Introduction: Freeze-thaw instability may contribute to preanalytical variation in blood-based biomarker studies. We investigated the effects of up to four freeze-thaw cycles on single molecule array immunoassays of serum neurofilament light chain and plasma total tau, amyloid β 1-40 (Aß40), and Aβ 1-42 (Aβ42). Methods: Individuals who had peripheral venepuncture during investigation of suspected neurodegenerative disease were recruited. After standardized preprocessing, 200 μL of plasma and serum aliquots were stored at -80°C within 60 minutes. Aliquots underwent one to four freeze-thaw cycles. Results: There was no significant difference across four freeze-thaw cycles for serum neurofilament light chain (n = 12), plasma total tau (n = 11), or plasma Aβ42 (n = 12). For plasma Aβ40 (n = 14), there were significant median reductions by ratios of .96 and .92 at the third and fourth cycles, respectively. Discussion: Up to four freeze-thaw cycles do not influence single molecule array blood biomarkers of neurofilament light chain, total tau, or Aβ42, with at most minor reductions in Aβ40

Heslegrave, A

Keshavan, A

Schott, JM

Zetterberg, H

UCL Discovery

Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 448-451Blood-Based BiomarkersStability of blood-based biomarkers of Alzheimer’s disease over multiplefreeze-thaw cyclesAshvini Keshavana,b,*, Amanda Heslegraveb,c, Henrik Zetterbergb,c,d,e, Jonathan M. SchottaaDementia Research Centre, Box 16 National Hospital for Neurology and Neurosurgery, London, UKbLeonard Wolfson Biomarkers Laboratory, Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UKcThe DRI Fluid Biomarker Laboratory, UK Dementia Research Institute at UCL, London, UKdDepartment of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg,Sahlgrenska University Hospital, M€olndal, SwedeneClinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, SwedenAbstract Introduction: Freeze-thaw instability may contribute to preanalytical variation in blood-basedThe authors have*Corresponding a483104.E-mail address: a.https://doi.org/10.10162352-8729/ 2018 Tlicense (http://creativebiomarker studies. We investigated the effects of up to four freeze-thaw cycles on single moleculearray immunoassays of serum neurofilament light chain and plasma total tau, amyloid b 1–40(Aß40), and Ab 1–42 (Ab42).Methods: Individuals who had peripheral venepuncture during investigation of suspected neurode-generative disease were recruited. After standardized preprocessing, 200 mL of plasma and serumaliquots were stored at280Cwithin 60 minutes. Aliquots underwent one to four freeze-thaw cycles.Results: Therewas no significant difference across four freeze-thaw cycles for serum neurofilament lightchain (n5 12), plasma total tau (n5 11), or plasma Ab42 (n5 12). For plasmaAb40 (n5 14), thereweresignificant median reductions by ratios of .96 and .92 at the third and fourth cycles, respectively.Discussion: Up to four freeze-thaw cycles do not influence single molecule array blood biomarkersof neurofilament light chain, total tau, or Ab42, with at most minor reductions in Ab40. 2018 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Keywords: Freeze-thaw; Blood; Biomarker; Simoa1. IntroductionPreanalytical factors may be an important source of vari-ation among studies investigating putative blood-based bio-markers of Alzheimer’s disease, in which replication acrossstudies has posed a significant challenge. Guidelines forstandardizing these preanalytic variables have stated that itis desirable to minimize the number of freeze-thaw cyclesto which samples are exposed [1]. The evidence for thiscomes predominantly from studies showing that more thanthree freeze-thaw cycles reduce plasma amyloid b 1–40and 1–42 (Ab40 and Ab42) concentrations by as much asdeclared that no conflict of interest exists.uthor. Tel.: (144) 2034 483011; Fax: (144) 2034keshavan@ucl.ac.uk/j.dadm.2018.06.001he Authors. Published by Elsevier Inc. on behalf of the Alzhecommons.org/licenses/by-nc-nd/4.0/).20% [2] and