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, 6 (10), 2297-2307

Preanalytical Blood Sample Workup for Cell-Free DNA Analysis Using Droplet Digital PCR for Future Molecular Cancer Diagnostics

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Preanalytical Blood Sample Workup for Cell-Free DNA Analysis Using Droplet Digital PCR for Future Molecular Cancer Diagnostics

Joost H van Ginkel et al. Cancer Med.

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.

Keywords: Cell-free DNA; droplet digital PCR; molecular cancer diagnostics; preanalytical workup.

Figures

Figure 1
Figure 1
Summary of materials and methods used during various experiments. Please note: this is a schematic overview of the experimental workflow. No exact experiments are depicted.
Figure 2
Figure 2
Comparison of cfDNA concentrations in paired blood samples in four different BCTs. All samples originated from healthy controls collected. In all experiments assay, 1 was used during ddPCR. The boxplots indicate cfDNA concentrations on the y‐axis, comparing serum with EDTA BCTs from 25 healthy controls on the x‐axis (A), and citrate, heparin, serum, and EDTA BCTs from eight other healthy controls (B). The crossing lines indicate medians, the upper and lower limits of the boxes indicate interquartile ranges (25th/75th percentiles), and whiskers represent minima and maxima. *< 0.05,***< 0.001.
Figure 3
Figure 3
Influence of storage time on cfDNA concentrations until centrifugation. Time points T 1T 6 are depicted on the x‐axes. Median cfDNA concentrations were depicted on the y‐axes for average yields of pooled EDTA samples after analysis using assay 1 (A), and paired EDTA samples from six healthy individuals after using assay 2 (B). In six blood samples collected in Streck and CellSave BCTs using assay 6, 7, 10–12 (C). At each consecutive time point, mean mutant and wild‐type cfDNA concentrations from samples of two other individuals were compared with the mean cfDNA concentration of the matching EDTA samples, as depicted by mutant/wild‐type fractions (y‐axis). QS QIAsymphony, MP MagnaPure.
Figure 4
Figure 4
Comparisons of centrifugation protocols A–C, and D and E. Comparisons were performed separately in centers A and B using assay 2 and 5, respectively. Absolute cfDNA concentrations (y‐axis) detected in individuals are depicted for each protocol.
Figure 5
Figure 5
Isolation methods in healthy individuals and cancer patients. Absolute droplets counts are shown on the y‐axes. For healthy individuals, wild‐type (A) and total‐positive droplet (B) yield using assay one were depicted. For cancer patients, the sum of mutant and wild‐type positive droplets (C), as well as total droplet yields (D) where depicted using assay 6–9 (*< 0.05).
Figure 6
Figure 6
DNA quantification after isolation of EDTA samples using assay 3. In order to perform linear regression, all ddPCR results were adhered to NanoDrop and Qubit quantification results assuming 3.3 pg DNA/haploid genome (x‐axes) and depicted as ng/μL (y‐axis). In total, 38 samples were quantified by NanoDrop (A), of which five results were negative values and excluded from analysis. Seventy‐eight samples were quantified using Qubit (B). R 2 represents goodness‐of‐fit of DNA quantification methods for ddPCR.

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