Circulating free DNA (cfDNA) consists of small fragments of genetic material found outside of cells, primarily in the bloodstream, but also in other bodily fluids like urine and cerebrospinal fluid. These molecules are not contained within a cell membrane and represent a biological snapshot of the body’s cellular turnover. The presence of cfDNA in blood plasma offers a non-invasive way to gather genetic information from various tissues without a traditional tissue biopsy. This material, which includes DNA shed from healthy cells, tumors, or a developing fetus, is used in medical diagnostics.
The Biology and Origin of cfDNA
Circulating free DNA enters the bloodstream mainly as a byproduct of natural cellular processes, particularly programmed cell death, known as apoptosis. During this controlled breakdown, enzymes systematically cleave the cell’s DNA into uniform, short fragments. The predominant size of these fragments is approximately 166 base pairs, corresponding to the length of DNA wrapped around a nucleosome, the structural unit that protects DNA within the nucleus.
A secondary source of cfDNA is cell necrosis, an uncontrolled form of cell death often associated with injury or disease. Necrosis tends to release larger, less uniformly digested DNA fragments into the circulation, sometimes exceeding 10,000 base pairs. In healthy individuals, most cfDNA originates from hematopoietic cells, such as blood cells, and the concentration is relatively low. The amount of cfDNA changes dramatically in the presence of diseases like cancer or during pregnancy, where specific, non-host DNA is introduced.
Primary Diagnostic Application: Non-Invasive Prenatal Testing
The most established and widely used application of cfDNA is Non-Invasive Prenatal Testing (NIPT). This test utilizes the fact that a small, measurable amount of cfDNA in a pregnant person’s blood originates from the placenta, sharing the fetus’s genetic makeup. This subset of DNA is called the cell-free fetal DNA (cffDNA) or the fetoplacental fraction.
The fetal fraction must reach a minimum concentration, typically around four percent, for the test to be reliable, which usually occurs by the tenth week of gestation. The total cfDNA sample is analyzed using massive parallel sequencing, which counts the number of DNA fragments originating from each chromosome. In a typical pregnancy, the number of fragments corresponding to each chromosome is proportional to its size.
If the fetus has an extra copy of a chromosome, such as in Trisomy 21 (Down Syndrome), the total number of DNA fragments aligning to that specific chromosome will be slightly higher than expected. This proportional deviation signals the presence of an aneuploidy. NIPT is highly accurate as a screening tool, demonstrating a sensitivity of over 99% for detecting Trisomy 21 and very low false-positive rates.
Despite its high accuracy, NIPT is classified as a screening test, not a definitive diagnosis; a positive result requires confirmation through invasive procedures like amniocentesis. The detection of this placental DNA allows for the screening of common chromosomal conditions, including Trisomy 21, Trisomy 18 (Edwards syndrome), and Trisomy 13 (Patau syndrome). The technology provides a safe and early method for assessing the risk of these conditions.
Emerging Role in Cancer Detection (Liquid Biopsy)
Another expanding field of cfDNA analysis is its use as a “liquid biopsy” for cancer detection and monitoring. The focus here is on circulating tumor DNA (ctDNA), a small fraction of cfDNA specifically released by tumor cells. The presence of ctDNA is significant because it carries the genetic mutations, translocations, and alterations specific to the tumor.
Analyzing ctDNA allows physicians to perform molecular profiling, identifying specific genetic mutations, such as those in the EGFR or KRAS genes, that guide the selection of targeted therapies. This non-invasive approach is valuable when a traditional tissue biopsy is difficult to obtain or when a tumor is heterogeneous. Liquid biopsy provides a real-time view of the tumor’s genetic evolution, which can change rapidly under therapeutic pressure.
Beyond initial diagnosis, ctDNA testing monitors a patient’s response to treatment; a decrease in ctDNA levels often correlates with successful therapy. It is also used to detect minimal residual disease (MRD) after surgery or chemotherapy, identifying cancer recurrence months before it is visible on imaging scans. The challenge is that ctDNA often represents a very small percentage of the total cfDNA, especially in early-stage cancer, requiring sensitive detection methods.
Collection, Analysis, and Current Limitations
The process of analyzing cfDNA begins with a standard blood draw, but subsequent handling is important due to the fragile nature and low concentration of the target DNA. To prevent contamination from normal genomic DNA, which would dilute the signal, the blood sample must be processed quickly to separate the plasma from the blood cells. This is typically achieved through a two-step centrifugation process, sometimes called double centrifugation.
Once the plasma is separated, the cfDNA is extracted and prepared for analysis, most commonly using Next-Generation Sequencing (NGS). Techniques like digital droplet PCR (ddPCR) are also employed, particularly in oncology, because they detect extremely low concentrations of mutated ctDNA. These methods count and sequence millions of individual DNA fragments to identify genetic signals of interest.
A primary limitation of cfDNA analysis is the low concentration of the target molecule (cffDNA or ctDNA), which can lead to false-negative results if below the assay’s detection limit. The lack of standardized protocols across different laboratories for blood collection, processing, and storage can introduce variability in test results. Contamination with DNA from white blood cells that lyse during processing is another hurdle, necessitating strict adherence to pre-analytical guidelines to ensure accuracy.