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. 2022 Aug;18(8):1484-1497.
doi: 10.1002/alz.12510. Epub 2021 Nov 29.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group

Inge M W Verberk  1 , Els O Misdorp  1 , Jannet Koelewijn  1 , Andrew J Ball  2 , Kaj Blennow  3   4 , Jeffrey L Dage  5 , Noelia Fandos  6 , Oskar Hansson  7 , Christophe Hirtz  8 , Shorena Janelidze  7 , Sungmin Kang  9 , Kristopher Kirmess  10 , Jana Kindermans  8 , Ryan Lee  9 , Matthew R Meyer  10 , Dandan Shan  2 , Leslie M Shaw  11 , Teresa Waligorska  11 , Tim West  10 , Henrik Zetterberg  3   4   12   13 , Rebecca M Edelmayer  14 , Charlotte E Teunissen  1
Affiliations

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group

Inge M W Verberk et al. Alzheimers Dement. 2022 Aug.

Abstract

Introduction: Pre-analytical sample handling might affect the results of Alzheimer's disease blood-based biomarkers. We empirically tested variations of common blood collection and handling procedures.

Methods: We created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze-thawing. We measured amyloid beta (Aβ)42 and 40 peptides with six assays, and Aβ oligomerization-tendency (OAβ), amyloid precursor protein (APP)699-711 , glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t-tau), and phosphorylated tau181.

Results: Collection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aβ and t-tau; t-tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations.

Discussion: We constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood-based biomarkers into the research and clinical settings.

Keywords: Alzheimer's disease; amyloid beta; plasma biomarkers; pre-analytics; stability; tau.

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Conflict of interest statement

Inge Verberk, Els Misdorp, Jannet Koelewijn, Jana Kindermans, and Teresa Waligorska report no financial disclosures nor conflicts of interest related to the present work or outside the submitted work. Andrew Ball and Dandan Shan are employees of Quanterix Corporation, and hold Quanterix Corporation stock and stock options. Simoa assay development work associated with this study was performed at and funded by Quanterix Corporation. Andrew Ball received a National Institutes of Health (NIH) Rapid Acceleration of Diagnostics grant (RADx 5534) for development/scaling of a SARS‐CoV‐2 assay, which was paid to the institution and was not associated with this study, and resulted in two provisional patent applications. Outside the submitted work, Kaj Blennow has served as a consultant or at advisory boards for Abcam, Axon, Biogen, JOMDD/Shimadzu, Lilly, MagQu, Prothena, Roche Diagnostics, and Siemens Healthineers, and as data monitoring committee for Julius Clinical and Novartis. All payments were made to him personally. Kaj Blennow is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. Jeffrey L. Dage is an employee and stockholder of Eli Lilly and Company, which also supports his costs for travel and attending of meetings. Subject matter relating to the p‐tau181 assay, methods, reagents, and/or compositions of matter set forth herein are subject to patents and/or patent applications of Eli Lilly and Company. Noelia Fandos is an employee of Araclon Biotech. Oskar Hansson has acquired research support (for the institution) from AVID Radiopharmaceuticals, Biogen, Eli Lilly, Eisai, GE Healthcare, Pfizer, and Roche. In the past 2 years, he has received consultancy/speaker fees from AC Immune, Alzpath, Biogen, Cerveau, and Roche. Christophe Hirtz has a collaboration contract with Shimadzu European Innovation Center including a PhD thesis. Shorena Janelidze is supported by research grants from Alzheimerfonden Kockska stiftelsen Stiftelsen för Gamla Tjänarinnor, and travel grants from the Strategic Research Area MultiPark (Multidisciplinary Research in Parkinson's disease) at Lund University. All payments were made to Lund University. Sungmin Kang and Ryan Lee are employees and stockholders of PeopleBio. Kristopher Kirmess, Matthew Meyer, and Tim West are employees of C2N Diagnostics. C2N has received funding in support of their assay from the following grant sources: NIH, BrightFocus Foundation, GHR Foundation. Leslie M. Shaw has grant support from NIH: NIA ADNI U19 AG024904; UPenn ADRC P30 AG010124; IIS grant support from Roche; serves on Advisory Boards for Biogen, Roche, FujiRebio, Siemens Healthineers. Henrik Zetterberg is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018‐02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG‐720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809‐2016862), the AD Strategic Fund and the Alzheimer's Association (#ADSF‐21‐831376‐C, #ADSF‐21‐831381‐C and #ADSF‐21‐831377‐C), the Olav Thon Foundation, the Erling‐Persson Family Foundation, Stiftelsen för Gamla Tjänarinnor, Hjärnfonden, Sweden (#FO2019‐0228), the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No 860197 (MIRIADE), and the UK Dementia Research Institute at UCL. Payments are made to the institution. He has served on scientific advisory boards and/or as consultant for Alector, Eisai, Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Pinteon Therapeutics, Nervgen, AZTherapies, CogRx, and Red Abbey Labs. These payments were made to him personally. He has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, and Biogen. Further, he is a chair of the Alzheimer's Association Global Biomarker Standardization Consortium and a chair of the Alzheimer's Association Biofluid‐Based Biomarker PIA (no payments), and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (payments made to him personally). Rebecca M. Edelmayer is a full‐time employee of the Alzheimer's Association. She serves on the external advisory board of the TREAT‐AD Consortium, NIH (2020‐Present; no payments made), and the MODEL‐AD Consortium, NIH (2019‐Present; no payments made). Charlotte Teunissen has a collaboration contract with ADx Neurosciences and Quanterix, and performed contract research for or received grants from the Alzheimer's Association for the present work, and from AxonNeurosciences, Biogen, Boehringer, Brainstorm Therapeutics, EIP Pharma, Esai, Janssen Prevention Center, Roche, Toyama and Vivoryon outside the submitted work. Research of Charlotte Teunissen is supported by the European Commission (Marie Curie International Training Network, grant agreement No 860197 (MIRIADE), and JPND), Health Holland, the Dutch Research Council (ZonMW), Alzheimer Drug Discovery Foundation, The Selfridges Group Foundation, Alzheimer Netherlands, Alzheimer Association. Further, she is a recipient of ABOARD, which is a public–private partnership receiving funding from ZonMW (#73305095007) and Health∼Holland, Topsector Life Sciences & Health (PPP‐allowance; #LSHM20106). More than 30 partners participate in ABOARD. ABOARD also receives funding from Edwin Bouw Fonds and Gieskes‐Strijbisfonds. Also, Charlotte Teunissen serves on the editorial board of Medidact Neurologie /Springer of the Neuromethods book series, on the editorial board of Alzheimer's Research and Therapy and of Neurology: Neuroimmunology & Neuroinflammation. All funding is paid to her institution.

