A new and emerging field in diagnostics, circulating cell-free DNA (cfDNA) analysis, has enormous potential for analyzing a variety of conditions such as diabetes, cardiovascular diseases, organ transplantation, autoimmune diseases, sepsis, trauma, and sports management. However, lack of standardization has limited cfDNA analysis in clinical settings. A special report in the May issue of  Clinical Chemistry outlines the preanalytical conditions and patient factors that affect cfDNA, offering proposed guidelines for analyzing this promising versatile biomarker.

Scientific literature has mainly focused on the potential clinical applications of cfDNA rather than basic knowledge. This has been especially true in establishing standard operating procedures (SOP) for cfDNA applications, according to Alain R. Thierry, PhD, senior investigator of the special report, and director of research at the Institute of Research in Oncology of Montpellier in Montpellier, France. A pioneer in analyzing cfDNA, Thierry has developed methods to precisely identify this biomarker and is coordinating clinical trials to examine applications of these methods. Through this work, he developed an expertise in preanalytical factors involving cfDNA.

Through the proposed guidance, Thierry hopes to assist researchers, clinical labs, private diagnostic sectors, and other stakeholders in establishing SOP for cfDNA applications.

“As with the discovery and development of any biomarker, preanalytical requirements and documentation are as important as analytical requirements,” Thierry and his co-authors noted. They cite five major challenges in the preanalytical phase of cfDNA analysis, including:

  • Blood has a heterogeneous content that impedes the isolation of cfDNA.
  • A complex biological material, blood is sensitive to an assay’s duration, especially in terms of enzymatic degradation or blood clotting.
  • Naked DNA is a labile macromolecule in a biological environment.
  • DNA from blood cells may contaminate cfDNA.
  • Some applications require detection of a small specific cfDNA fraction, such as tumor mutant cfDNA or fetal cfDNA.

The authors made specific recommendations with respect to blood collection, plasma preparation, and cfDNA extraction methods and    storage, and quality control and transport of blood tubes. As an example, to prevent lysis of leukocytes when using tubes containing the anticoagulant K2EDTA, “plasma isolation should be of the shortest duration possible,” they emphasized, recommending a maximum window of 4 hours.

Thierry recommended that labs analyzing cfDNA “should strictly follow these guidelines to design stringent specific SOP based on their diagnostic objectives.”

Limited data is available on the biological and demographic factors that influence the release of cfDNA. “Higher yields of cfDNA, for instance, were found in healthy men compared with healthy women, in current smokers compared with never-smokers, in older individuals, in menopausal women, in certain ethnicities, in fasting individuals, in those with increasing body mass index, and after intense physical exercise,” summarized Thierry and his colleagues. Certain types of therapies may also impact the accuracy of cfDNA test results.

“For interpreting cfDNA signals and their associations with clinical manifestations of diseases, more research on the fundamental biology of cfDNA would help to further define the standardization of preanalytical conditions,” they recommended.

Pick up May’s Clinical Chemistry to get more details on the best SOP practices for dealing with preanalytical conditions when analyzing cfDNA.