Enter Note Done

Session


Keywords: Cell-free DNA; Diagnostics; NIPT; Cancer; Clinical testing

Authors:
Y. Lu; K. Ooi; K. Shi; T. Fu; E. Chan

Affiliation: Anchor Molecular Inc, Pleasanton, California.


The usage of PCR-based or NGS-based liquid biopsy assays for circulating tumor DNA (ctDNA) detection is growing rapidly in clinical labs. However, inconsistent results are often observed especially when assays from different developers are compared. One reason might be due to lack of thorough understanding of the role of plasma factors on cfDNA extraction since the extraction step contributes significantly to assay variation. To address this issue, Anchor Molecular has developed a proprietary technology that specifically removes DNA from plasma without affecting the composition of the plasma. The DNA content in the treated plasma was measured to be less than 0.01ng/mL. The composition of all other components such as proteins and lipids remain the same as in normal patient plasma. Extraction Quality control samples based on this DNA-depleted plasma were used to evaluate the effect of plasma factors on extraction and sensitivity and specificity of cfDNA assay.
These controls are made by either synthetic DNA or native DNA fragments derived from cell lines spiked into DNA-depleted plasma. The recovery and stability of these DNAs were studied by extractions followed by quantitative PCR (qPCR). Specificity of cfDNA extraction was studied by spiking both low- and high-molecular-weight DNA fragments into DNA-depleted plasma followed by extraction by five different cfDNA extraction kits. The purified DNAs were analyzed using Bioanalyzer 2100. The sensitivity of cfDNA extraction was examined by extractions of the spiked low concentration of target DNA fragments with different commercial kits followed by qPCR. The observed and expected concentrations of extracted DNA-depleted, plasma-spiked DNAs were shown to be linearly correlated, mimicking the spiked DNAs in normal plasma.
Both synthetic DNA and fragmented genomic DNA showed at least 125 days of real-time stability at 2-8?C. The specificity study showed that different kits exhibited varying preferences in extracting DNAs of different sizes. In the sensitivity study, differences in extraction efficiency and precision were observed among different kits.
The data demonstrated that plasma factors influence cfDNA extraction. Quality controls samples based on DNA-free plasma can be effectively used for monitoring the entire ctDNA assay process including the extraction step.