Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Vox Sang. 2020 Oct;115(7):586-594. doi: 10.1111/vox.12923. Epub 2020 Apr 28.
Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR.
Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6 years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at -25°C.
We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA.
Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes.
对于游离胎儿和总 DNA 的非侵入性产前检测，优化的样本储存条件至关重要。我们研究了在-25°C 下储存血浆样本和提取 cfDNA 长达 0-6 年对 qPCR 的影响。
在-25°C 下储存 0-6 年后，计算血浆储存前后游离胎儿和总 cfDNA 产量和胎儿分数。进行稀释实验以研究 PCR 抑制。使用或不使用蛋白酶 K 提取以检查蛋白质解离。通过立即测试等分试样，以及在-25°C 下储存 18 个月和 3 年后，研究了提取 cfDNA 的储存情况。
我们观察到在储存 2-3 年后，血浆中可扩增的胎儿和总 DNA 水平显著增加，并且在储存 3 年后胎儿分数略有下降。在储存超过 2 年的血浆样本中进行 DNA 提取时，cfDNA 的检测不依赖于蛋白酶 K，这表明随着时间的推移，蛋白质从 DNA 中丢失，这可能是观察到的 DNA 产量增加的原因。储存的、提取的 cfDNA 中测量的胎儿和总 DNA 量以及胎儿分数均增加。
在-25°C 下长期储存后，血浆中可轻松检测到游离胎儿和总细胞游离 DNA。然而，由于测量的 DNA 量和胎儿分数存在较大差异，因此在使用储存的血浆和提取的 cfDNA 进行测试开发或验证目的时，可能需要谨慎。
