Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Sato, Akemi; Nakashima, Chiho; Abe, Tomonori; Kato, Junichi; Hirai, Mitsuharu; Nakamura, Tomomi; Komiya, Kazutoshi; Kimura, Shinya; Sueoka, Eisaburo; Sueoka‐Aragane, Naoko

doi:10.18632/oncotarget.25881

Cited by 29 publications

(26 citation statements)

References 32 publications

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“…Most papers describe cfDNA at size 160-170 bp, and long fragment cfDNA has been thought to be contamination by genomic DNA. We previously reported a pre-analytical procedure for assessing cfDNA, in which we confirmed that long fragment cfDNA appeared in samples stored under inappropriate conditions [8]. However, long fragment cfDNA of 5 Kb was definitely observed in patients with advanced lung cancer even under proper conditions, as described in the present paper and our previous paper [7].…”

Section: Discussionsupporting

confidence: 89%

Origin of circulating free DNA in patients with lung cancer

et al. 2020

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Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.

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Section: Discussionsupporting

confidence: 89%

Origin of circulating free DNA in patients with lung cancer

et al. 2020

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“…Increased concentrations could be due to cell lysis that may be a source of pre-analytical error. Additional studies in dogs evaluating the quality as well as the quantity of cfDNA are warranted to determine if citrate plasma maintains cfDNA quality better than does EDTA, as has been described for people 47. Such additional studies might also evaluate whether there is an impact of diagnosis or disease severity on the sensitivity of biomarker detection in distinct sample types.…”

Section: Discussionmentioning

confidence: 99%

Effect of sample type on plasma concentrations of cell‐free DNA and nucleosomes in dogs

Goggs

2019

Veterinary Record Open

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Cell-free DNA (cfDNA) and nucleosomes are two biomarkers of cell death and neutrophil extracellular trap formation that are increased in dogs with sepsis, immune-mediated haemolytic anaemia, cancer and following trauma and have diagnostic and prognostic values. cfDNA and nucleosomes are typically measured in plasma samples using DNA-specific fluorophores and ELISA assays, respectively, but their concentrations may be affected by pre-analytical variables such as sample type. The present study aimed to investigate the influence of sample type on the plasma cfDNA and nucleosome concentrations of a heterogeneous group of dogs presenting to an emergency room. Triplicate samples were collected into K2-ethylenediamine tetraacetic acid, 3.2% citrate and a specialised DNA stabilisation tube (Streck BCT), processed rapidly and frozen for batch analysis. Biomarker concentrations were compared between sample types by calculation of Spearman’s correlation coefficients, and with Deming regression, Bland-Altman plots and the Friedman test. Overall, biomarker concentrations were highly correlated between the three sample types. The most concordant results were obtained using citrate samples and the DNA stabilisation tube. Matched cfDNA concentrations between the different sample types were significantly different but there was no significant difference between the nucleosome concentrations in any of the sample types. The present study suggests that cfDNA and nucleosomes can be successfully measured in various sample types, but distinct sample types do not produce interchangeable results. This argues for use of a consistent sample type within studies and suggests standardisation may be useful for the field.

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“…For this reason, the use of plasma/serum pools instead of single blood samples for long-term storage experiments might be a good alternative. Recently, it was demonstrated that plasma samples with a low mutant AF degrade faster during storage than samples with a high mutant AF, supporting the assumption that individual samples behave very differently [93]. The storage of extracted cfDNA in a dry form (such as paper-based matrices) as compared to the usual storage in a liquid might be a viable alternative as shown by Heider and colleagues [149].…”

Section: Long-term Storage Of Cell-free Matrices and Freeze-thaw Cyclesmentioning

confidence: 88%

“…In recent years, several companies developed a range of DNA isolation kits of which only the "dedicated" kits were recommended for the extraction of cfDNA [192]. In several papers, a variety of different kits were compared [84,85,93,[193][194][195][196][197][198][199][200][201][202][203][204]. From these results, one can conclude that bead-based isolation methods worked somewhat better than membrane-based kits and that the former ones are especially useful for the isolation of low-molecularweight DNA.…”

Section: Dna Isolationmentioning

confidence: 99%

Pre-analytical issues in liquid biopsy – where do we stand?

Fleischhacker

Schmidt

2020

Journal of Laboratory Medicine

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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.

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Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Cited by 29 publications

References 32 publications

Origin of circulating free DNA in patients with lung cancer

Origin of circulating free DNA in patients with lung cancer

Effect of sample type on plasma concentrations of cell‐free DNA and nucleosomes in dogs

Pre-analytical issues in liquid biopsy – where do we stand?

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