also reduce cerebrospinal fluid (CSF) Ab42 con-centration [3] as quantified on immunoassay platforms.There is some conflicting evidence on the stability in CSFof total tau, which in one report remained stable over up tosix freeze-thaw cycles [3], but in another study, both CSF to-tal tau and phospho-tau-181 concentrations as measured byenzyme-linked immunosorbent assay reduced over threefreeze-thaw cycles [4]. Conversely, CSF neurofilament lightchain (NFL) remains stable over up to four freeze-thaw cy-cles [5]. However, there has been no systematic examinationof the stability of these biomarkers in blood. More recently,the Single molecule array (Simoa) HD-1 analyzer, a highlysensitive digital immunoassay platform, has been developed,which is capable of measuring these and other biomarkerswith sensitivity in the femtomolar range [6]. In this study,we examined the effects of up to four freeze-thaw cycleson serum NFL and plasma total tau, Ab40, and Ab42.imer’s Association. This is an open access article under the CC BY-NC-NDA. Keshavan et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 448-451 4492. Methods2.1. ParticipantsWerecruited individuals seen at the specialist cognitive disor-ders clinics at the National Hospital for Neurology and Neuro-surgery, who were having diagnostic lumbar puncture withpaired venous blood testing for investigation of a suspectedneurodegenerative condition. At the time of clinical sampling,all individuals donated an additional blood sample for research,andbasic demographic datawere recorded.All participants gavewritten informed consent, or for thosewhowere deemed lackingin capacity to do so by the assessing clinician, proxy consentwasobtained from a consultee. The study was conducted in accor-dance with local clinical research regulations and was approvedby the local ethics committee (reference: 12/0344).2.2. Sample handlingThe plasma was collected in 8-mL ethylenediaminetetra-acetic acid tubes and serum in 6.5-mL serum separator tubegel separator tubes by peripheral venepuncture with a tourni-quet, using either a 21G or 23G butterfly needle and BDVacutainer collecting system. Participants were not in-structed to fast, but all blood samples for each participantwere taken between 9AMand 1 PMon a single venepuncture.Plasma and serumwere centrifuged at 1800! g for 5minutesat room temperature. Plasmawas centrifuged within a medianof 20 minutes after sampling, whereas serum was allowed toclot for a median of 15 minutes before centrifugation. The su-pernatant was pipetted into 200-mL aliquots into identicalpolypropylene tubes, for freezingat280C,within 60minutesof sampling, in keeping with current guidelines [1].2.3. Freeze-thaw cyclesEach additional freeze-thaw cycle after the first consistedof 1 hour of direct thaw to room temperature, followed by1 hour of direct replacement into 280 C. After the finalthaw, samples were transferred to 1.5-mL polypropylenecentrifuge tubes and centrifuged as per the kit-manufacturer’srecommendation at 13,000! g for 10minutes, and the super-natant of each samplewas pipetted onto the plate for analysis.For each biomarker, all aliquots from the same participantwere assayed using the same batch of reagents in the same run.2.4. Blood fraction choiceTo examine the effects of freeze-thaw cycle number, wechose to measure NFL in serum as we and others have shownthat NFL may be measured reliably in serum to show differ-ences between disease groups among neurodegenerative dis-eases [7,8] and that there is a linear relationship betweenplasma and serum NFL within individuals, with serumreturning higher values when paired samples are analyzedon the same run (Supplementary Fig. S1A). Similarly, wechose to measure total tau, Ab40, and Ab42 in plasma as itreturns higher values than serum for these biomarkers(Supplementary Fig. S1B–D).2.5. AnalysisCommercially available Simoa total tau, NFL, Ab40, andAb42 kits (Quanterix) were used according to the manufac-turer’s instructions. The principles of these