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Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Rothwell DG, Smith N, Morris D, Leong HS, Li Y, Hollebecque A, Ayub M, Carter L, Antonello J, Franklin L, Miller C, Blackhall F, Dive C, Brady G - Mol Oncol (2015)

Bottom Line: However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed.We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw.Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples.

View Article: PubMed Central - PubMed

Affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK. Electronic address: dominic.rothwell@cruk.mancehster.ac.uk.

No MeSH data available.


Related in: MedlinePlus

A. Graph showing increase in cfDNA levels in plasma from EDTA blood left at room temperature for up to 96 h post-draw. B. Schematic of EDTA and CellSave cfDNA stability study. C. cfDNA yields from 20 HNV blood samples collected in EDTA or CellSave and processed either 4 h or 96 h post-draw. No significant difference in overall yields between the 4 h EDTA, 4 h CellSave and 96 h CellSave samples with a highly significant increase in cfDNA yield following 96 h in EDTA.
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fig1: A. Graph showing increase in cfDNA levels in plasma from EDTA blood left at room temperature for up to 96 h post-draw. B. Schematic of EDTA and CellSave cfDNA stability study. C. cfDNA yields from 20 HNV blood samples collected in EDTA or CellSave and processed either 4 h or 96 h post-draw. No significant difference in overall yields between the 4 h EDTA, 4 h CellSave and 96 h CellSave samples with a highly significant increase in cfDNA yield following 96 h in EDTA.

Mentions: Our objective was to evaluate the ‘real life’ utility of CellSave preserved whole blood collection for analysis of cfDNA and CTCs as applied to blood samples obtained in multiple sites and shipped to a centralised laboratory for analysis. This had the wider goal of developing a standardised protocol to facilitate the generation of consistent, molecular analysis of both cfDNA and CTCs in clinical samples. To determine the effect of WBC lysis on cfDNA yields following long term storage (>24 h) of whole blood in EDTA, we isolated plasma from blood within 1 h of collection in a standard EDTA vacutainers tube and then at 24, 48, 72 and 96 h post-draw. Following isolation, the cfDNA yield was determined using the RNAseP real-time PCR assay (Figure 1A). Increasing amounts of cfDNA were detected over time, with almost a 3-fold increase seen by 24-h post-draw, increasing to over 60 fold by 96 h, which could reduce the ability to detect the ctDNA fraction within clinical samples.


Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Rothwell DG, Smith N, Morris D, Leong HS, Li Y, Hollebecque A, Ayub M, Carter L, Antonello J, Franklin L, Miller C, Blackhall F, Dive C, Brady G - Mol Oncol (2015)

A. Graph showing increase in cfDNA levels in plasma from EDTA blood left at room temperature for up to 96 h post-draw. B. Schematic of EDTA and CellSave cfDNA stability study. C. cfDNA yields from 20 HNV blood samples collected in EDTA or CellSave and processed either 4 h or 96 h post-draw. No significant difference in overall yields between the 4 h EDTA, 4 h CellSave and 96 h CellSave samples with a highly significant increase in cfDNA yield following 96 h in EDTA.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834815&req=5

fig1: A. Graph showing increase in cfDNA levels in plasma from EDTA blood left at room temperature for up to 96 h post-draw. B. Schematic of EDTA and CellSave cfDNA stability study. C. cfDNA yields from 20 HNV blood samples collected in EDTA or CellSave and processed either 4 h or 96 h post-draw. No significant difference in overall yields between the 4 h EDTA, 4 h CellSave and 96 h CellSave samples with a highly significant increase in cfDNA yield following 96 h in EDTA.
Mentions: Our objective was to evaluate the ‘real life’ utility of CellSave preserved whole blood collection for analysis of cfDNA and CTCs as applied to blood samples obtained in multiple sites and shipped to a centralised laboratory for analysis. This had the wider goal of developing a standardised protocol to facilitate the generation of consistent, molecular analysis of both cfDNA and CTCs in clinical samples. To determine the effect of WBC lysis on cfDNA yields following long term storage (>24 h) of whole blood in EDTA, we isolated plasma from blood within 1 h of collection in a standard EDTA vacutainers tube and then at 24, 48, 72 and 96 h post-draw. Following isolation, the cfDNA yield was determined using the RNAseP real-time PCR assay (Figure 1A). Increasing amounts of cfDNA were detected over time, with almost a 3-fold increase seen by 24-h post-draw, increasing to over 60 fold by 96 h, which could reduce the ability to detect the ctDNA fraction within clinical samples.

Bottom Line: However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed.We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw.Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples.

View Article: PubMed Central - PubMed

Affiliation: Nucleic Acid Biomarker Laboratory, Clinical Experimental Pharmacology Group, CR-UK Manchester Institute, University of Manchester, M20 4BX, UK. Electronic address: dominic.rothwell@cruk.mancehster.ac.uk.

No MeSH data available.


Related in: MedlinePlus