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 regeneration1,2. 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 oncology5 and prenatal6 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:
- There’s not much of it
- It requires specific DNA extraction techniques
- 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 plasma7 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 10 hours or even up to 14 days9,12.
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:
- Recover cfDNA from plasma instead of serum.
- Use blood cell stabilising tubes to collect and store blood when more than 24 hours are expected to pass from blood draw to plasma isolation.
- Conduct an initial centrifugation step at 800-1000g for 10 minutes to separate the plasma from the blood cell portion.
- Centrifuge the isolated plasma at 10,000-16,000g for 10 minutes to remove any leftover blood cells and cell debris.
- Store plasma at -20/-80°C, avoiding repeated freeze/thaw cycles as much as possible.
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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.