assays aresimilar; a detailed account of the basis of Simoa technologyhas been provided by Wilson and colleagues [6].All measurements were conducted on the same automatedHD-1 analyzer (Quanterix). For serum NFL, plasma Ab40,and plasma Ab42, all samples were assayed in duplicate.For plasma tau, all samples were assayed in triplicate becauseof kit availability. The mean of the replicates for each samplewas calculated and represented graphically. Results wereincluded in the analysis if the coefficient of variation acrossreplicates was ,15%. The number of individuals includedwas therefore as follows: serum NFL, 12; plasma total tau,11; plasma Ab40, 14; and plasma Ab42, 12.2.6. StatisticsA Friedman analysis of variance (ANOVA) test (nonpara-metric repeated measures) was used to assess differenceswithin individuals across four freeze-thaw cycles (SPSS Sta-tistics, version 24). Where statistically significant differ-ences were found at the P 5 .05 level, the median ratio ofthe concentration at a specific freeze-thaw cycle to the con-centration at the first cyclewas calculated across individuals.3. ResultsFig. 1 shows the measured concentration of each analyteagainst the number of freeze-thaw cycles.The Friedman ANOVA test showed a two-tailed signifi-cance of ..05 for a change in the means for serum NFL,plasma total tau, and plasma Ab42 over four cycles.For plasma Ab40, the median concentration ratio betweenthe third and first cycles was .96 (interquartile range, .92–.99; Friedman ANOVA P5 .015). The median concentrationratio between the fourth and first cycles was .92 (interquar-tile range, .90–.96; Friedman ANOVA P 5 .001), and thissurvived the exclusion of the one clear outlier individual.4. DiscussionWeshow that for up to four freeze-thawcycles, each consist-ing of at least 1 hour of thawing to room temperature, in aliquotvolumes of 200mL, there is no consistent trend for change in theconcentrations of serumNFL, plasma total tau, or plasmaAb42as measured using the Simoa digital immunoassay platform.We found some evidence for a small reduction in plasmaAb40 after the third and further after the fourth cycle. This latterfinding is in keeping with the experience of Lachno and col-leagues, who used a Luminex bead-based immunoassay plat-form and a 1-hour thaw per cycle to demonstrate a reductionSerum NFL Plasma total tauPlasma Aß40 Plasma Aß4210203040501 2 3 4pg/mlNumber of freeze-thaw cycles1234561 2 3 4pg/mlNumber of freeze-thaw cycles1001502002503003501 2 3 4pg/mlNumber of freeze-thaw cycles5101520251 2 3 4pg/mlNumber of freeze-thaw cyclesA BC DFig. 1. Concentration of the biomarker versus number of freeze-thaw cycles for (A) serum NFL, n5 12; (B) plasma total tau, n5 11; (C) plasma Ab40, n5 14;and (D) Plasma Ab42, n 5 12. Each colored line represents an individual. Abbreviations: Ab, amyloid b; NFL, neurofilament light chain.A. Keshavan et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 448-451450in plasmaAb 1–40 from the fourth cycle in five individuals [2].Here,we show, in a larger sample size, a statistically significant,albeit very small, reduction by amedian ratio of 0.96 at the thirdcycle and 0.92 at the fourth cycle relative to the first. The latterreduction survived removal of an outlier individual for whomthere was a ratio of 0.38 between the concentrations at fourthand first cycles but no obvious technical difference identifiedin sample treatment. It is not clear whether the results fromthis outlier were in any way related to the freeze-thaw effect,but in keeping with the group results, we suggest that samplesin which plasma Ab40 is to be measured should be restrictedto a maximum of two freeze-thaw cycles.The conditions chosen in this study are consistent withthe aliquot volumes and treatment recommended by currentconsensus guidelines [1] and mimic the likely conditions inlarge-cohort studies on blood-based biomarkers of neurode-generative disease, in which minimizing sample volumes isdesirable and cross-validation between laboratories