Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma

Abstract

Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1h at 4°C, or 4days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1h of blood storage at 4°C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.