Medina Diaz Inga, Nocon Annette, Mehnert Daniel H, Fredebohm Johannes, Diehl Frank, Holtrup Frank
Research and Development, Sysmex Inostics GmbH, Hamburg, Germany.
PLoS One. 2016 Nov 10;11(11):e0166354. doi: 10.1371/journal.pone.0166354. eCollection 2016.
Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.
Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).
Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.
Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.
要使液体活检检测广泛可用，需要一种能在常温下运送全血，同时保持游离DNA（cfDNA）群体完整性和血细胞稳定性，以防止循环肿瘤DNA（ctDNA）被野生型基因组DNA稀释的概念。评估了Streck游离DNA BCT血液采集管（cfDNA BCT）的细胞和DNA稳定特性，以确定它们是否可与高度灵敏的突变检测技术联合使用。
将健康供体或晚期结直肠癌（CRC）患者的静脉血采集到cfDNA BCT和标准K2EDTA管中。在制备血浆和提取DNA之前，将试管在不同温度下保存不同时间。使用qPCR分析分离出的cfDNA的短片段和长片段的总DNA产量，并使用BEAMing和血浆安全测序（Safe-SeqS）分析突变变化。
在cfDNA BCT中采集健康个体的全血并在室温下保存长达5天，不会影响DNA产量和突变背景水平（n = 60）。在cfDNA BCT中采集的正常血液样本中加入的低频突变DNA以及CRC患者血液样本中的突变循环肿瘤DNA，在室温下保存3天后均能被可靠检测到。然而，在≤10°C和40°C下长时间保存的血液样本显示正常基因组DNA水平升高，细胞血浆界面异常大，血浆体积降低。
在cfDNA BCT中运送数天的全血可用于使用BEAMing和Safe-SeqS的下游液体活检检测。由于运送温度是一个关键因素，必须特别注意保持规定的室温范围，以获得可靠的突变检测结果。
