Pre-Analytical Evaluation of Streck Cell-Free DNA Blood Collection Tubes for Liquid Profiling in Oncology

Abstract

Excellent pre-analytical stability is an essential precondition for reliable molecular profiling of circulating tumor DNA (ctDNA) in oncological diagnostics. Therefore, in vitro degradation of ctDNA and the additional release of contaminating genomic DNA from lysed blood cells must be prevented. Streck Cell-Free DNA blood collection tubes (cfDNA BCTs) have proposed advantages over standard K₂EDTA tubes, but mainly have been tested in healthy individuals. Blood was collected from cancer patients (n = 53) suffering from colorectal (n = 21), pancreatic (n = 11), and non-small-cell lung cancer (n = 21) using cfDNA BCT tubes and K₂EDTA tubes that were processed immediately or after 3 days (BCTs) or 6 hours (K₂EDTA) at room temperature. The cfDNA isolated from these samples was characterized in terms of yield using LINE-1 qPCR; the level of gDNA contamination; and the mutation status of KRAS, NRAS, and EGFR genes using BEAMing ddPCR. CfDNA yield and gDNA levels were comparable in both tube types and were not affected by prolonged storage of blood samples for at least 3 days in cfDNA BCTs or 6 hours in K₂EDTA tubes. In addition, biospecimens collected in K₂EDTA tubes and cfDNA BCTs stored for up to 3 days demonstrated highly comparable levels of mutational load across all respective cancer patient cohorts and a wide range of concentrations. Our data support the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days for reliable cfDNA and mutation analyses.

Keywords: cell-free DNA (cfDNA); circulating tumor DNA (ctDNA); liquid profiling; pre-analytics; precision medicine.

Figure 1

Experiment layout and study cohorts. (A) Experimental setup for evaluating the performance of cfDNA BCTs and K₂EDTA tubes. Plasma samples from all patients were evaluated for cfDNA yield and mutational load using qPCR and BEAMing, respectively. Cohorts II and III included an additional storage condition (K₂EDTA, 2 h at RT) to access nuclease activity with DNA quantification. (B) Analyzed mutations for CRC, pancreatic cancer, and NSCLC cohorts.

Figure 2

Evaluation of cfDNA yield. Plasma from CRC patients (A), pancreatic cancer patients (B), and NSCLC patients (C) was prepared after different whole-blood storage times at room temperature (18–22 °C) in K₂EDTA tubes and cfDNA BCTs. CfDNA concentration in each plasma sample was determined with qPCR amplification using a 96 bp multi-copy LINE-1 fragment. Error bars indicate standard deviation.

Figure 3

Effect of storage time on cfDNA sample quality. The level of genomic DNA contamination in plasma samples from (A) CRC patients, (B) pancreatic cancer patients, and (C) NSCLC patients following 2 h of RT blood storage in K₂EDTA tubes, 2 h of RT storage in cfDNA BCTs, and 3 days of RT storage in cfDNA BCTs, was assessed by calculating the ratio between the measured amounts of long (402 bp) and short (96 bp) LINE-1 cfDNA fragments (y-axis). CfDNA BCTs were able to stabilize cfDNA and WBCs over 3 days of storage at RT. Statistical analysis was performed using Dunnett’s multiple comparisons test, and the Tukey method was used to create whiskers and outliers (black dots). No statistically significant differences were observed for all tested conditions (p > 0.05). Error bars indicate standard deviation.

Figure 4

Correlation of BEAMing mutation test results between collection tubes. (A) Blood from CRC, pancreatic cancer, and NSCLC patients (n = 53) was collected in K₂EDTA tubes and cfDNA BCTs, and samples were analyzed for mutational load using BEAMing after the indicated storage time at RT. Mutant fractions of all detectable mutation sets are plotted for both cfDNA BCT conditions (x-axis) and matched K₂EDTA reference (y-axis). A total of 16 mutations were found in 15 of the 53 samples (28.3%), with one patient harboring a mutation in two genes. Mutant allele frequencies in cfDNA ranged from 0.026% to 41.6% and showed very comparable results between the tube conditions, even for low-positive samples. (B) Blood samples from NSCLC (n = 21) and pancreatic cancer patients (n = 11) were collected in K₂EDTA tubes and processed at 2 h and 6 h following phlebotomy. BEAMing analysis revealed very comparable results, suggesting that K₂EDTA tubes are sufficient for storage of samples for up to 6 h after collection. Good correlations were obtained for both pancreatic cancer and NSCLC samples, with R² values of 0.9923 and 0.9957, respectively.