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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 EMAIL logo
Published/Copyright: May 31, 2022
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 60 Issue 8

Abstract

Objectives

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.

Methods

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.

Results

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.

Conclusions

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

Keywords: cfDNA; circulating cell-free DNA; cirDNA; ctDNA; haemolysis; long-term; plasma; pre-analytical; short-term; 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

Acknowledgments

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).

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2021-1152).

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

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https://doi.org/10.1515/cclm-2021-1152
Keywords for this article
cfDNA; circulating cell-free DNA; cirDNA; ctDNA; haemolysis; long-term; plasma; pre-analytical; short-term; storage

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Transdermal measurement of cardiac troponins: the future is now
  4. Reviews
  5. Perinatal presepsin assessment: a new sepsis diagnostic tool?
  6. Hypertriglyceridemia, a causal risk factor for atherosclerosis, and its laboratory assessment
  7. Opinion Paper
  8. The novelties of the regulation on health technology assessment, a key achievement for the European union health policies
  9. General Clinical Chemistry and Laboratory Medicine
  10. Performance of four regression frameworks with varying precision profiles in simulated reference material commutability assessment
  11. Comparison of six regression-based lot-to-lot verification approaches
  12. Failure Mode and Effects Analysis (FMEA) at the preanalytical phase for POCT blood gas analysis: proposal for a shared proactive risk analysis model
  13. Evaluation of a pneumatic tube system carrier prototype with fixing mechanism allowing for automated unloading
  14. Analytical performance of eight enzymatic assays for ethanol in serum evaluated by data from the Belgian external quality assessment scheme
  15. Vitamin D metabolism in living kidney donors before and after organ donation
  16. Validation of steroid ratios for random urine by mass spectrometry to detect 5α-reductase deficiency in Vietnamese children
  17. Evaluation of serum neurofilament light in the early management of mTBI patients
  18. Assessment of urine sample quality by the simultaneous measurement of urinary γ-glutamyltransferase and lactate dehydrogenase enzyme activities: possible application to unravel cheating in drugs of abuse testing
  19. Reference Values and Biological Variations
  20. Age and sex specific reference intervals of 13 hematological analytes in Chinese children and adolescents aged from 28 days up to 20 years: the PRINCE study
  21. Cancer Diagnostics
  22. Prostate health index (PHI) as a reliable biomarker for prostate cancer: a systematic review and meta-analysis
  23. A comparison of the faecal haemoglobin concentrations and diagnostic accuracy in patients suspected with colorectal cancer and serious bowel disease as reported on four different faecal immunochemical test systems
  24. Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form
  25. Cardiovascular Diseases
  26. Analytical and clinical performance evaluation of a new high-sensitivity cardiac troponin I assay
  27. Infectious Diseases
  28. Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples
  29. Letters to the Editors
  30. Improving D-dimer testing appropriateness by controlling periodicity of retesting: prevention is better than cure
  31. Biological variation of serum cholinesterase catalytic concentrations
  32. Three-month ad interim analysis of total anti-SARS-CoV-2 antibodies in healthy recipient of a single BNT162b2 vaccine booster
  33. Fibrin strands in peripheral blood smear: the COVID-19 era
  34. Fragments of alpha-1-antitrypsin in patients with severe COVID-19 and bacterial pulmonary sepsis
  35. Comparison of thyroid stimulating hormone, free thyroxine, total triiodothyronine, thyroglobulin and peroxidase antibodies measurements by two different platforms
  36. Effect of different incubation times on the detection of factor VIII inhibitor in acquired hemophilia A
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