National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China.
Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China.
Clin Chem. 2019 Jun;65(6):761-770. doi: 10.1373/clinchem.2018.299602. Epub 2019 Mar 14.
Noninvasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) is widely used. However, biomimetic quality control materials that have properties identical to clinical samples and that are applicable to a wide range of methodologies are still not available to support assay development, internal quality control, and proficiency testing.
We developed a set of dual enzyme-digested NIPT quality control materials (DENQCMs) that comprise simulated human plasma and mixtures of mother cell line-derived cfDNA based on DNA fragmentation factor digestion (D-cfDNA) and the matched child cell line-derived cfDNA based on micrococcal nuclease digestion (M-cfDNA). Serially diluted samples positive for trisomies 21, 18, and 13 were included in the materials. To evaluate the biomimetics, DENQCMs were analyzed using random massively parallel sequencing (MPS), targeted MPS, and imaging single DNA molecule methods, and the estimated fetal fractions (FFs) were compared with expected FFs. Genome-wide analysis of cfDNA fragmentation patterns was performed to confirm their biological characteristics.
The genetic status of each DENQCM was correctly detected by 4 routine NIPT assays for the samples with FFs >5%. The chromosome Y-based and single-nucleotide polymorphism-based estimations of FFs were linearly related to those expected FFs. The MPS results exhibited a concordance of quality metrics between DENQCMs and maternal plasma, such as GC contents of cfDNA and unique read ratios.
The DENQCMs are universally applicable for different platforms. We propose DENQCMs as an approach to produce matched maternal and fetal cfDNA that will be suitable for the preparation of quality control materials for NIPT.
基于游离细胞 DNA（cfDNA）的非侵入性产前检测（NIPT）已被广泛应用。然而，仍然缺乏具有与临床样本相同特性且适用于广泛方法学的仿生质量控制材料，以支持检测开发、内部质量控制和能力验证。
我们开发了一组双酶消化的 NIPT 质量控制材料（DENQCMs），这些材料包含模拟人血浆和基于 DNA 片段化因子消化（D-cfDNA）的母体细胞系衍生 cfDNA 混合物，以及基于微球菌核酸酶消化（M-cfDNA）的匹配胎儿细胞系衍生 cfDNA。材料中包括经连续稀释的唐氏综合征 21、18 和 13 三体阳性样本。为了评估仿生特性，使用随机大规模平行测序（MPS）、靶向 MPS 和成像单 DNA 分子方法对 DENQCMs 进行了分析，并将估计的胎儿分数（FF）与预期的 FF 进行了比较。对 cfDNA 片段化模式进行了全基因组分析，以确认其生物学特征。
对于 FF>5%的样本，4 种常规 NIPT 检测方法均正确检测到每个 DENQCM 的遗传状态。基于染色体 Y 和基于单核苷酸多态性的 FF 估计与预期的 FF 呈线性相关。MPS 结果显示，DENQCMs 与母体血浆之间的质量指标具有一致性，例如 cfDNA 的 GC 含量和独特读取比。
DENQCMs 可普遍适用于不同的平台。我们建议使用 DENQCMs 来产生匹配的母体和胎儿 cfDNA，这将适合 NIPT 质量控制材料的制备。
