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.

中文翻译：

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全基因组研究采血管对无细胞DNA甲基化基因组的影响

背景：从液体活检组织中分离出来的cfDNA的甲基化模式，在疾病的诊断和监测中引起了极大的兴趣。我们评估了采血管的类型以及抽血和血浆制备之间的时间间隔对基于亚硫酸氢盐的cfDNA甲基化谱图的影响。方法：从三个健康的志愿者受试者（BD Vacutainer K2E EDTA喷雾管，Streck-Free DNA BCT管，PAXgene Blood ccfDNA管，Roche Cell-Free DNA Collection管和Biomatrica LBgard管）一式三份抽取15条血液。在血浆制备之前，样品应立即处理或在室温下保存24或72小时。DNA片段大小通过毛细管电泳评估。对从这些血浆样品中分离的无细胞DNA进行了亚硫酸氢盐还原法测序。我们评估了血管和时间延迟对几个质量控制指标的影响。结果：所有保存管在评估的质量指标上表现相似。此外，在EDTA管中经过72小时的时间延迟后，观察到cfDNA浓度及其衍生自NK细胞的分数显着增加。结论：cfDNA的甲基化模式在不同的保存试管之间是可靠且可重现的。尽快（最好在24小时之内）加工的EDTA管是最具成本效益的。如果无法立即处理，则保留管是有效的替代方法。结果：所有保存管在评估的质量指标上表现相似。此外，在EDTA管中经过72小时的时间延迟后，观察到cfDNA浓度及其衍生自NK细胞的分数显着增加。结论：cfDNA的甲基化模式在不同的保存试管之间是可靠且可重现的。尽快（最好在24小时内）加工的EDTA管是最经济的。如果无法立即处理，则保留管是有效的替代方法。结果：所有保存管在评估的质量指标上表现相似。此外，在EDTA管中经过72小时的时间延迟后，观察到cfDNA浓度及其衍生自NK细胞的分数显着增加。结论：cfDNA的甲基化模式在不同的保存试管之间是可靠且可重现的。尽快（最好在24小时内）加工的EDTA管是最经济的。如果无法立即处理，则保留管是有效的替代方法。cfDNA的甲基化模式在不同的保存管之间是可靠且可重现的。尽快（最好在24小时之内）加工的EDTA管是最具成本效益的。如果无法立即处理，则保留管是有效的替代方法。cfDNA的甲基化模式在不同的保存管之间是可靠且可重现的。尽快（最好在24小时之内）加工的EDTA管是最具成本效益的。如果无法立即处理，则保留管是有效的替代方法。