National Measurement Institute, Australia (NMIA), Lindfield, New South Wales, Australia.
National Measurement Institute, Australia (NMIA), Lindfield, New South Wales, Australia.
J Mol Diagn. 2022 Sep;24(9):1041-1049. doi: 10.1016/j.jmoldx.2022.06.003. Epub 2022 Jul 11.
Standardization of molecular diagnostics is fundamental for effective application of genetic analyses in personalized medicine. The amount of DNA extracted from a specimen can have a significant impact on diagnostic accuracy, especially in cases where the diagnostic variant has a low concentration such as cancer. Blood and tissue samples were supplied to genetic laboratories to assess the reproducibility of extraction methodologies; DNA was extracted using participants' routine procedures and returned to the external quality assessment provider. The amount of DNA was measured by two independent analytical techniques, fluorescence intensity of intercalating dye and digital PCR; DNA quality was evaluated by DNA integrity number scores. The amount of DNA extracted varied widely between and within participants and for different blood volumes, indicating that consistent diagnostic quality is challenging even within a single test center. The median digital PCR-measured amount of DNA was on average six times higher than the intercalating dye measurements obtained in this study, indicating the possibility that the latter quantitative method may significantly underestimate the amount of DNA, thus making it not fit for purpose. Standardization of genetic diagnostic tests will require a significant improvement in the reproducibility of DNA extraction; this could be achieved if suppliers and users of DNA extraction kits validate their extraction methodology using reliable quantitative measurements or reference materials.
分子诊断的标准化是将遗传分析有效应用于个性化医学的基础。从标本中提取的 DNA 量可能对诊断准确性有重大影响，特别是在诊断变体浓度较低的情况下，如癌症。血液和组织样本被提供给遗传实验室，以评估提取方法的重现性；使用参与者的常规程序提取 DNA，并将其返还给外部质量评估提供商。通过两种独立的分析技术——嵌入染料的荧光强度和数字 PCR 来测量 DNA 量；通过 DNA 完整性编号分数来评估 DNA 质量。从参与者体内和之间提取的 DNA 量以及不同的血液量差异很大，这表明即使在单个测试中心内，一致的诊断质量也是具有挑战性的。平均而言，数字 PCR 测量的 DNA 量比本研究中获得的嵌入染料测量值高出六倍，这表明后一种定量方法可能会大大低估 DNA 量，因此不适合使用。遗传诊断测试的标准化将需要显著提高 DNA 提取的重现性；如果 DNA 提取试剂盒的供应商和用户使用可靠的定量测量或参考材料来验证其提取方法，则可以实现这一目标。
