Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics

Cancer Medicine - Tập 6 Số 10 - Trang 2297-2307 - 2017
Joost H. van Ginkel1, Daan A. van den Broek2, Joyce van Kuik1, Dorothé Linders2, Roel de Weger1, Stefan M. Willems1, Manon M. H. Huibers1
1Department of Pathology, University Medical Center, Utrecht, The Netherlands
2Department of Clinical Chemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands

Tóm tắt

AbstractIn current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell‐free DNA (cfDNA) concentrations of several wild‐type targets and BRAF and EGFR‐mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future.

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Cancer Medicine

Tập 6 Số 10

2297-2307

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