Revolutionizing Healthcare Sector: Harnessing Power of Cell-Free DNA for Innovative Applications

‘’The discovery of DNA is like finding the key with which we can unlock the secrets of life’’ James Watson

The discovery of cell-free DNA (cfDNA) has opened the door for ground-breaking advancements in the medical field. Scientists and researchers are exploring the amazing potential of cell-free DNA (cfDNA) for a wide range of cutting-edge applications, marking a revolutionary step towards transforming the healthcare sector.

Here the question arises: what is cfDNA and what role does it play? Cell-free DNA (cfDNA), also referred to as circulating DNA, are degraded fragments of extracellular DNA that are circulated in body fluids such as blood plasma, urine, cerebrospinal fluid, pleural fluid, saliva, etc.

It was first identified by Mandel and Metais in the human circulation system in 1948. The molecular properties of cfDNA are also linked to its pathogenic significance. However, their release mechanisms determine their integrity (size), plasma concentration, and genetic and epigenetic profiling, which includes mutations and copy number variations.

They are usually smaller, at 166 bp, based on their source of origin. In healthy people, the concentration of cfDNA in blood plasma usually ranges from 1 to 10 ng/mL. On the other hand, the concentration can be noticeably higher in diseased people.


Moreover, cfDNA has two basic types: mitochondrial (mt-cfDNA) and nuclear (n-cfDNA). Both differ in their structural features, which may indicate alternative biological stability mechanisms. These DNA fragments can be formed from the intracellular to the extracellular region by a variety of processes.

The following mechanisms can cause cfDNA to be released into the bloodstream in both healthy and diseased cases: necrosis (premature cell death), apoptosis (programmed cell death), NETosis (formation of neutrophil extracellular traps), extracellular vesicles, erythroblast enucleation (a process by which the immature red blood cells break up to remove the nucleus), and exogenous sources.

Different molecular-based techniques have been utilised in the analysis of cfDNA from samples, e.g., fluorescence-based methods, polymerase chain reaction (PCR), especially droplet digital PCR, real-time PCR, microarrays, next-generation sequencing, etc.


Due to its beneficial and unique characteristics, cfDNA can serve as a potential biomarker in a wide variety of theranostic applications in cases like genetic and cardiovascular diseases, neurological and hematological disorders, autoimmune diseases, cancer, infection, pharmacogenomics, forensic and transplant rejection, etc.

By using chromosomal microarray-based cfDNA testing, analysis of large amounts of cfDNA fragments can be carried out, which is used for prenatal screening for Down syndrome and other aneuploidies.

Physicians have modified sequencing-related cfDNA testing to identify organ rejection in patients with heart and lung transplants. For the detection of allograft damage or rejection, donor-derived cfDNA can be identified circulating in the peripheral blood of the recipient.

cfDNA can be rapidly and non-invasively used as a biomarker for early screening and diagnosis, to monitor cancer progression, and to predict the response of treatment to different types of cancer, including hepatocellular carcinoma, melanoma, gastric, thyroid, colorectal, prostate, lung, pancreatic, breast cancer, etc. Researchers have designed mt-cfDNA-based biomarkers to predict the severity of coronary artery lesions and diagnose acute coronary syndrome.

In pharmacogenomics, cfDNA analysis can be applied for personalised therapies based on a patient’s genetic profile, thus increasing drug effectiveness and reducing side effects. In addition to that, cfDNA analysis can be employed in forensic cases to recognize people or establish genetic links.

Pakistan’s Perspective

In Pakistan, there has been a significant amount of research conducted on cfDNA, indicating an increasing interest in the respective field and its potential for diagnostic and prognostic applications for a variety of medical disorders.

The Biochemistry Department of the Riphah International University of Islamabad has carried out comparative research on non-invasive prenatal beta-thalassemia testing utilizing chorionic villus samples and cell-free fetal DNA from maternal plasma.

Similarly, the Biochemistry Department of Ziauddin University of Karachi has conducted cfDNA analysis for the detection of different chromosomal abnormalities in pregnant females in Pakistan. QuaideAzam University of Islamabad has done an evaluation of cell-free mitochondrial DNA in various viral infections as an indicator of the severity of illness. (Shahid et al., 2020).


Despite having a lot of plus points, certain factors remain significant challenges for cfDNA analysis, such as limited accuracy and sensitivity, false positives or false negative results, complex interpretation of data, and the high cost of advanced cfDNA testing techniques, which may prevent some populations or healthcare systems from adopting them.

Future Indication and Conclusion

With its growing multitude of applications, cell-free DNA (cfDNA) has the potential to completely transform the healthcare industry in the future.

It is expected that cfDNA’s significant diagnostic value will extend beyond non-invasive prenatal testing and cancer detection as technical improvements continue to solve existing obstacles. Because of its adaptability, cfDNA can be used in individualised and targeted therapy methods by enabling early identification and monitoring of a variety of conditions. Its incorporation into standard clinical practice may also improve prediction for disease, direct treatment choices, and result in more efficient medical procedures.

An in-depth understanding of the significance of cfDNA in the health sector will probably open up new avenues for research and development, bringing in an era of precision medicine and better patient outcomes.