Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5030.