There’s been a revolution. It comes in the form of cell free DNA (cfDNA), which is changing patient pathways for the better across the globe. Due to its nature, cfDNA is considered as an excellent biomarker and source of genetic information.

Cell free DNA is thought to originate predominantly from cells that undergo programmed cell death (otherwise known as apoptosis) as part of the natural process of tissue regeneration 1,2. DNA released in this manner is systematically fragmented in small pieces (average length of 166 bp) and can therefore seep through cell and tissue layers eventually ending up in the circulatory system3,4.

Currently in genetic diagnostics, especially in the oncology 5 and prenatal 6 fields, cfDNA originating from tumour cells (known as circulating tumour DNA or ctDNA) and placental tissue (i.e. cell free fetal DNA or cffDNA) is sequenced to find tumour mutations and fetal genetic disorders, respectively.

It’s not easy though

Recovery and analysis of cfDNA does not come without its challenges. There are three main problems to overcome:

1. There’s not much of it
2. It requires specific DNA extraction techniques
3. It can get “contaminated” with genomic DNA

Problem one: there is not much of it. In fact it ranges from 1.6 to 23.7 ng/ml of plasma 7 and it is avidly degraded by DNAse enzymes present in our bloodstreams with a half-life of approximately 16 minutes8.

Problem two: it requires DNA extraction techniques capable of recovering very small fragments of DNA9.

Problem three: it is at risk of being “contaminated” by genomic DNA released from white blood cells which start degrading over time from the point of blood draw10.

This final aspect is particularly problematic when blood samples are collected far away from the centre of testing and need to be shipped nationally, or even internationally, across long distances. Therefore, following the correct pre-analytical procedures for blood collection and processing is necessary to ensure the recovery of high-purity cfDNA and, consequentially, a higher diagnostic testing success rate11.

Additionally, specifically dedicated blood collection tubes containing a fixing agent capable of “freezing” blood cells and therefore causing a delay in cell degradation have also been developed to minimize the genomic DNA “contamination” issue9,12.

What is best laboratory practice?

Multiple studies have been conducted to investigate the best laboratory procedures for processing blood samples upon arrival at testing centres. These found that plasma deliver better quality cfDNA compared to serum, probably due to the increase in white blood cell degradation during the clotting procedure13,14.

An analysis on quantity and quality of cfDNA extracted from plasma separated from the blood cell portion at different intervals found that “contamination” from white blood cell DNA is visible at 24 hours post blood draw when using standard K2EDTA tubes; while use of blood cell stabilising tubes delays this event to 72 hours 10 or even up to 14 days 9,12.

Other factors, such as storage conditions at 4°C or room temperature of collected blood 10,13; use of different types of fixing agent within blood cell stabilising tubes 9; as well as freezing/thawing of extracted cfDNA 13, do not affect cfDNA quantity or quality. However, freezing/thawing of plasma for up to three times was found to cause a slight degradation of larger cfDNA fragments towards smaller fragment sizes, albeit leaving the overall cfDNA quantity unchanged 13.

A significant increase in genomic DNA “contamination” from white blood cells was seen when performing a single centrifugation step at low speed to isolate plasma from the blood cell portion. Instead, using a combination of low speed centrifugation, removal of plasma and additional high speed centrifugation yielded the best quality of cfDNA 9,15.

What are the best conditions for cfDNA downstream applications?

A review of the studies conducted to find the best pre-analytical conditions in which to collect/store blood and isolate/store plasma for cfDNA downstream applications can be summarised in the following recommendations:

Want to know more? Our team of experts are on hand to answer your questions.

References:
1 Chan KCA. Size Distributions of Maternal and Fetal DNA in Maternal Plasma. Clin Chem 2004; 50: 88–92.
2 Underhill HR, Kitzman JO, Hellwig S et al. Fragment Length of Circulating Tumor DNA. PLoS Genet 2016; 12: e1006162.
3 Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing. Clin Chem 2010; 56: 1279–1286.
4 Lo YMD, Chan KCA, Sun H et al. Maternal Plasma DNA Sequencing Reveals the Genome-Wide Genetic and Mutational Profile of the Fetus. Sci Transl Med 2010; 2: 61ra91-61ra91.
5 Diaz LA, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol Off J Am Soc Clin Oncol 2014; 32: 579–586.
6 Daley R, Hill M, Chitty LS. Non-invasive prenatal diagnosis: progress and potential. Arch Dis Child – Fetal Neonatal Ed 2014; 99: F426–F430.
7 Breitbach S, Tug S, Helmig S et al. Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma. PLoS ONE 2014; 9: e87838.
8 Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999; 64: 218–224.
9 van Ginkel JH, van den Broek DA, van Kuik J et al. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics. Cancer Med 2017; 6: 2297–2307.
10 Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield. PLoS ONE 2011; 6: e25202.
11 Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YD. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001; 47: 1607–1613.
12 van Dessel LF, Beije N, Helmijr JCA et al. Application of circulating tumor DNA in prospective clinical oncology trials – standardization of preanalytical conditions. Mol Oncol 2017; 11: 295–304.
13 Chan KCA, Yeung S-W, Lui W-B, Rainer TH, Lo YMD. Effects of preanalytical factors on the molecular size of cell-free DNA in blood. Clin Chem 2005; 51: 781–784.
14 Wong FCK, Sun K, Jiang P et al. Cell-free DNA in maternal plasma and serum: A comparison of quantity, quality and tissue origin using genomic and epigenomic approaches. Clin Biochem 2016; 49: 1379–1386.
15 Sherwood JL, Corcoran C, Brown H, Sharpe AD, Musilova M, Kohlmann A. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC). PloS One 2016; 11: e0150197.