Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

Rasmussen, Louise; Herzog, Mariëlle; Rømer, Eva; Micallef, Jake; Bulut, Orhan; Wilhelmsen, Michael; Christensen, Ib Jarle; Nielsen, Hans Jørgen

doi:10.1080/00365513.2016.1190862

Cited by 9 publications

(6 citation statements)

References 17 publications

(19 reference statements)

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“…More recently the effects of pre-analytical variables were investigated to determine the durability of circulating nucleosomes. Stasis, contamination with white cells, within-day variation, varying time before centrifugation, colonoscopy and surgical trauma had no significant influence on the level of 5-methylcytosine DNA (5mC) or H3K9me3 in circulating nucleosomes [ 117 ]. The 5mC and H3K9me3 levels were significantly lower in cancer patients compared to healthy individuals [ 117 ].…”

Section: Quantifying Ptm Of Circulating Nucleosomes As Biomarkersmentioning

confidence: 99%

Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

McAnena

Brown

Kerin

2017

Cancers

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Traditionally the stratification of many cancers involves combining tumour and clinicopathological features (e.g., patient age; tumour size, grade, receptor status and location) to inform treatment options and predict recurrence risk and survival. However, current biomarkers often require invasive excision of the tumour for profiling, do not allow monitoring of the response to treatment and stratify patients into broad heterogeneous groups leading to inconsistent treatment responses. Here we explore and describe the benefits of using circulating biomarkers (nucleosomes and/or modifications to nucleosomes) as a non-invasive method for detecting cancer and monitoring response to treatment. Nucleosomes (DNA wound around eight core histone proteins) are responsible for compacting our genome and their composition and post-translational modifications are responsible for regulating gene expression. Here, we focus on breast and colorectal cancer as examples where utilizing circulating nucleosomes as biomarkers hold real potential as liquid biopsies. Utilizing circulating nucleosomes as biomarkers is an exciting new area of research that promises to allow both the early detection of cancer and monitoring of treatment response. Nucleosome-based biomarkers combine with current biomarkers, increasing both specificity and sensitivity of current tests and have the potential to provide individualised precision-medicine based treatments for patients.

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Section: Quantifying Ptm Of Circulating Nucleosomes As Biomarkersmentioning

confidence: 99%

Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

McAnena

Brown

Kerin

2017

Cancers

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“…In several prior studies, comparisons between single components or steps were published which are part of preanalytical processes used for ccfDNA exploitation. These comparisons included evaluations of sample type [39,44,45], different collection tubes [46][47][48], centrifugation conditions [49][50][51], or ccfDNA extraction chemistries [22,33]. The previous studies gave insights into certain aspects of the workflow, but their setups are not aligned with ISO 20186-3, have only a limited sample size, deal with challenging sample material, and/or use a complex and difficult to reproduce experimental setup [32,33].…”

Section: Plos Onementioning

confidence: 99%

Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal

et al. 2021

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The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

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“…These are interesting observations, but unfortunately the study population is too small (11 subjects in total) to draw any significant conclusions. Rasmussen and co-workers analyzed plasma and serum samples taken from healthy volunteers 3 times a day (8 am, 12 am and 3 pm) for the quantity of nucleosomes containing 5-methylcytosine (5mC) DNA or the histone modification H3K9Me3, but did not find any fluctuation [44]. When they looked for differences in these markers in serum samples obtained on different days (day 1, 8, 15, 22 and 29), the intra-individual variation was 12.7% and 11.5% for 5mC and H3K9Me3, respectively.…”

Section: Basic Principles Of Cell-free Nucleic Acids: Release Half-lmentioning

confidence: 99%

“…(It is known that the change of position from supine to sitting or vice versa has an effect on some laboratory parameters [135], but so far we do not know whether this is true for cell-free nucleic acids as well.) -Does tourniquet stasis have an influence on the quantity/quality of cell-free nucleic acids (there is only one report which analyzed the amount of nucleosomes harboring either methylated DNA or a histone modification after blood draw with/without stasis and no difference was detected [44])? -Does the needle size used for blood drawing (apart from being big enough to prevent hemolysis) have an effect?…”

Section: Factors Which Can Impact the Quantity/quality Of Cfdnamentioning

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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Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

Abstract: Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation.

Cited by 9 publications

References 17 publications

Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

Circulating Nucleosomes and Nucleosome Modifications as Biomarkers in Cancer

Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal

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

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