Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

Abstract

Objectives: Making 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.

Methods: Venous 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).

Results: Collection 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.

Conclusion: Whole 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.

Fig 1. Experimental study cohorts.

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.

Fig 2. Analysis of cfDNA yield and genomic DNA release for study cohort I.

(A) DNA yield was assessed for cfDNA from blood samples stored at room temperature (18°C– 22°C) in K₂EDTA tubes vs cfDNA BCTs (healthy donors, n = 60). Plasma was prepared after indicated storage conditions. Extracted DNA was analyzed for overall yield by qPCR amplifying a 96 bp LINE-1 fragment. (B) Illustration of the DNA yield ratio between long (402 bp) and short (96 bp) LINE-1 fragments (n = 60). Increased ratios compared to K₂EDTA reference would indicate genomic DNA release. Shown are box plots with 1.5 × interquartile range (IQR) applied to create whiskers and outliers. Statistical analysis using one-way ANOVA revealed no significant difference between conditions.

Fig 3. Mutation background analysis of study cohort I using BEAMing and Safe-SeqS.

(A) Mutation background was assessed using BEAMing for 6 KRAS mutations (c.34G>A, c.34G>C, c.34G>T, c.35G>A, c.183A>T, c.437C>T) in presumably healthy wild-type donors (n = 60). Samples were stored at room temperature. Shown are scatter dot plots with mean values (horizontal line). One-way ANOVA statistics revealed no statistical significance between conditions. (B) Safe-SeqS was applied to analyze base changes in five c-KIT amplicons from healthy donor blood samples stored in K₂EDTA tubes and cfDNA BCTs at RT for the indicated time (n = 15). In total, 553 bases were analyzed per sample (171 As (31%), 105 Cs (19%), 110 Gs (20%) and 167 Ts (30%)). §: 1 event detected for K₂EDTA 2 h C>G; #: 0 events detected for cfDNA BCT 5 d G>C.

Fig 4. Detectability of low frequency mutations in study cohort II.

Blood from healthy donors (n = 8) was spiked with synthetic double-stranded mutant DNA fragments of different allele frequencies in a background of 60,000 genome equivalents of fragmented human genomic wild-type DNA and subsequently stored at RT. DNA was extracted and analyzed by BEAMing after the indicated storage time. Mean and standard deviation of the detected mutant fraction is shown. (A) PIK3CA c.3140A>G spike (0.1%), (B) EGFR c.2369C>T spike (0.5%), (C) KRAS c.34G>A spike (1%).

Fig 5. Correlation of mutation analysis results from CRC samples collected in K₂EDTA and cfDNA BCTs (study cohort III).

Blood from CRC patients (n = 21) was collected in K₂EDTA tubes and cfDNA BCTs. Samples were processed after the indicated storage time and analyzed using the OncoBEAM RAS assay (33 RAS mutations). Mutant fractions for all RAS-positive events are plotted for both cfDNA BCT conditions vs matched K₂EDTA reference (n = 7 per condition).

Fig 6. Effect of extreme storage temperatures on plasma separation and DNA yield in study cohort IV.

(A) Representative image of cfDNA BCTs centrifuged after 3 days of storage at RT, 4°C and 40°C. RT storage resulted in expected plasma separation with defined buffy coat layer and clear yellow plasma fraction. Extreme temperature conditions resulted in an expanded cellular interface layer or hemolytic plasma at 4°C or 40°C, respectively. (B) Effect of extreme temperatures on genomic DNA release (402 bp LINE-1 qPCR fragment). Statistically significant differences between K₂EDTA and cfDNA BCT storage conditions determined by one-way ANOVA are marked with ** (p ≤ 0.01). Shown are box plots with 1.5 x IQR applied to create whiskers. (C) Obtained mean plasma volume with SD for indicated storage conditions.

Fig 7. Genomic DNA release and obtained plasma volume after 3 days of storage within temperature range recommended by the manufacturer (study cohort V).

Blood collected in cfDNA BCTs (n = 8) was stored for 3 days at the indicated temperature and subsequently analyzed using the genomic DNA release assay based on the 402:96 bp LINE-1 ratio (A). Shown are box plots with 1.5 x IQR applied to create whiskers. Statistically significant differences from the reference condition (20°C) were determined by one-way ANOVA and are marked with an * (p ≤ 0.05). (B) Obtained mean plasma volume with SD for indicated storage conditions.