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Licensed Unlicensed Requires Authentication Published by De Gruyter October 17, 2017

Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays

  • Qiujin Shen , Johan Björkesten , Joakim Galli , Daniel Ekman , John Broberg , Niklas Nordberg , Annika Tillander , Masood Kamali-Moghaddam EMAIL logo , Gunnel Tybring and Ulf Landegren EMAIL logo



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.


Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 °C or 22 °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 °C and thawing at 22 °C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.


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 °C or 22 °C, respectively. Some increases became noticeable after 8 h delay at 4 °C but already after 1 h at 22 °C. For samples stored at 4 °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 °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 °C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.


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.

Corresponding authors: Associate Prof. Masood Kamali-Moghaddam, PhD, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala Biomedical Center, Husargatan 3, Uppsala, Sweden, Phone: +46 18 4714454, Fax: +46 18 4714808; and Prof. Ulf Landegren, PhD, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala Biomedical Center, Husargatan 3, Uppsala, Sweden, Phone: +46 18 4714910, Fax: +46 18 4714808

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: Swedish Research Council VR: no. 829-2009-6285 (; European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013): no. 313010 (BBMRI-LPC), no. 294409 (ProteinSeq); Marie Curie ITN (FP7/2007–2013): no. 316929 (GastricGlycoExplorer).

  3. Employment or leadership: DE is an employer of Olink Bioscience; JB and NN are employers of Olink Proteomics.

  4. Honorarium: None declared.

  5. Competing interests: UL is a founder and shareholder of Olink Proteomics and Olink Bioscience, having rights to the proximity ligation and extension technology. The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


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Supplemental Material:

The online version of this article offers supplementary material (

Received: 2017-7-24
Accepted: 2017-9-11
Published Online: 2017-10-17
Published in Print: 2018-3-28

©2018 Walter de Gruyter GmbH, Berlin/Boston

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