Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24 h after venipuncture
Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24 h after venipuncture
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DOI：
10.1016/j.clinbiochem.2013.07.020
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发表时间：
2013-12-01
影响因子：
2.8
通讯作者：
Faas, Brigitte H. W.
中科院分区：
文献类型：
--
作者：
Buysse, Karen;Beulen, Lean;Faas, Brigitte H. W.
目的：母体血浆中的循环胎儿游离 DNA (ccffDNA) 是无创产前检测 (NIPT) 的一个有吸引力的来源。静脉穿刺后 24 小时，总游离 DNA 量显着增加，导致血样中 ccffDNA 分数相对减少。在本研究中，我们评估了延长处理时间对大规模并行测序 (MPS) 检测非整倍体可靠性的下游影响。 设计和方法：将怀有正常和 21 三体性 (121) 胎儿的孕妇的全血收集到常规 EDTA 抗凝管中，并在静脉穿刺后 6 小时、24 小时和 48 小时内进行处理。通过 MPS 使用 Z 得分计算和基于 X 染色体读数的 ccffDNA 百分比进一步分析所有三个不同时间点的样本。结果：两个 T21 样本在所有时间点均被正确识别。然而，48 小时后，我们注意到 Z 分数出现了更高的偏差。尽管先前已显示血浆样本中 ccffDNA 的百分比在静脉穿刺后 24 小时显着下降，但基于 MPS 结果的百分比在 6、24 或 48 小时后并未显示出显着下降。结论：从静脉穿刺后 24 小时处理的血浆样本中提取的 ccffDNA 的质量和数量足够高，足以通过 MPS 进行可靠的下游 NIPT 分析。此外，我们表明，确定实际用于 NIPT 的样本部分中 ccffDNA 的百分比非常重要，因为下游程序可能会影响胎儿或母体部分。 (C) 2013 年加拿大临床化学家协会。由爱思唯尔公司出版。保留所有权利。
Objectives: Circulating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24 h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS).Design and methods: Whole blood from pregnant women carrying normal and trisomy 21 (121) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6 h, 24 and 48 h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads.Results: Both T21 samples were correctly identified as such at all time-points. However, after 48 h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24 h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48 h.Conclusions: The quality and quantity of ccffDNA extracted from plasma samples processed up to 24 h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction. (C) 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.