Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

Abstract

Aim: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3).

Materials and methods: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis and contamination with white-cells and platelets. Study 2: influence of within-day variations. Study 3: influence of day-to-day variation. Study 4: influence of temperature during handling and storage, and of neoplastic disease. Study 5: influence of colonoscopy. Study 6: influence of the surgical trauma. 5mC and H3K9Me3 measurements were performed using enzyme-linked immunosorbent assays.

Results: Stasis, white-cell and platelet contamination, within-day variations, varying storage time before centrifugation, colonoscopy, and surgical trauma had no significant influence on levels of 5mC or H3K9Me3. Day-to-day variations of 12.7% and 11.5% (intra-individual) and 98.1% and 60.8% (inter-individual) were shown for 5mC and H3K9Me3, respectively. Levels of 5mC or H3K9Me3 were significantly higher in samples stored at room temperature until centrifugation compared to samples stored on ice. Patients with cancer had significantly lower levels of 5mC or H3K9Me3 compared to levels in healthy individuals.

Conclusion: Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation.

Keywords:

• Biomarkers
• colorectal neoplasms
• DNA methylation
• early detection of cancer
• epigenomics
• histone code
• tumor

Disclosure statement

MH and JM are employed by Belgian Volition SA, Namur, Belgium. The other authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding information

This study was financially supported from Belgian Volition SA, Namur, Belgium and The Augustinus Foundation, The Beckett Fund, The Inger Bonnén Fund, The Hans & Nora Buchard Fund, The Walter Christensen Family Fund, The Aase & Ejnar Danielsen Fund, The Erichsen Family Fund, The Sofus Carl Emil Friis Fund, The Torben & Alice Frimodt Fund, The Eva & Henry Frænkel Fund, The Gangsted Fund, The H Foundation, The Erna Hamilton Fund, The Sven & Ina Hansen Fund, The Henrik Henriksen Fund, Foundation Jochum, The KID Fund, The Kornerup Fund, The Dagmar Marshall Fund, The ‘Midtjyske Bladfund’, The Axel Muusfeldt Fund, The Arvid Nilsson Fund, The Obel Family Fund, The Krista & Viggo Petersen Fund, The Willy & Ingeborg Reinhard Fund, The Kathrine & Vigo Skovgaard Fund, The Toyota Fund, The Vissing Fund, The Wedell-Wedellsborg Fund and Hvidovre University Hospital (The Capital Region of Denmark).