Preanalytical variables that affect the outcome of cell-free DNA measurements

Fragments of cell-free DNA (cfDNA) in human body fluids often carry disease-specific alterations and are now widely recognized as ideal biomarkers for the detection and monitoring of genomic disorders, especially cancer, that are normally difficult to examine noninvasively. However, the conversion of promising research findings into tools useful in routine clinical testing of cancer has been a slow-moving process. A major reason is that the diagnostic sensitivity and specificity of cfDNA-based clinical assays are negatively impacted by a combination of suboptimal and inter-institutional differences in preanalytical procedures. The most prominent factors include: (i) a poor understanding of the biological factors that determine the characteristics of the cfDNA population in a biospecimen prior to collection, (ii) inattention to how cfDNA with different structures and physical properties are affected differently by a given preanalytical step, and (iii) the sheer number of possible conditions that can be selected from for each preanalytical step along with a continually expanding menu of commercial products that often show varying degrees of bias and efficiency. The convergence of these variables makes it difficult for research groups and institutions to reach a consensus on optimal preanalytical procedures and a challenging task to establish widely applied standards, which ultimately hamper the development of cfDNA assays that are fit for broad clinical implementation. In this review, we follow a systematic approach to explore the most confounding preanalytical factors that affect the outcome of cfDNA measurements.

人体体液中的无细胞脱氧核糖核酸（cell-free DNA, cfDNA）片段通常携带疾病特异性改变，如今已被广泛认定为无创检测及监测基因组疾病（尤其是癌症）的理想生物标志物——此类疾病往往难以通过无创方式进行检查。然而，将颇具前景的研究成果转化为可应用于癌症常规临床检测的工具，进程始终较为缓慢。其中一项核心阻碍在于，基于cfDNA的临床检测方法的诊断灵敏度与特异性，会因预分析流程欠佳以及不同机构间流程差异的共同影响而受损。最突出的影响因素包括：（i）对样本收集前决定生物标本中cfDNA群体特征的生物学因素缺乏充分认知；（ii）未关注具有不同结构与物理特性的cfDNA，在同一预分析步骤中所受影响存在差异；（iii）每项预分析步骤可选择的实验条件数量繁多，且商业化产品种类持续扩充，不同产品往往存在不同程度的偏倚与效率差异。这些变量相互交织，使得各研究团队与机构难以就最优预分析流程达成共识，也难以建立广泛应用的行业标准，最终阻碍了适配大规模临床应用的cfDNA检测方法的开发。在本综述中，我们将采用系统性研究方法，探究影响cfDNA检测结果的最具干扰性的预分析因素。