Abstract 4552: Quality assessment of cfDNA from urine and plasma using fragment size analysis

Abstract INTRODUCTION: The majority of liquid biopsy approaches are based on cell-free DNA (cfDNA) from plasma. In recent times, urine cfDNA is getting more attention as it has been shown to provide complementary information to blood cfDNA, which could increase sensitivity with respect to detection of pathogenic genetic variants. However, cfDNA degradation and genomic DNA (gDNA) contamination can impact the cfDNA profile and prohibit reliable molecular analysis of these analytes. Therefore, quality assessment of cfDNA eluates from both body fluids before use in sensitive downstream assays is essential. In this study, we present quality assessments based on capillary electrophoresis using samples stabilized and isolated with verified and standardized preanalytical workflows using the PAXgene Urine Liquid Biopsy Set (RUO) and the PAXgene Blood ccfDNA Tube (CE-IVD). METHODS: Urine and blood samples from apparently healthy, consented individuals were collected and stabilized in the PAXgene Urine Liquid Biopsy Set and the PAXgene Blood ccfDNA Tube (PreAnalytiX). Unstabilized samples from the same individuals were collected and processed in parallel. The samples were stored for several days on the lab bench at room temperature (15-25°C) or incubated at varying temperatures mimicking real world storage and/or transport scenarios. After storage, urine and blood samples were centrifuged to remove cells and cfDNA was isolated from the urine supernatant or plasma either using automated or manual methods. The concentration of cfDNA was measured using the Qubit dsDNA HS Quantification Assay Kit on the Qubit Fluorometer (Thermo Fisher Scientific). Fragment profiles were analyzed using the QIAxcel DNA High Sensitivity Kit on the QIAxcel Connect instrument (both QIAGEN). For analysis of cfDNA from urine, the maximum fragment size shown was manually set to 5000 bp in the ScreenGel software (QIAGEN). RESULTS: cfDNA isolated from the PAXgene Blood ccfDNA Tube showed the typical, predominantly mononucleosomal fragment length independent of whether the blood was stored or processed directly, while unstabilized blood showed contamination with gDNA. Urine cfDNA isolated from samples stabilized with the PAXgene Urine Liquid Biopsy Set showed that in addition to mono- and di-nucleosome DNA, longer fragments could be detected with and without storage. In unstabilized urine, DNA degrades rapidly, resulting in reduced fragment sizes compared to stabilized urine already on the day of urine collection without storage. After storage of unstabilized urine for three days, only degraded cfDNA could be detected. CONCLUSIONS: In this study we could demonstrate that stabilization of cfDNA in urine and blood are essential to preserve the native profiles of this analyte. Standardized preanalytical workflows provided by PreAnalytiX and state of the art quality control via the QIAxcel instrument should be considered for development of reliable downstream analysis. Citation Format: Thorsten Voss, Daniela Mancarella, Christina Goebel. Quality assessment of cfDNA from urine and plasma using fragment size analysis [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 4552.