Abstract 4573: Verified preanalytical workflow for urine cfDNA including the PAXgene Urine Liquid Biopsy Set allows transport and storage at varying temperatures and is unaffected by blood interference

Abstract INTRODUCTION: Urine has become an important standalone source for biomarkers research using cell-free DNA (cfDNA). In addition, urine cfDNA has been shown to provide complementary information to blood cfDNA. However, cfDNA degradation, dilution by genomic DNA (gDNA) released from body cells and bacteria can impact the cfDNA profile and prohibit transport and storage of urine, making reliable urine molecular analysis impossible. Furthermore, interfering substances, such as blood, could affect the preanalytical workflow including the cfDNA isolation and hence, affect the research outcome. In this study, we tested the PAXgene Urine Liquid Biopsy Set (RUO) for urine collection and cfDNA stabilization in a standardized preanalytical workflow with challenging sample conditions and show that it enabled reliable analysis of cfDNA. METHODS: Stabilized urine from apparently healthy, consented individuals was collected in the PAXgene Urine Collection Cup. Female urine specimens were spiked with cell-free male urine supernatant and, where indicated, with 1% v/v female blood. Urine was stabilized using the PAXgene Urine Liquid Biopsy Tube or was left unstabilized. Urine samples were stored for 3 days or 10 days at room temperature (RT, 15-25°C) on the lab bench or incubated for up to 10 days at varying temperatures mimicking real-world transport scenarios. Two temperature profiles were tested: Cold profile (4 hours (h) at RT , followed by 3 days (d) at 5°C, followed by 6 d at 12°C and 8 h at RT ) and warm profile (3 d at 25°C, followed by 12 h at 37°C, followed by 1 d at 25°C, followed by 5 d at 5°C and 10 h at RT). Additional urine samples were transported via air from Germany to the USA. Where indicated, transported samples underwent dropping and vibration testing. cfDNA was isolated from the urine supernatant either using automated or manual QIAGEN kits. Autosomal and male-specific targets were quantified by qPCR on the QIAGEN RGQ instrument. RESULTS: Stabilization with the PAXgene Urine Liquid Biopsy Tube increased the cfDNA yield isolated from urine. Furthermore, it minimized the loss of the cfDNA target of interest, minimized the dilution of the cfDNA target of interest by gDNA release and minimized bacterial growth during urine storage at challenging warm and cold weather temperature profiles and real-life urine transport. Blood concentration of 1% v/v in urine did not affect the yield of cfDNA isolated from the PAXgene Urine Liquid Biopsy Tube. CONCLUSION: The optimized and standardized preanalytical workflow for cfDNA analysis from urine using the PAXgene Urine Liquid Biopsy Set enabled urine storage and transport and reliable analysis of urine cfDNA under challenging research settings. Furthermore, 1% v/v blood in urine did not interfere with cfDNA isolation and detection, underlining the robustness of the verified preanalytical workflow. Citation Format: Daniela Mancarella, Laura Sofie Witthaus, Moritz Meyer, Julia Rutsch, Moritz Rath, Lisa Erkelenz, Thorsten Voss. Verified preanalytical workflow for urine cfDNA including the PAXgene Urine Liquid Biopsy Set allows transport and storage at varying temperatures and is unaffected by blood interference [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4573.