Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter May 31, 2022

Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form

Nicole Laurencia Yuwono ORCID logo, Mollie Ailie Acheson Boyd, Claire Elizabeth Henry, Bonnita Werner, Caroline Elizabeth Ford and Kristina Warton ORCID logo



Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in sample quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation.


We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood samples after 16 months of storage and tested the effect of varying DNA extraction protocol parameters.


Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis.


The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage.

Corresponding author: Dr. Kristina Warton, Gynaecological Cancer Research Group, Adult Cancer Program, School of Women’s and Children’s Health, Faculty of Medicine and Health, Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia, Phone: +61293851457, Fax: +61293851510, E-mail:

Funding source: Beth Yarrow Memorial Award in Medical Science

Award Identifier / Grant number: NA

Funding source: CAMILLA AND MARC

Award Identifier / Grant number: NA

Funding source: Gynaecological Oncology Fund

Award Identifier / Grant number: NA

Funding source: Translational Cancer Research Network

Award Identifier / Grant number: NA

Funding source: Australian Research Training Program

Award Identifier / Grant number: NA

Funding source: Ovarian Cancer Research Foundation

Award Identifier / Grant number: GA-2018-14


We would like to acknowledge and thank the volunteers for their blood donation. We also thank Professor Susan Ramus for her advice on study design as well as Dr. Kylie-Ann Mallitt and Dr. Nancy Briggs for statistics consultation.

  1. Research funding: Nicole Yuwono and Bonnie Werner was supported by the Australian Government Research Training Program (RTP) Stipend through The University of New South Wales and Translational Cancer Research Network PhD Scholarship Top Up Award, via the Cancer Institute NSW. Nicole Yuwono was further supported by Beth Yarrow Memorial Award in Medical Science and UNSW Completion Scholarship. Claire Henry was supported by the Gynaecological Oncology Fund of the Royal Hospital for Women, Sydney. Dr. Kristina Warton was supported by Ovarian Cancer Research Foundation (GA-2018-14) and CAMILLA AND MARC. The funders had no role in the decision to publish or preparation of the manuscript.

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

  3. Competing interests: Dr. Kristina Warton holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the University of New South Wales Human Research Ethics Committee (#HC17020).


1. Wan, JCM, Massie, C, Garcia-Corbacho, J, Mouliere, F, Brenton, JD, Caldas, C, et al.. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 2017;17:223–38. in Google Scholar PubMed

2. Trigg, RM, Martinson, LJ, Parpart-Li, S, Shaw, JA. Factors that influence quality and yield of circulating-free DNA: a systematic review of the methodology literature. Heliyon 2018;4:e00699. in Google Scholar PubMed PubMed Central

3. Sozzi, G, Roz, L, Conte, D, Mariani, L, Andriani, F, Verderio, P, et al.. Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays. J Natl Cancer Inst 2005;97:1848–50. in Google Scholar PubMed

4. Sato, A, Nakashima, C, Abe, T, Kato, J, Hirai, M, Nakamura, T, et al.. Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy. Oncotarget 2018;9:31904–14. in Google Scholar PubMed PubMed Central

5. Clausen, FB, Barrett, AN, Advani, HV, Choolani, M, Dziegiel, MH. Impact of long-term storage of plasma and cell-free DNA on measured DNA quantity and fetal fraction. Vox Sang 2020;115:586–94. in Google Scholar PubMed

6. Breitbach, S, Tug, S, Helmig, S, Zahn, D, Kubiak, T, Michal, M, et al.. Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS One 2014;9:e87838. in Google Scholar PubMed PubMed Central

7. Warton, K, Graham, L-J, Yuwono, N, Samimi, G. Comparison of 4 commercial kits for the extraction of circulating DNA from plasma. Cancer Genetics 2018;228-9:143–50. in Google Scholar PubMed

8. Wan Azman, WN, Omar, J, Koon, TS, Tuan Ismail, TS. Hemolyzed specimens: major challenge for identifying and rejecting specimens in clinical laboratories. Oman Med J 2019;34:94–8. in Google Scholar PubMed PubMed Central

9. Appierto, V, Callari, M, Cavadini, E, Morelli, D, Daidone, MG, Tiberio, P. A lipemia-independent NanoDrop(®)-based score to identify hemolysis in plasma and serum samples. Bioanalysis 2014;6:1215–26. in Google Scholar PubMed

