Abstract 673: Protein Plus BCT: Ensuring reliable protein biomarker analysis in liquid biopsies by minimizing pre-analytical variations

Abstract While plasma protein biomarkers show promise for cancer early detection, diagnosis, prognosis, and monitoring, pre-analytical variation can affect sample integrity and limit the reliability of proteomic analysis. Sample collection, storage, and processing conditions can alter the proteome through ex vivo blood cell activation and lysis, which releases proteins that can obscure relevant in vivo abundances. This can significantly impact cancer biomarkers, many of which are expressed in white blood cells and platelets. Here, we use both immunoassay- and LC-MS-based proteomics to show that Protein Plus BCT minimizes proteome alterations during whole blood storage and plasma processing. To this end, plasma was isolated using single and double spin protocols from blood collected into EDTA and Protein Plus BCT immediately after draw or after 3 days of storage at either 2 °C, room temperature (22 °C), 30 °C, or 37 °C. When samples were analyzed on the Ella platform for oncology-relevant biomarkers, including TGF-β (relevant in CRC), we observed that the abundance of TGF-β increased by 5- and 4.3-fold on day 3 compared to draw time in plasma isolated from EDTA using single and double spin protocols, respectively. Conversely, there were minimal changes in abundance for plasma isolated from Protein Plus BCT, with no more than 1.5-fold change from day 3 to draw time observed for either single or double-spin processing. When samples were prepared for bottom-up proteomic analysis, we noted 6 times more proteins were significantly elevated in abundance for single spin plasma at draw time for EDTA compared to Protein Plus BCT. This trend persisted after 3 days of whole blood storage, with 11 times more proteins being elevated in abundance for single spin plasma for EDTA compared to Protein Plus BCT, further demonstrating the ability of Protein Plus BCT to maintain proteome stability across diverse sample storage and handling conditions. Specifically, we saw that EDTA samples demonstrated at least a 3 Log2 fold change increase in S100A8 concentration, a potential cancer biomarker, on day 3 relative to draw time levels regardless of sample processing (single vs double spin) or storage temperature (2 °C, 22 °C, 30 °C, or 37 °C), whereas we saw no increase in abundance in samples collected into Protein Plus BCT on day 3 except for samples stored at 37 °C, which showed a 0.5 Log2 fold change increase. Taken together, these data indicate that Protein Plus BCT preserves sample integrity and minimizes changes to the proteome, relative to EDTA, by reducing the impact of extended storage, temperature variations, and sample handling on accurate analysis. This offers researchers and assay developers flexibility in sample transport, storage, and processing. Protein Plus BCT is for Research Use Only. Not for use in diagnostic procedures. Protein Plus BCT should only be used for research or the development of new assays. Citation Format: Rachel M. Miller, Sama Mehta, Jing Li. Protein Plus BCT: Ensuring reliable protein biomarker analysis in liquid biopsies by minimizing pre-analytical variations [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 673.