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Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Olink Biosci, Uppsala, Sweden..
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2018 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 4, p. 582-594Article in journal (Refereed) Published
Abstract [en]

Background: A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles.

Methods: Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 degrees C or 22 degrees C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 degrees C and thawing at 22 degrees C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.

Results: Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 degrees C or 22 degrees C, respectively. Some increases became noticeable after 8 h delay at 4 degrees C but already after 1 h at 22 degrees C. For samples stored at 4 degrees C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 degrees C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 degrees C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.

Conclusions: Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH , 2018. Vol. 56, no 4, p. 582-594
Keywords [en]
biobank, protein detection, proteome, proximity extension assay (PEA), sample collection and handling
National Category
Clinical Laboratory Medicine
Identifiers
URN: urn:nbn:se:uu:diva-350276DOI: 10.1515/cclm-2017-0648ISI: 000426657400016PubMedID: 29040064OAI: oai:DiVA.org:uu-350276DiVA, id: diva2:1205345
Funder
Swedish Research Council, 829-2009-6285EU, European Research Council, 313010, 294409Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2019-11-03Bibliographically approved
In thesis
1. Dried blood sampling and digital readout to advance molecular diagnostics
Open this publication in new window or tab >>Dried blood sampling and digital readout to advance molecular diagnostics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.

Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.

Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care.   

Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1615
Keywords
molecular diagnostics, dried blood sample, DBS, digital readout, digital enumeration, DNA detection methods, proximity extension assay, protein stability, genotyping, rare mutations, cell free DNA, multiplex protein measurement.
National Category
Medical Biotechnology Biochemistry and Molecular Biology Medical Laboratory and Measurements Technologies
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-396325 (URN)978-91-513-0810-4 (ISBN)
Public defence
2019-12-20, A1:111a, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-28 Created: 2019-11-03 Last updated: 2019-11-28

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Shen, QiujinBjörkesten, JohanKamali-Moghaddam, MasoodLandegren, Ulf

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