10. Kirschner, MB, Edelman, JJ, Kao, SC, Vallely, MP, Van Zandwijk, N, Reid, G. The impact of hemolysis on cell-free microRNA biomarkers. Front Genet 2013;4. in Google Scholar PubMed PubMed Central

11. Streleckiene, G, Forster, M, Inciuraite, R, Lukosevicius, R, Skieceviciene, J. Effects of quantification methods, isolation kits, plasma biobanking, and hemolysis on cell-free DNA analysis in plasma. Biopreserv Biobanking 2019;17:553–61. in Google Scholar PubMed

12. Nishimura, F, Uno, N, Chiang, PC, Kaku, N, Morinaga, Y, Hasegawa, H, et al.. The effect of in vitro hemolysis on measurement of cell-free DNA. J Appl Lab Med 2019;4:235–40. in Google Scholar PubMed

13. Murugesan, K, Hogan, CA, Palmer, Z, Reeve, B, Theron, G, Andama, A, et al.. Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. J Clin Microbiol 2019;57:e00782–19.10.1128/JCM.00782-19Search in Google Scholar PubMed PubMed Central

14. Barrett, AN, Thadani, HA, Laureano-Asibal, C, Ponnusamy, S, Choolani, M. Stability of cell-free DNA from maternal plasma isolated following a single centrifugation step. Prenat Diagn 2014;34:1283–8. in Google Scholar PubMed

15. Shishido, SN, Welter, L, Rodriguez-Lee, M, Kolatkar, A, Xu, L, Ruiz, C, et al.. Preanalytical variables for the genomic assessment of the cellular and acellular fractions of the liquid biopsy in a cohort of breast cancer patients. J Mol Diagn 2020;22:319–37. in Google Scholar PubMed PubMed Central

16. Holdenrieder, S, Von Pawel, J, Nagel, D, Stieber, P. Long-term stability of circulating nucleosomes in serum. Anticancer Res 2010;30:1613–5.Search in Google Scholar

17. Pinzani, P, Salvianti, F, Orlando, C, Pazzagli, M. Circulating cell-free DNA in cancer. In: Biassoni, R, Raso, A, editors. Quantitative real-time PCR: methods and protocols. New York, NY: Springer New York; 2014: 133–45 pp.10.1007/978-1-4939-0733-5_13Search in Google Scholar PubMed

18. Alborelli, I, Generali, D, Jermann, P, Cappelletti, MR, Ferrero, G, Scaggiante, B, et al.. Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study. Cell Death Dis 2019;10:534. in Google Scholar PubMed PubMed Central

19. Devonshire, AS, Whale, AS, Gutteridge, A, Jones, G, Cowen, S, Foy, CA, et al.. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 2014;406:6499–512. in Google Scholar PubMed PubMed Central

20. Garcia, J, Dusserre, E, Cheynet, V, Bringuier, PP, Brengle-Pesce, K, Wozny, AS, et al.. Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study. Oncotarget 2017;8:87980–96. in Google Scholar PubMed PubMed Central

21. Underhill, HR, Kitzman, JO, Hellwig, S, Welker, NC, Daza, R, Baker, DN, et al.. Fragment length of circulating tumor DNA. PLoS Genet 2016;12:e1006162. in Google Scholar PubMed PubMed Central

22. Cristiano, S, Leal, A, Phallen, J, Fiksel, J, Adleff, V, Bruhm, DC, et al.. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature 2019;570:385–9. in Google Scholar PubMed PubMed Central

23. Jiang, P, Chan, CWM, Chan, KCA, Cheng, SH, Wong, J, Wong, VW-S, et al.. Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci Unit States Am 2015;112:E1317. in Google Scholar PubMed PubMed Central

24. Mouliere, F, Chandrananda, D, Piskorz, AM, Moore, EK, Morris, J, Ahlborn, LB, et al.. Enhanced detection of circulating tumor DNA by fragment size analysis. Sci Transl Med 2018;10:eaat4921. in Google Scholar PubMed PubMed Central

Supplementary Material

The online version of this article offers supplementary material (

Received: 2021-10-28
Accepted: 2022-05-16
Published Online: 2022-05-31
Published in Print: 2022-07-26

© 2022 Walter de Gruyter GmbH, Berlin/Boston

Scroll Up Arrow