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Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis

Authors :
Zhuoyang Wang
Charu Aggarwal
Stephanie S. Yee
Aseel Abdalla
Jacob Till
Taylor A. Black
Robyn T. Sussman
Jacquelyn J. Roth
Kojo S.J. Elenitoba-Johnson
Erica L. Carpenter
Wei-Ting Hwang
Hareena Sangha
Caren Gentile
Jeffrey C. Thompson
Mark H. O'Hara
Source :
J Mol Diagn
Publication Year :
2021
Publisher :
Elsevier BV, 2021.

Abstract

Circulating cell-free DNA (ccfDNA) is used increasingly 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 real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (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. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

Details

ISSN :
15251578
Volume :
23
Database :
OpenAIRE
Journal :
The Journal of Molecular Diagnostics
Accession number :
edsair.doi.dedup.....ef407627d2b0842277092e087e0e4988
Full Text :
https://doi.org/10.1016/j.jmoldx.2021.08.007