Guidelines for the Preanalytical Conditions for Analyzing Circulating Cell-Free DNA

Abstract

Circulating cell-free DNA (cfDNA) isolated from blood has been identified as a potential biomarker in numerous fields, and has been the object of intensive research over the past decade, although its original discovery dates back 60 years. While it is already used routinely in commercial and clinical practice in oncology and prenatal testing, other potential applications have emerged, including for diabetes, cardiovascular diseases, organ transplantation, autoimmune diseases, sepsis, trauma, and sport management. As with the discovery and development of any biomarker, preanalytical requirements and documentation are as important as analytical requirements. Except for the case of noninvasive prenatal testing and prenatal diagnosis, the implementation of cfDNA in a clinical setting remains limited because of the lack of standardization of cfDNA analysis. In particular, only a few attempts have been made to collect and pool scientific data on the relevant preanalytical factors, and no standard operating procedure has yet been set. For this report, we have performed a thorough and systematic search via MEDLINE® for relevant preanalytical variables and patient factors. These form the basis of the guidelines we propose for analyzing nuclear cfDNA.

Blood contains circulating cell‐free respiratory competent mitochondria

“…Fresh blood was collected in EDTA tubes. Plasma was isolated by a single centrifugation, performed at 1200 g for 10 minutes at 4°C …”

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

“…To achieve proper results with various genetic tests, it is important to establish a method to extract a high yield of cfDNA. Many researchers have investigated the optimal conditions for each step, encompassing sample collection, handling, and storage to maximize the recovery of cfDNA [7][8][9][10].…”

Hypoxia differently modulates the release of mitochondrial and nuclear DNA

“…DNA molecules of nuclear and mitochondrial origins are found in the extracellular compartment, in vitro in the media of cell culture 42,43 and in vivo in the physiological fluids. 44,45 While the analysis of circulating DNA from plasma is now optimised and standardised, 37 only poor experimental works have been performed with using cell culture. To accurately observe the effect of hypoxia on cells, we first carried out a study on standardising cell culture conditions to avoid any bias.…”

“…Sample treatment and cfDNA extraction All methods were performed according to the pre-analytical guidelines previously established by our group. 37 Supernatants from cultured cancer cell lines or mice plasma were harvested in Eppendorf tubes, centrifuged to remove all cells (1200 × g; 10 min) and then frozen until use. After thawing, samples were centrifuged at 16,000 × g for 10 min at 4°C to remove cellular debris and organelles.…”