Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows

Clin Chem. 2020 Jan 1;66(1):149-160. doi: 10.1373/clinchem.2019.306837.


Background: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making.

Methods: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID ( for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites.

Results: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT.

Conclusions: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Specimen Collection
  • Cell Line, Tumor
  • Cell-Free Nucleic Acids / chemistry
  • Cell-Free Nucleic Acids / metabolism*
  • Cell-Free Nucleic Acids / standards
  • Circulating Tumor DNA / blood
  • DNA Mutational Analysis
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Nucleotide Sequencing / standards
  • Humans
  • Neoplasms / genetics
  • Neoplasms / pathology
  • Nucleosomes / genetics
  • Polymorphism, Single Nucleotide
  • Pre-Analytical Phase
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Tumor Suppressor Protein p53 / genetics


  • Cell-Free Nucleic Acids
  • Circulating Tumor DNA
  • Nucleosomes
  • TP53 protein, human
  • Tumor Suppressor Protein p53