Open AccessPreanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics
Author(s) -
Ginkel Joost H.,
Broek Daan A.,
Kuik Joyce,
Linders Dorothé,
Weger Roel,
Willems Stefan M.,
Huibers Ma M. H.
Publication year - 2017
Publication title -
cancer medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.403
H-Index - 53
ISSN - 2045-7634
DOI - 10.1002/cam4.1184
Subject(s) - digital polymerase chain reaction , dna , microbiology and biotechnology , cold pcr , whole blood , real time polymerase chain reaction , nucleic acid , cell free fetal dna , dna extraction , polymerase chain reaction , cancer , locked nucleic acid , centrifugation , biology , molecular diagnostics , lung cancer , genomic dna , medicine , bioinformatics , pathology , mutation , immunology , gene , biochemistry , genetics , point mutation , oligonucleotide , pregnancy , fetus , prenatal diagnosis
In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ct DNA ) is an emerging practice. Since ct DNA levels in blood are low, highly sensitive Droplet Digital PCR (dd PCR ) can be used for detecting rare mutational targets. In order to perform dd PCR 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 dd PCR 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 (cf DNA ) concentrations of several wild‐type targets and BRAF and EGFR ‐mutant ct DNA concentrations quantified by dd PCR were primary outcome measurements. Highest cf DNA concentrations were measured in blood collected in serum tubes. No significant differences in cf DNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cf DNA concentrations were detected after DNA isolation with the Quick cf DNA Serum & Plasma Kit, while plasma isolation using the QIA amp Circulating Nucleic Acid Kit yielded the most consistent results. Dd PCR results on cf DNA 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.