Through use of length-dependent qPCR, OncoBEAM enhanced digital PCR, and Plasma-Safe-SeqS NGS, several questions regarding the optimum handling and shipping conditions are answered
A 2016 publication from Sysmex Inostics, ‘Performance of Streck cfDNA blood collection tubes for liquid biopsy testing’ (Medina Diaz et al. PLoS One, November 2016, DOI:10.1371/journal.pone.0166354) has proven to be very popular, with over 18,000 views and 48 citations. It is unique in its approach in evaluating a method for transfer of blood samples prior to liquid biopsy analysis, utilizing both highly-sensitivity OncoBEAM enhanced digital PCR in addition to Plasma-Safe-SeqS, our sensitive NGS-based method, for background mutations introduced through preservation and storage.
Venous blood samples from healthy donors as well as colorectal cancer (CRC) patients were collected in Streck Blood Collection Tubes (BCT) as well as standard K2EDTA tubes, then stored at different temperatures for various lengths of time before plasma separation and DNA purification. Length-specific qPCR was used to measure cfDNA compared to contaminating genomic DNA, and high-sensitivity enhanced digital PCR OncoBEAM and NGS-based Plasma-Safe-SeqS technology was used to look for introduction of mutations. Additionally, some spike-in low-level synthetic mutant DNA was also added and measured.
The number of healthy donors for the five study cohorts totaled 103; an overview of the experimental conditions and analysis methodologies is in figure 1 below and available online.
Figure 1 from Medina Diaz et al. available online here. Per its figure legend:
Experimental setup for cfDNA BCT vs K2EDTA performance experiments. Cohort I: Time point experiments at room temperature including DNA quantification and mutation analysis using BEAMing and Safe-SeqS. Cohort II: BEAMing analysis of blood samples spiked with synthetic double-stranded mutant DNA fragments at different allele frequencies. Cohort III: BEAMing analysis of samples collected from colorectal cancer (CRC) patients. Cohort IV: Experiment evaluating effects of extreme storage temperatures on DNA quantity. Cohort V: Experimental evaluation of recommended temperature range.
The effect of time on cfDNA in Streck Blood Collection Tubes
In Study Cohort I, 60 healthy samples were compared between K2EDTA and BCT storage at room temperature for 3 and 5 days, and no significant differences were detected in the quantity and quality of the cfDNA samples.
The effect of temperature on cfDNA in Streck Blood Collection Tubes
Of particular interest is ‘Study Cohort IV’, where BCT storage at temperatures slightly outside the official manufacturer-recommended temperature range (e.g. 6 °C to 37 °C); 4 °C, RT and 40 °C for up to 5 days was used in this experiment, and visual differences between the samples were apparent after the first centrifugation step. RT-stored samples (stored at both the 3 and 5 day intervals) had a clear plasma fraction with a defined buffy coat; the 4°C samples had an expanded cellular interface layer (affecting 20-50% of the plasma fraction), while the 40 °C samples had a darker, hemolytic plasma fraction.
Length-dependent qPCR indicated a much higher ratio in these temperature-stressed samples, indicating much higher genomic DNA contamination compared to reference.
The authors conclude “exposure to extreme temperatures outside the recommended range of 6-37 °C needs to be avoided for cfDNA BCTs in order to prevent dilution of potential mutant cfDNA molecules with wild-type genomic DNA released from WBCs (white blood cells).” See figure 2.
Figure 2: Whisker-plot data derived from Medina Diaz et al. PLOS One 2016 showing the effect of storage temperature outside the manufacture-recommended temperature range, and photograph of a clear buffy coat layer under RT conditions, an increased interface cell layer at 4 °C, and hemolytic plasma at 40 °C.
No spurious low-level mutations introduced
Going back to ‘Study Cohort I’, these 60 samples were analyzed by both OncoBEAM enhanced digital PCR for six KRAS mutations, as well as by Plasma-Safe-SeqS NGS-based mutation detection for five c-KIT amplicons.
Figure 3, a slightly different representation of Figure 3 in the Medina Diaz et al reference. A) Enhanced digital PCR (BEAMing) data analyzing 6 mutations across 60 samples B) Targeted NGS (Plasma-Safe-SeqS) data analyzing >500 bases across 15 samples.
As you can see from Figure 3A, with an analytical cutoff of the OncoBEAM technology at 0.02% none of the five KRAS mutations assayed for were observed above the cutoff. In Figure 3B, the base change pattern observed with Plasma-Safe-SeqS did not change in any of the conditions tested.
A few key take-away conclusions
Through work like this Sysmex Inostics has established recognized and standardized conditions for blood collection and transport for ultra-sensitive ctDNA detection for liquid biopsy testing. Certainly K2EDTA-collected plasma can be spun down within four hours and stored at -70°C, however the use of Streck BCT collection tubes allows for flexibility in transport, as long as the 6°C to 37°C temperature boundaries are observed for up to 5 days.
Medina Diaz I, Nocon A, Mehnert DH, Fredebohm J, Diehl F, Holtrup F (2016) Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing. PLoS ONE 11(11): e0166354. https://doi.org/10.1371/journal.pone.0166354