requirement . reduction in variation is most critical for csf concentrations around the cutoff points especially if these guide decision making in individual patients . efforts for standardisation and the establishment of international reference laboratories and reference values will ultimately increase the reliability of the assays . this will provide a basis to include these biomarkers assays more prominently in the diagnostic workup of alzheimer 's disease .
large variation in assay performance and outcomes of csf a1 - 42 , total tau ( tau ) , and phosphorylated tau ( ptau ) ( at amino acid 181 ) levels is observed between laboratories . 
 the aim of this study was to assess the differences in assay procedures between several experienced international laboratories , as potential sources of error . 
 14 groups performed the a42 , tau , and ptau assays according to the guidelines of the manufacturer . 
 differences in analytical procedures between the laboratories were monitored . 
 at least 23 items in assay procedures were identified that varied between the laboratories , including procedures for washing , pipetting , incubation , finishing , and sample handing . in general , the inter- and intra - assay variation between the groups was generally below 10% for all three assays . 
 we concluded that 17 international centers that use the same assays for a42 , tau and ptau on a regular basis do not uniformly adhere to the procedures recommended by the manufacturer . for harmonization of intercenter results of these biomarkers standardization of protocols 
 is highly needed .