Circulating Cell-Free DNA Part 1

Blog Staff


cfDNA has been the talk of the town – now more than ever. The number of publications published on cfDNA per year has increased over 15-fold in the last decade (PubMed).

cfDNA, the extracellular DNA that circulates freely in the blood and other bodily fluids, is released from sources such as apoptotic cells in healthy conditions1, necrotic cells in cancer,1 or from fetal cells in maternal blood.2 cfDNA fragments are about 170 bps in length and have a short half-life of about 15 minutes to a few hours.3 Its discovery and utility for various research and clinical applications has revolutionized the field of genomics. Several characteristics of cfDNA, such as concentration, stability and integrity, are significantly different in cancer cells compared to normal cells.4 Studies have also shown that a fraction of cfDNA, called circulating tumor DNA (ctDNA), can be used to determine somatic mutations, copy number variations (CNV) and epigenetic alterations that are characteristic of cancer cells.5,6,7 Analysis of cfDNA in maternal blood can also provide information about chromosomal aneuploidies such as trisomy 13, trisomy 8 and trisomy 21 in the fetus.8 Therefore, the ability to accurately measure and quantify cfDNA as a biomarker has enormous potential for the detection and real-time monitoring of cancer (liquid biopsy) and in prenatal screening (non-invasive prenatal testing, NIPT).

However, despite showing great promise as a tool for precision medicine, the use of cfDNA has not realized its peak yet. Two reasons can account for this:

  • Amount of cfDNA: cfDNA makes up only a fraction of the DNA present in plasma (3-6%).9 Therefore, isolating it is rather challenging, and needs specialized techniques.
  • Stability, integrity and purity of cfDNA: Methods such as digital PCR or next-generation sequencing have been used to detect and analyze cfDNA; however, they are unforgiving in terms of the quality and purity of cfDNA required as an input. It is difficult to properly preserve and stabilize cfDNA post-collection and prevent contamination from high molecular weight genomic DNA for long periods of time. Contaminating DNA can interfere with PCR and sequencing efforts, biasing results toward wild-type alleles.5

Given how expensive and time consuming downstream analytical techniques are and how precious the sample is, it is extremely critical to optimize preanalytical factors that may affect cfDNA quality to ensure the results obtained are reliable. In Part 2 of this series, we will discuss these preanalytical factors in detail and provide some tips on how to avoid them.

Roche offers cell-free DNA collection tubes for the collection, stabilization and transportation of whole blood specimens, cfDNA-based non-invasive prenatal test to screen for possible chromosomal conditions in a pregnancy, and ctDNA analysis kits for liquid biopsy assays for oncology research. Check them out.


  1. Jung et al. 2010. Cell-free DNA in the blood as a solid tumor biomarker – a critical appraisal of the literature. Clinica Chemica Acta; 411:1611-1624.
  2. Lo et al. 1997. Presence of fetal DNA in maternal plasma and serum. Lancet; 350:485-487.
  3. Salvi et al. 2016. Cell-free DNA as a diagnostic marker for cancer: current insights. Onco Targets Ther; 9:6549-6559.
  4. Volckmar et al. 2018. A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosomes Cancer; 57:123-139.
  5. Markus et al. 2018. Evaluation of pre-analytical factors affecting plasma DNA analysis. Nature; 8:7375.
  6. Bettegowda et al. 2014. Detection of circulating tumor DNA in early- and late-stage human malignancies. Science translational medicine; 6: 224ra224.
  7. Dawson et al. 2013. Analysis of circulating tumor DNA to monitor metastatic breast cancer. The New Engl J of Medicine; 8: 1199-1209.
  8. Chiu et al. 2008. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. PNAS; 105(51):20458-20463.
  9. Ordonez et al. 2013. Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma. BioMed Res Internatl; doi:10.1155/2013/195363