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

Joost H Van Ginkel,Dorothé Linders,Manon M H Huibers,Stefan M Willems,Roel De Weger,Daan A Van Den Broek,Joyce Van Kuik

doi:10.1002/cam4.1184

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.

Highlights

Current cancer diagnostics is often performed on molecular pathology findings from biopsy material
In all 25 cases, cell-free DNA (cfDNA) concentrations were significantly highest in serum samples compared to paired ethylenediaminetetraacetic acid (EDTA) samples (204.0 [67.7–532.0] vs. 18.4 [12.7–21.4], P < 0.001) (Fig. 2A)
This study shows that droplet digital PCR (ddPCR) results of cfDNA quantification strongly depend on preanalytical blood sample workup, comprising an extensive multistep process

Summary

Introduction

Current cancer diagnostics is often performed on molecular pathology findings from biopsy material This is an invasive technique and not always possible to perform. Blood samples are easy to obtain and contain cell-free DNA (cfDNA) including circulating tumor DNA (ctDNA). These DNA fragments carry patient-specific genetic targets, and can be used as a diagnostic, prognostic or predictive biomarker. No standardized approach exists for the use of liquid biopsy in conjunction with ddPCR, which has only recently been introduced into molecular diagnostics and clinical research. This could explain inconsistencies in blood sample workup using ddPCR on liquid biopsies

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