Abstract
Purpose of ReviewLiquid biopsy analyses based on circulating cell-free nucleic acids, circulating tumor cells or other diseased cells from organs, and exosomes or other microvesicles in blood offer new means for non-invasive diagnostic applications. The main goal of this review is to explain the importance of preserving whole blood specimens after blood draw for use as liquid biopsies, and to summarize preservation solutions that are currently available.Recent FindingsDespite the great potential of liquid biopsies for diagnostics and disease management, besides non-invasive prenatal testing (NIPT), only a few liquid biopsy applications are fully implemented for routine in vitro diagnostic testing. One major barrier is the lack of standardized pre-analytical workflows, including the collection of consistent quality blood specimens and the generation of good-quality plasma samples therefrom. Broader use of liquid biopsies in clinical routine applications therefore requires improved pre-analytical procedures to enable high-quality specimens to obtain unbiased analyte profiles (DNA, RNA, proteins, etc.) as they are in the patient’s body.SummaryA growing number of stabilizing reagents and dedicated blood collection tubes are available for the post-collection preservation of circulating cell-free DNA (ccfDNA) profiles in whole blood. In contrast, solutions for the preservation of circulating tumor cells (CTC) that enable both, enumeration and molecular analyses are rare. Solutions for extracellular vesicle (EV) populations, including exosomes, do not yet exist.
Highlights
Circulating cell-free DNA was first described by Mandel and Metais in 1948 [1]
Luk et al compared anticoagulated via EDTA or Citrate (ACD) and K2-EDTA tubes, Cell-Free DNA and Cell-Free RNA blood collection tube (BCT) (Streck), and Cyto-Chex BCT (Streck) in a prostate circulating tumor cells (CTC) setting, analyzing androgen receptor variant 7 (ARv7) with a droplet digital PCR (ddPCR) assay after CTC enrichment on the AutoMaCS Pro Separator (Miltenyi, Bergisch Gladbach, Germany) [92]. They found that the CTC AR v7 transcript remains stable in EDTA or ACD-anticoagulated blood for 48 h at room temperature, whereas mRNA detection in all the three Streck BCT tubes dropped directly after blood collection and was impossible to measure after any transport time
Several providers of blood collection tubes with stabilization reagents for liquid biopsy applications have claims for Circulating cell-free DNA (ccfDNA) and CTC preservation (e.g., Streck Cell-Free DNA BCT and Biomatrica LBgard Blood Tube), but as long as there is no convincing preservation and enrichment concept available for parallel isolation of all required liquid biopsy components from 1 blood sample, there seems to be no alternative to using different tubes for different purposes
Summary
Circulating cell-free DNA (ccfDNA) was first described by Mandel and Metais in 1948 [1]. Existence of cell-free fetal DNA (cffDNA) circulating in the maternal bloodstream was initially published by Lo and colleagues [5]. They were among the first to recognize the potential of using ccfDNA for noninvasive prenatal testing (NIPT), such as determination of rhesus factor [6], and. Other subtypes of CTCs have been described which lose their epithelial origin and undergo epithelial to mesenchymal transition (EMT) and tumor stemlike phenotype changes. Such cells are potentially negative for cytokeratins but express EMT markers [19, 20]. In addition to the prognostic value of CTC identification and counting in cancer progression, molecular characterization of CTCs can guide treatment decisions and improve patient outcome
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