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.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: Swedish Research Council VR: no. 829-2009-6285 (BBMRI.se); 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).
Employment or leadership: DE is an employer of Olink Bioscience; JB and NN are employers of Olink Proteomics.
Honorarium: None declared.
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.
1. van Ommen G-J, Törnwall O, Bréchot C, Dagher G, Galli J, Hveem K, et al. BBMRI-ERIC as a resource for pharmaceutical and life science industries: the development of biobank-based Expert Centres. Eur J Hum Genet 2015;23:893–900.10.1038/ejhg.2014.235Search in Google Scholar PubMed PubMed Central
2. Betsou F, Lehmann S, Ashton G, Barnes M, Benson EE, Coppola D, et al. Standard preanalytical coding for biospecimens; defining the sample PREanalytical code. Cancer Epidemiol Biomarkers Prev 2010;19:1004–11.10.1158/1055-9965.EPI-09-1268Search in Google Scholar PubMed
3. Lehmann S, Guadagni F, Moore H, Ashton G, Barnes M, Benson E, et al. Standard preanalytical coding for biospecimens: review and implementation of the sample PREanalytical Code (SPREC). Biopreserv Biobank 2012;10:366–74.10.1089/bio.2012.0012Search in Google Scholar PubMed PubMed Central
4. Tuck MK, Chan DW, Chia D, Godwin AK, Grizzle WE, Krueger KE, et al. Standard operating procedures for serum and plasma collection. J Proteome Res 2010;8:113–7.10.1021/pr800545qSearch in Google Scholar PubMed PubMed Central
5. Rai AJ, Gelfand CA, Haywood BC, Warunek DJ, Yi J, Schuchard MD, et al. HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 2005;5:3262–77.10.1002/9783527609482.ch3Search in Google Scholar
6. Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 2002;1:845–67.10.1074/mcp.A300001-MCP200Search in Google Scholar
7. Assarsson E, Lundberg M, Holmquist G, Björkesten J, Thorsen SB, Ekman D, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One 2014;9:e95192.10.1371/journal.pone.0095192Search in Google Scholar PubMed PubMed Central
8. Lundberg M, Eriksson A, Tran B, Assarsson E, Fredriksson S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Res 2011;39:e102.10.1093/nar/gkr424Search in Google Scholar PubMed PubMed Central
9. Larssen P, Wik L, Czarnewski P, Eldh M, Lof L, Goran Ronquist K, et al. Tracing cellular origin of human exosomes using multiplex proximity extension assays. Mol Cell Proteomics 2017;16:1–30.10.1074/mcp.M116.064725Search in Google Scholar PubMed PubMed Central
10. Björkesten J, Enroth S, Shen Q, Wik L, Hougaard D, Cohen A, et al. Stability of proteins in dried blood spot biobanks. Mol Cell Proteomics 2017;16:1286–96.10.1074/mcp.RA117.000015Search in Google Scholar PubMed PubMed Central
11. Banfi G, Salvagno GL, Lippi G. The role of ethylenediamine tetraacetic acid (EDTA) as in vitro anticoagulant for diagnostic purposes. Clin Chem Lab Med 2007;45:565–76.10.1515/CCLM.2007.110Search in Google Scholar
12. Hsieh SY, Chen RK, Pan YH, Lee HL. Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling. Proteomics 2006;6:3189–98.10.1002/pmic.200500535Search in Google Scholar
13. Ellervik C, Vaught J. Preanalytical variables affecting the integrity of human biospecimens in biobanking. Clin Chem 2015;61:914–34.10.1373/clinchem.2014.228783Search in Google Scholar
14. Holland NT, Smith MT, Eskenazi B, Bastaki M. Biological sample collection and processing for molecular epidemiological studies. Mutat Res 2003;543:217–34.10.1016/S1383-5742(02)00090-XSearch in Google Scholar
15. Newhall KJ, Diemer GS, Leshinsky N, Kerkof K, Chute HT, Russell CB, et al. Evidence for endotoxin contamination in plastic Na+-heparin blood collection tube lots. Clin Chem 2010;56:1483–91.10.1373/clinchem.2006.144618Search in Google Scholar
16. Aziz N, Irwin MR, Dickerson SS, Butch AW. Spurious tumor necrosis factor-alpha and interleukin-6 production by human monocytes from blood collected in endotoxin-contaminated vacutainer blood collection tubes. Clin Chem 2004;50:2215–6.10.1373/clinchem.2004.040162Search in Google Scholar
17. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal 1999;13:133–40.10.1002/(SICI)1098-2825(1999)13:3<133::AID-JCLA8>3.0.CO;2-0Search in Google Scholar
18. Lengellé J, Panopoulos E, Betsou F. Soluble CD40 ligand as a biomarker for storage-related preanalytic variations of human serum. Cytokine 2008;44:275–82.10.1016/j.cyto.2008.08.010Search in Google Scholar
19. Betsou F, Gunter E, Clements J, De Souza Y, Goddard KA, Guadagni F, et al. Identification of evidence-based biospecimen quality-control tools: a report of the international society for biological and environmental repositories (ISBER) biospecimen science working group. J Mol Diagnostics 2013;15:3–16.10.1016/j.jmoldx.2012.06.008Search in Google Scholar
20. Lee J-E, Kim J-W, Han B-G, Shin S-Y. Impact of whole-blood processing conditions on plasma and serum concentrations of cytokines. Biopreserv Biobank 2016;14:51–5.10.1089/bio.2015.0059Search in Google Scholar
21. De Jongh R, Vranken J, Vundelinckx G, Bosmans E, Maes M, Heylen R. The effects of anticoagulation and processing on assays of IL-6, sIL-6R, sIL-2R and soluble transferrin receptor. Cytokine 1997;9:696–701.10.1006/cyto.1997.0217Search in Google Scholar PubMed
22. Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464–9.10.1016/j.clinbiochem.2012.01.012Search in Google Scholar PubMed
23. Tanner M, Kent N, Smith B, Fletcher S, Lewer M. Stability of common biochemical analytes in serum gel tubes subjected to various storage temperatures and times pre-centrifugation. Ann Clin Biochem 2008;45:375–9.10.1258/acb.2007.007183Search in Google Scholar PubMed
24. Chaigneau C, Cabioch T, Beaumont K, Betsou F. Serum biobank certification and the establishment of quality controls for biological fluids: examples of serum biomarker stability after temperature variation. Clin Chem Lab Med 2007;45:1390–5.10.1515/CCLM.2007.160Search in Google Scholar PubMed
25. Lee JE, Kim SY, Shin SY. Effect of repeated freezing and thawing on biomarker stability in plasma and serum samples. Osong Public Health Res Perspect 2015;6:357–62.10.1016/j.phrp.2015.11.005Search in Google Scholar PubMed PubMed Central
26. Mitchell BL, Yasui Y, Li CI, Fitzpatrick AL, Lampe PD. Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects. Cancer Inform 2005;1:98–104.10.1177/117693510500100110Search in Google Scholar
27. de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunol 2009;10:52.10.1186/1471-2172-10-52Search in Google Scholar PubMed PubMed Central
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2017-0648).
©2018 Walter de Gruyter GmbH, Berlin/Boston