Abstract 2341: Impact of delayed processing on cell-free (cf) DNA quantity and quality using different cfDNA-stabilizing blood tubes: a Connect for Cancer Prevention Pilot Study Cohort

Abstract Background: Prospective evaluations of cfDNA/liquid biopsy assays are critical to assess their potential for improved early detection of cancer. Important knowledge gaps related to blood collection, preservation, and processing practices must be addressed for adequate assay execution.2 The NCI Connect for Cancer Prevention Cohort Study (Connect) aims to study new approaches in early detection. Connect conducted pilot studies evaluating the STRECK Cell-Free DNA BCT (DNA), Streck Nucleic Acid (NA), and PAXgene tubes to determine the impact of processing time on cfDNA quantity and quality utilizing quality control assays. Methods: Plasma samples were collected from 40 healthy doners using Streck DNA, Streck NA, and PAXgene tubes through the NCI Frederick Research Donor Program. Half of the samples from each tube set were processed at room temperature immediately and the other half after five days. Three assays were performed to compare cfDNA quantity and quality: (1) Qubit/Spectramax assay measuring total DNA concentration (ng/uL); (2)TapeStation measuring %cfDNA and average fragment size (bp); and (1) an EFIRM Alu assay measuring Alu element concentration (copies/mL). Descriptive analyses were performed to compare metric concentrations between processing time points, with and without samples identified as outliers. Results: After five-day processing, hemolysis for the PAXgene tubes (50.0%) and the Streck DNA tubes (40.0%), but not the Streck NA tubes (0.0%). No hemolysis was observed before immediate processing. Qubit/Spectramax and TapeStation assays showed that all three tube types had samples with normal fragment size between 50-700bp, over 90% of with normal total DNA concentration of less than 1.00ng/uL, and a majority with more than 60% of cfDNA. Streck DNA had the most outliers with less than 60% cfDNA (n=10) compared to PAXgene (n=9) and Streck NA (n=1). Streck DNA also had more outliers with 1.00 or greater total DNA concentration (n=4) compared to PAXgene (n=2) and Streck NA (n=0). EFIRM Alu results will be available during conference presentation. Conclusions: Streck DNA, Streck NA, and PAXgene showed variable cfDNA quality and quantity performance. The Streck NA tube had the lowest number of samples with hemolysis and the lowest number of outliers for %cfDNA and total DNA concentration. With respect to preanalytical variables of prolonged processing time and tube type, the Streck Nucleic acid seems most suited at maintaining cfDNA quantity and quality. Citation Format: Jazmyn Bess, Domonique Hopkins, Norma Diaz Mayoral, Heather Cooley, Angela de Palatis, David Wong, David Elashoff, Fang Wei, Feng Li, Mohammad Aziz, Chris Karlovich, Eric Greenbank, Stephanie Weinstein, Christian Abnet, Marie-Josephe Horner, Amanda Black, Michelle Brotzman, Kathleen Wyatt, Lynn Sorbara, Matthew R. Young, Mia Gaudet, Nicolas Wentzensen. Impact of delayed processing on cell-free (cf) DNA quantity and quality using different cfDNA-stabilizing blood tubes: a Connect for Cancer Prevention Pilot Study Cohort [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 2341.