School of Women's and Children's Health, Lowy Cancer Research Centre, University of New South Wales, Kensington, NSW, 2052, Australia.
Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia.
Mol Diagn Ther. 2017 Oct;21(5):563-570. doi: 10.1007/s40291-017-0284-x.
Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size.
Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution.
While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR.
Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.
在临床环境中采集用于循环 DNA 研究的血液样本时，通常无法立即处理样本。为了避免白细胞裂解后长时间储存血液所带来的问题，现已开发出含有防腐剂的稳定血样管，防止细胞裂解。我们评估了 Streck BCT 管和 PAXgene ccfDNA 管以及标准 EDTA 采血管在 DNA 产量和片段大小方面的表现。
将血液采集到 EDTA、Streck BCT 或 PAXgene ccfDNA 管中，在 4°C 下储存 1 小时，或在室温下储存 4 天。使用 QIAamp 循环核酸试剂盒提取 DNA，并在琼脂糖凝胶上可视化或通过 qPCR 定量。使用 Alu 重复元件的 247 碱基和 115 碱基扩增子的比值来推断大小分布。
室温下储存 4 天后，EDTA 管血液样本中的血浆 DNA 增加了约 10-20 倍，而 Streck BCT 管和 PAXgene ccfDNA 管则保持了稳定的血浆 DNA 浓度。与 EDTA 管相比，Streck BCT 和 PAXgene ccfDNA 管在 4°C 下储存 1 小时后，DNA 产量略有下降。按照 Streck 管产品说明书推荐的方法，将 DNA 提取方案中的蛋白消化步骤延长至 60 分钟，可逆转这种下降。琼脂糖凝胶上提取的 DNA 的可视化显示，在室温下储存 4 天后，EDTA 管采集的样本含有丰富的高分子量 DNA，其呈梯状部分片段化。在室温下储存 4 天的 Streck BCT 管样本中也观察到高分子量 DNA 略有增加，但这并未反映在 qPCR 测量的大、小 Alu 片段比值的变化上。
含有防止细胞裂解的防腐剂的管可以扩展临床环境下的血液采集范围；然而，不同管型采集的样本之间的细微差异突出了标准化方案的要求，以及对样本处理的关注。
