ABSTRACT

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 bisulphite-based cfDNA methylation profiling. Fifteen 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 bisulphite 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. 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. 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.

摘要

从液体活检中分离出的 cfDNA 甲基化模式在疾病的诊断和监测方面引起了极大的兴趣。我们评估了采血管类型以及抽血和血浆制备之间的时间延迟对基于亚硫酸氢盐的 cfDNA 甲基化分析的影响。从三名健康志愿者受试者中抽取 15 管血液（BD Vacutainer K2E EDTA 喷雾管、Streck 无细胞 DNA BCT 管、PAXgene Blood ccfDNA 管、罗氏无细胞 DNA 采集管和 Biomatrica LBgard 血液管，一式三份）。样品在血浆制备前立即处理或在室温下储存 24 或 72 小时。通过毛细管电泳评估 DNA 片段大小。对从这些血浆样品中分离的无细胞 DNA 进行了减少代表性的亚硫酸氢盐测序。我们评估了血管和时间延迟对几个质量控制指标的影响。所有保存管在评估的质量指标上表现相似。此外，在 EDTA 管中延迟 72 小时后，观察到 cfDNA 浓度及其源自 NK 细胞的部分显着增加。cfDNA 的甲基化模式在不同的保存管之间是稳健且可重复的。尽快处理 EDTA 管，最好在 24 小时内处理，是最具成本效益的。如果无法立即处理，保存管是有效的替代品。在 EDTA 试管中延迟 72 小时后，观察到 cfDNA 浓度及其源自 NK 细胞的部分显着增加。cfDNA 的甲基化模式在不同的保存管之间是稳健且可重复的。尽快处理 EDTA 管，最好在 24 小时内处理，是最具成本效益的。如果无法立即处理，保存管是有效的替代品。在 EDTA 试管中延迟 72 小时后，观察到 cfDNA 浓度及其源自 NK 细胞的部分显着增加。cfDNA 的甲基化模式在不同的保存管之间是稳健且可重复的。尽快处理 EDTA 管，最好在 24 小时内处理，是最具成本效益的。如果无法立即处理，保存管是有效的替代品。