In cancer, a portion of circulating cfDNA comes from tumor cells. This tumor-derived fraction is called circulating tumor DNA (ctDNA). Because ctDNA contains cancer-related genetic and epigenetic changes of the tumor cells, it can be used to study tumor biology, support treatment decisions, monitor treatment response and resistance, track minimal residual disease, and help with early cancer detection1.
cfDNA-based liquid biopsy is becoming more widely used because it is generally well tolerated and far less invasive than traditional tissue sampling. Therefore, understanding where cfDNA originates and the variables that impact its detection is essential for generating accurate and biologically meaningful insights.
Where Is Cell-Free DNA Found?
cfDNA can be detected in a variety of biofluids, including blood, urine, cerebrospinal fluid (CSF), saliva, and pleural effusions. Among these, blood remains the most widely used and well-characterized source for cfDNA analysis, because it is accessible and routinely collected as part of clinical patient care. Blood is also highly biologically rich, reflecting molecular contributions from tissues and organs throughout the body, whereas other biofluids often provide more localized biological signals.
cfDNA is a low-abundance analyte, with concentrations typically ranging from 1 to 10 ng/mL in healthy individuals2. At those levels, pre-analytical variables can have a major effect on sample quality and downstream analysis. Blood collection tube type, processing time, and plasma preparation methods can all influence the amount and integrity of cfDNA that can be recovered.
Blood Collection Tube Type
Whole blood is typically collected in ethylenediaminetetraacetic acid (EDTA) or Cell-Free DNA BCT® (Streck) tubes, which contain additives that help preserve blood cell integrity after collection to minimize the release of contaminating genomic DNA into plasma. Following collection, blood is centrifuged to generate double-spun plasma (DSP), the cell-free fraction used for cfDNA analysis.
The choice of collection tube determines how the blood sample remains suitable for processing to isolate cfDNA. Streck tubes contain a preservative that stabilizes nucleated blood cells and can maintain sample stability for more than 48 hours before processing. EDTA tubes, by comparison, are less stable and need to be processed as quickly as possible to limit genomic DNA contamination from blood cells. In Discovery’s workflow, EDTA-collected samples are processed within two hours of collection and Streck-collected samples within 48 hours to maximize cfDNA recovery while minimizing genomic DNA contamination.
In our evaluation, when EDTA-collected samples are processed promptly, comparable levels of cfDNA can be isolated from DSP prepared using either tube type.
For cfDNA studies with specific program requirements, we can prospectively collect blood using custom collection tube types to meet downstream assay requirements.
Single vs. Double-Spun Plasma
Single-spun plasma contains cfDNA, but it can also carry substantial amounts of contaminating cellular and genomic DNA that may interfere with downstream analysis. For this reason, double-spun plasma is generally preferred for cfDNA work as the second centrifugation step helps remove residual blood cells and cellular debris, improving the ability to detect low-abundance cfDNA.
However, many existing plasma collections, particularly those generated for studies not originally designed for cfDNA analysis, may only be available as single-spun plasma. We have found that single-spun plasma samples collected from solid tumor patients can be processed again to generate DSP that is more suitable for cfDNA analysis. Subjecting single-spun plasma to an additional centrifugation step to produce DSP effectively removes the contaminating material resulting in a higher concentration of detectable cfDNA, as seen in Figure 2, thereby expanding the utility of existing plasma collections for downstream cfDNA applications.
