Abstract
Abstract It is well documented that in the chain from sample to the result in a clinical laboratory, the pre-analytical phase is the weakest and most vulnerable link. This also holds for the use and analysis of extracellular nucleic acids. In this short review, we will summarize and critically evaluate the most important steps of the pre-analytical phase, i.e. the choice of the best control population for the patients to be analyzed, the actual blood draw, the choice of tubes for blood drawing, the impact of delayed processing of blood samples, the best method for getting rid of cells and debris, the choice of matrix, i.e. plasma vs. serum vs. other body fluids, and the impact of long-term storage of cell-free liquids on the outcome. Even if the analysis of cell-free nucleic acids has already become a routine application in the area of non-invasive prenatal screening (NIPS) and in the care of cancer patients (search for resistance mutations in the EGFR gene), there are still many unresolved issues of the pre-analytical phase which need to be urgently tackled.
Highlights
New European Union regulations on medical devices and in vitro diagnostic medical devices entered into force and will apply fully for in vitro diagnostics in May 2022 [1]
Even if the analysis of cell-free nucleic acids has already become a routine application in the area of non-invasive prenatal screening (NIPS) and in the care of cancer patients, there are still many unresolved issues of the pre-analytical phase which need to be urgently tackled
Their conclusion “that plasma nucleases play only a partial role in the removal of circulating fetal DNA in most subjects” might not be correct as the ethylenediaminetetraacetic acid (EDTA) inhibits nuclease action. bIt is not clear why purified Candida albicans DNA behaves differently when compared with DNA from other organisms demonstrating a much higher stability in plasma. cIn other reports, it was shown that the majority of cell-free nucleic acids is taken up by the liver and the importance of DNase activity in the removal is not clear, especially as DNA released in complexes with proteins, lipids or other molecules might be protected from degradation
Summary
New European Union regulations on medical devices and in vitro diagnostic medical devices entered into force and will apply fully for in vitro diagnostics in May 2022 [1]. A literature survey on the analysis of circulating tumor DNA (ctDNA) issued by the American Society of Clinical Oncology and the College of American Pathologists stated that “some ctDNA assays are clinically useful for certain types of advanced tumors” [3]. This type of analysis does not yet hold any clinical utility for ctDNA analysis in early-stage cancer, therapy monitoring or residual disease detection. The first meeting on Circulating Nucleic Acids in Plasma and Serum (CNAPS) that was organized 20 years ago by the founders of the liquid biopsy field Drs Stroun and Anker, already called for a standardization of the numerous pre-analytical issues. We will focus on papers dealing with cell-free nucleic acids and exclude work done on circulating tumor cells
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