Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing.

Inga Medina Diaz,Daniel H Mehnert,Frank Diehl,Frank Holtrup,Johannes Fredebohm,Annette Nocon,Kelvin Yuen Kwong Chan

doi:10.1371/journal.pone.0166354

Abstract

ObjectivesMaking liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.MethodsVenous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).ResultsCollection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.ConclusionWhole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.

Highlights

The accumulation of genetic and epigenetic alterations is responsible for the transformation of normal human cells into cancer cells
Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from colorectal cancer (CRC) patients collected in cell-free DNA (cfDNA) BCTs were reliably detected after 3 days of storage at room temperature
Do cfDNA BCTs allow the stabilization of cfDNA and prevent release of additional wild-type DNA from blood cells over the time period of a typical shipment process? Second, does the cell stabilizer affect the downstream ability to amplify the cfDNA? Third, does the cell stabilizing agent induce DNA damage resulting in false positive mutation results or does it affect the detectability of rare mutations? And fourth, what happens to the blood sample and subsequent analysis if storage and transportation conditions deviate from room temperature? To answer these questions, we evaluated whole blood stored in cfDNA BCTs and standard K2EDTA blood collection tubes side-by-side by real-time quantitative PCR as well as the highly sensitive BEAMing and Plasma Safe-Sequencing technologies [10,16]

Summary

Introduction

The accumulation of genetic and epigenetic alterations is responsible for the transformation of normal human cells into cancer cells. Cell-Free DNA BCT tubes (cfDNA BCTs) commercialized by Streck (La Vista, NE) are designed to provide these sample stability features for up to 14 days at temperatures between 6 ̊C and 37 ̊C [13] These tubes have already been widely applied for noninvasive prenatal testing (NIPT) where the average fetal fraction in the maternal plasma is 10– 15% when measured between gestational weeks 10 and 20 [14]. We evaluated whole blood stored in cfDNA BCTs and standard K2EDTA blood collection tubes side-by-side by real-time quantitative PCR as well as the highly sensitive BEAMing and Plasma Safe-Sequencing technologies [10,16] We employed these techniques to investigate blood samples from healthy volunteers with and without artificial mutant DNA spike-ins as well as from patients with colorectal tumors

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Conclusion