Figures

FIGURE 1
FIGURE 1
Effect of pre‐analytical sample handling variations on Aβ42 levels, measured by different technologies. Values measured in the experimental conditions were normalized against the values measured in the reference conditions, which is EDTA plasma, centrifuged at 1800 × g at room temperature, after a standing time of 30 minutes at room temperature, immediately followed by aliquoting and −80°C storage. In 1. Sampling, levels in Na‐citrate and Li‐heparin were undetectable with the Euroimmun ELISA assay. In 5. Storage, 2‐week 2–8°C condition, the recovery was 41% for Simoa N4PE, which is below the y‐axis limit. Horizontal, dashed reference lines were set at 90% and 110%, to visualize a > 10% change of the median under an experimental condition compared to the reference condition. Aβ, amyloid beta; EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme‐linked immunosorbent assay; FT, freeze–thaw cycle; Li‐heparin, lithium–heparin; MS, mass spectrometry; Na‐citrate, sodium–citrate; RT, room temperature
FIGURE 2
FIGURE 2
Effect of pre‐analytical sample handling variations on Aβ40 values, measured by different technologies. Values measured in the experimental conditions were normalized against the values measured in the reference conditions, which is EDTA plasma, centrifuged at 1800 × g at room temperature, after a standing time of 30 minutes at room temperature, immediately followed by aliquoting and −80°C storage. In 5. Storage, 2‐week 2–8°C condition, the recovery was 27% for the MALDI MS assay, which is below the y‐axis limit. Horizontal, dashed reference lines were set at 90% and 110%, to visualize a > 10% change of the median under an experimental condition compared to the reference condition. Aβ, amyloid beta; EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme‐linked immunosorbent assay; FT, freeze–thaw cycle; Li‐heparin, lithium–heparin; MALDI, matrix‐assisted laser desorption/ionization; MS, mass spectrometry; Na‐citrate, sodium–citrate; RT, room temperature
FIGURE 3
FIGURE 3
Effect of pre‐analytical sample handling variations on biomarkers APP699‐711, OAβ, GFAP, NfL, t‐tau, and p‐tau181. Values measured in the experimental conditions were normalized against the values measured in the reference conditions, which is EDTA plasma, centrifuged at 1800 × g at room temperature, after a standing time of 30 minutes at room temperature, immediately followed by aliquoting and −80°C storage. Horizontal, dashed reference lines were set at 90% and 110%, to visualize a > 10% change of the median under an experimental condition compared to the reference condition. APP, amyloid precursor protein; FT, freeze‐thaw cycle; GFAP, glial fibrillary acidic protein; Li‐heparin, lithium–heparin; Na‐citrate, sodium–citrate; NfL, neurofilament light; OAβ, amyloid beta oligomerization tendency; p‐tau181, tau phosphorylated at threonine 18; RT, room temperature; t‐tau, total tau
FIGURE 4
FIGURE 4
Recommended plasma handling standardized operating procedure (SOP). The SOP was constructed according to results obtained in systematic pre‐analytical experiments. Deviating from this SOP might result in less reliable Aβ42 and Aβ40 measurements. For reliable total tau measurement, a maximum processing time of 1 hour might be necessary. Longer delays than 24 hours for centrifugation or 2 weeks for storage might be possible, but this was not investigated in this study. Aβ, amyloid beta; RT, room temperature; wk, week
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