Circulating cell-free DNA (cfDNA) refers to fragmented genetic material found in bodily fluids, such as blood plasma. This DNA circulates freely throughout the body, not contained within cells. While naturally present in healthy individuals, its levels often increase in various medical conditions. CfDNA is a non-invasive tool with potential in medical diagnostics and monitoring.
Understanding Circulating Cell-Free DNA
Cell-free DNA consists of small fragments, typically 120 to 220 base pairs long, with a common size of around 170 base pairs, corresponding to the length of DNA wrapped around a nucleosome. These fragments are released into the bloodstream primarily when cells undergo natural death processes, such as apoptosis (programmed cell death) and necrosis (cell death due to injury). While most cfDNA in healthy individuals originates from hematopoietic cells like leukocytes, some cfDNA can also be actively secreted.
Once released, cfDNA remains detectable in blood plasma and serum. Its stability in circulation, with a half-life ranging from 15 minutes to 2.5 hours, allows for its detection and analysis. The concentration of cfDNA can vary significantly among individuals, from 1 to 100,000 fragments per milliliter of plasma. This presence and stability make cfDNA a valuable source of genetic information for medical applications.
Diagnostic Applications of Circulating Cell-Free DNA
The analysis of cfDNA has opened new avenues for non-invasive diagnostics across several medical fields. This approach, often termed “liquid biopsy,” allows for genetic insights without invasive tissue samples.
Non-Invasive Prenatal Testing (NIPT)
Non-invasive prenatal testing (NIPT), also known as cell-free DNA screening, uses cfDNA from a pregnant person’s blood to screen for fetal chromosomal abnormalities. Fetal cfDNA, originating from the placenta, constitutes approximately 2-20% of the total cfDNA in maternal blood and can be detected as early as 7-10 weeks of gestation. This allows for screening conditions such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13) without the risks of invasive procedures like amniocentesis or chorionic villus sampling. Fetal cfDNA is rapidly cleared from maternal circulation within hours after delivery, making it specific to the pregnancy.
Cancer Detection and Monitoring (Liquid Biopsy)
In oncology, cfDNA serves as a tool for cancer detection, monitoring treatment response, and identifying disease recurrence. A subset of cfDNA, known as circulating tumor DNA (ctDNA), is derived from tumor cells and carries tumor-specific genetic mutations and epigenetic changes. CtDNA can be used for early cancer detection, even before symptoms appear, and for tracking therapy effectiveness. For instance, tests utilizing cfDNA methylation patterns have received regulatory approval for colorectal cancer screening. This approach offers a dynamic way to assess tumor biology and guide personalized treatment strategies, providing real-time insights into disease progression.
Transplant Monitoring
Donor-derived cell-free DNA (dd-cfDNA) is an emerging biomarker for monitoring organ transplant recipients for rejection. When a transplanted organ experiences injury or rejection, dying donor cells release their DNA fragments into the recipient’s bloodstream. Elevated levels of dd-cfDNA correlate with various types of transplant rejection, including antibody-mediated and T-cell mediated rejection. This non-invasive method offers a safer, more convenient alternative to traditional invasive biopsies for assessing allograft health and aiding early diagnosis of rejection episodes.
The Process of Analyzing Circulating Cell-Free DNA
Analyzing cfDNA typically begins with a standard blood draw, as plasma is the preferred sample type to minimize contamination from genomic DNA released by white blood cells. Careful handling of the blood sample is necessary to ensure cfDNA integrity, including specific collection and storage protocols.
Once collected, cfDNA is isolated from the plasma or serum component of the blood, usually through a two-step centrifugation process to remove cellular debris and prevent contamination. Extraction methods are then used to concentrate the low-abundance cfDNA fragments. The extracted cfDNA is then quantified using sensitive PCR-based methods, given the low yields typically ranging from 1 to 30 ng/mL of plasma.
Polymerase Chain Reaction (PCR) is employed to detect specific cfDNA targets, while Next-Generation Sequencing (NGS) allows for a broader, more comprehensive analysis of genetic alterations or patterns. Despite these advanced techniques, challenges remain, including the naturally low concentration and fragmented nature of cfDNA. This necessitates highly sensitive assays and careful pre-analytical procedures to ensure accurate and reliable results.