Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

Background The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulfite-based cfDNA methylation profiling.

Methods 15 tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulfite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics.

Results All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes.

Conclusion The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.EGA study EGAS00001004271

背景：从液体活检样本中分离得到的循环游离DNA（cell-free DNA, cfDNA）的甲基化谱，在疾病诊断与病情监测领域正受到广泛关注。本研究评估了采血管类型以及采血至血浆制备的时间间隔，对基于亚硫酸氢盐的cfDNA甲基化谱分析的影响。

方法：本研究从3名健康志愿者中共采集15份血液样本，分别采用BD Vacutainer K2E EDTA喷雾采血管、Streck无细胞DNA BCT采血管、PAXgene血液ccfDNA采血管、罗氏无细胞DNA采集管以及Biomatrica LBgard血液采血管，每组设置3个重复。样本分为两组：一组立即进行处理，另一组在室温下储存24小时或72小时后再进行血浆制备。通过毛细管电泳法对DNA片段大小进行评估，并对从上述血浆样本中分离得到的无细胞DNA开展简化代表性亚硫酸氢盐测序。本研究评估了采血管类型与时间间隔对多项质量控制指标的影响。

结果：所有受试保存采血管在各项评估的质量指标上表现相近。此外，在EDTA采血管中储存72小时后，可观察到cfDNA浓度以及其中来源于自然杀伤细胞（natural killer cells, NK）的片段占比出现显著升高。

结论：不同类型保存采血管之间，cfDNA甲基化谱均具有良好的稳定性与可重复性。EDTA采血管若能尽快处理（建议在24小时内完成），成本效益比最高。若无法立即完成样本处理，其他类型的保存采血管均可作为有效的替代方案。

本研究隶属于欧洲基因组表型组档案（EGA）研究项目EGAS00001004271