will beessential. The ability to subaliquot samples will allow forsharing of samples across centers while reducing concernsabout the effects of small numbers of short-durationfreeze-thaw cycles on measured analyte concentrations.AcknowledgmentsThe authors are grateful to all the study participants; to DrRoss Paterson who helped with the study cohort and tocollect the samples; to Jamie Toombs, Martha Foiani, andElena Veleva who processed the samples before initial stor-age; and to Dr Jennifer Nicholas for statistical advice.This work was funded as part of a Leonard Wolfson Exper-imental Neurology Clinical Fellowship awarded to A.K.Supplementary dataSupplementary data related to this article can be found athttps://doi.org/10.1016/j.dadm.2018.06.001.A. Keshavan et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 10 (2018) 448-451 451RESEARCH IN CONTEXT1. Systematic review: The authors reviewed the litera-ture using PubMed. There are several publicationson preanalytical factors including the effects offreeze-thaw cycles on core cerebrospinal fluid(CSF), biomarkers of Alzheimer’s disease (AD), andCSF neurofilament light chain (NFL), but there hasbeen no systematic examination of the effects ofmultiple freeze-thaw cycles of these biomarkers inblood despite the existence of a growing literature onthe use of these blood biomarkers of AD in largecohorts.2. Interpretation: This work demonstrates that up tofour freeze-thaw cycles on aliquot volumes of200 mL do not affect measured concentrations ofserumNFL or plasma total tau and Ab42. Beyond twofreeze-thaw cycles, plasma Ab40 may drop signifi-cantly in a few samples.3. Future directions: Sample sharing across researchcenters may be facilitated by the ability to freezeand thaw small aliquots up to the limits describedin this article.References[1] O’Bryant SE, Gupta V, Henriksen K, Edwards M, Jeromin A, Lista S,et al. Guidelines for the standardization of preanalytic variables forblood-based biomarker studies in Alzheimer’s disease research. Alz-heimers Dement 2015;11:549–60.[2] Lachno DR, Emerson JK, Vanderstichele H, Gonzales C, Martenyi F,Konrad RJ, et al. Validation of a multiplex assay for simultaneous quan-tification of amyloid-beta peptide species in human plasma with utilityfor measurements in studies of Alzheimer’s disease therapeutics. J Alz-heimers Dis 2012;32:905–18.[3] Schoonenboom NS, Mulder C, Vanderstichele H, Van Elk EJ,Kok A, Van Kamp GJ, et al. Effects of processing and storageconditions on amyloid beta (1-42) and tau concentrations in cere-brospinal fluid: implications for use in clinical practice. ClinChem 2005;51:189–95.[4] Simonsen AH, Bahl JM, Danborg PB, Lindstrom V, Larsen SO,Grubb A, et al. Pre-analytical factors influencing the stability of cere-brospinal fluid proteins. J Neurosci Methods 2013;215:234–40.[5] Koel-Simmelink MJ, Vennegoor A, Killestein J, Blankenstein MA,Norgren N, Korth C, et al. The impact of pre-analytical variables onthe stability of neurofilament proteins in CSF, determined by a novelvalidated SinglePlex Luminex assay and ELISA. J Immunol Methods2014;402:43–9.[6] Wilson DH, Rissin DM, Kan CW, Fournier DR, Piech T, Campbell TG,et al. The simoa HD-1 analyzer: a novel fully automated digital immu-noassay analyzer with single-molecule sensitivity and multiplexing. JLab Autom 2016;21:533–47.[7] Rohrer JD,Woollacott IO,DickKM,BrotherhoodE,GordonE, FellowsA,et al. Serum neurofilament light chain protein is ameasure of disease inten-sity in frontotemporal dementia. Neurology 2016;87:1329–36.[8] Weston PSJ, Poole T, Ryan NS, Nair A, Liang Y, Macpherson K, et al.Serum neurofilament light in familial Alzheimer disease: a marker ofearly neurodegeneration. Neurology 2017;89:2167–75.

Stability of blood-based biomarkers of Alzheimer's disease over multiple freeze-thaw cycles.

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Stability of blood-based biomarkers of Alzheimer's disease over multiple freeze-thaw cycles.

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