Circulating cell-free DNA (ccfDNA) is increasingly used as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by qPCR, Qubit, and ddPCR. For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Utilizing a high speed (16,000g) or slower speed (4,100g) second centrifugation step showed no difference in ccfDNA yield (qPCR p=0.208, Qubit p=0.468, ddPCR p=0.509) nor ctDNA VAF (p=0.438). A 2- versus 3-spin centrifugation protocol also showed no difference in ccfDNA yield (qPCR p=0.317, Qubit p=0.439, ddPCR p=0.160) or ctDNA VAF (p=0.496). A higher yield was observed from fresh versus frozen plasma by qPCR (p=0.001) and Qubit (p<0.001), whereas a higher yield was observed for frozen versus fresh by ddPCR (p=0.010), and yet no difference was observed in ctDNA VAF (p=0.219). Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
Copyright © 2021. Published by Elsevier Inc.