Circulating Cell-Free DNA Part 2

Blog Staff


4 critical factors that can make or break your experiment

Although the potential of cfDNA for various screening and diagnostic applications such as liquid biopsy1 and non-invasive prenatal testing2 has created quite some excitement, the use of cfDNA is still fraught with a number of issues. Lack of standardized protocols and parameters for sample handling, reproducibility in results, and other technical difficulties are some of the challenges that remain to be tackled. In a previous post, we mentioned factors that that may influence its broad adoption. In this article, we’ll discuss preanalytical factors affecting cfDNA in detail and explore various options on how best to optimize them.

1. Stability and contamination

cfDNA can be obtained from both plasma and serum, but plasma has been the primary sample source for most oncology studies as serum is susceptible to a higher degree of contamination from genomic DNA released from white blood cells3. Break down of red blood cells, also known as hemolysis, can be another source of contamination as well. The type of cfDNA collection tubes used can have an impact on the preservation of plasma and on the ability to isolate circulating tumor DNA (ctDNA) subsequently for molecular diagnostics.4 Collection tubes with anticoagulants, such as the di- or tri-potassium salts of EDTA (K2EDTA and K3EDTA), help preserve sample integrity. K2EDTA collection tubes are recommended by the International Council for Standardization in Hematology (ICSH) and the Clinical and Laboratory Standards Institute (CLSI) as appropriate for PCR-based molecular diagnostics.5 Apart from anticoagulants, other stabilizing agents are also added to preserve the integrity of nucleated white blood cells until downstream analysis. Therefore, it may be worthwhile to find out if the stability agent in the collection tube is compatible with your assay and review stability data before making your purchasing decision.

2. Temperature and storage

Since it usually takes a few days between blood collection and sample preparation, care must be taken to control for factors that can affect cfDNA during transportation or storage of blood samples. The temperature at which the tubes are handled during transit and storage is an important factor that can affect the quality of cfDNA.1,5 Therefore, it is important to note the recommended storage temperatures from the cfDNA collection tube manufacturer. While at it, also find out if there is data on cfDNA stability during temperature excursion or long storage times.

3. Transportability and safe handling

Even though this may seem trivial, transportability is indeed an important consideration since blood-draw and testing may not be performed on the same site. cfDNA collection tubes made with fragile material like glass, despite having some advantages, pose a risk of breakage and injury, not to mention a broken tube with blood would be a health hazard as well. cfDNA tubes made of polyethylene terephthalate (PET) make handling and transportation safer while retaining the advantages of glass and provide a viable alternative. Consider the material with which your tubes are made of and see if it is safe and durable for your purposes.

4. Compatibility with other parts of your workflow

The reagents in the tube used to stabilize and preserve cfDNA could interfere with specific downstream applications. For example, anticoagulants, such as heparin, have been shown to cause PCR inhibition, thus disrupting downstream molecular analysis.6 Contaminating high molecular weight DNA from lysed peripheral blood cells during processing can interfere with tagmentation-based sequencing and PCR.7 Therefore, it’s important to consider what the collection tubes have with reference to what your downstream needs are.

Finally, if you’re trying to choose among tubes offered by different manufacturers, find out how good their service and support is. Purchasing from a trusted vendor with extensive sample prep expertise can go a long way in helping out with your experiments.

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. 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.
  2. Lo et al. 1997. Presence of fetal DNA in maternal plasma and serum. Lancer; 350(9076):485-7.
  3. Messaoudi et al. 2013. Circulating cell free DNA: preanalytical considerations. Clinica Chimica Acta.424:222-230.
  4. Alidousty et al. 2017. Comparison of blood collection tubes from three different manufacturers for the collection of cell-free DNA for liquid biopsy mutation testing. J of Mol Diagn; 19(5):801-804.
  5. Parpart-Li et al. 2016. The effect of preservative and temperature on the analysis of circulating tumor DNA. Clinical cancer research; doi: 10.1158/1078-0432.CCR-16-1691
  6. Beutler E, Gelbart T, Kuhl W. 1990 Interference of heparin with the polymerase chain reaction. Biotechniques;9:166.
  7. Markus et al. 2018. Evaluation of pre-analytical factors affecting plasma DNA analysis. Nature; 8:7375