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

Abstract

In 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.

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

“…However, fluorometry can overestimate cfDNA concentration as it does not differentiate HMW DNA fragments [38]. Although previous studies have demonstrated the correlation of fluorometric quantification and real-time PCR [39,40], an electrophoresis-based instrument provides a better method considering that genomic DNA contamination is often observed after cfDNA isolation. In addition, urinary cfDNA is known to be more fragmented and variably sized compared to plasma [25,41].…”

Intra‐individual variation of circulating tumour DNA in lung cancer patients

“…The plasma was thawed on ice and centrifuged at 13 000 g for 10 min prior to DNA extraction (van Ginkel et al , ). DNA was extracted from between 2.8 and 4 mL of EDTA plasma using the QIAamp® Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.…”

Quantification of Cell-Free DNA: A Comparative Study of Three Different Methods

“…Using fluorescent probes that directly bind to dsDNA, the fluorometric quantification is an approach that compensates for the disadvantage of the spectrophotometry-based methods (e.g., NanoDrop) [19]. Several previous studies have described that the fluorometric quantification is highly correlated to qPCR quantification measurements and dPCR results [20][21][22]. The fragment lengths of isolated cfDNA usually are A major limitation of this study is that the repro ducibility of each platform could not be demonstrated as we…”