Abstract 4616: Detection of phosphorylated biomarkers in clinical blood samples: a flow cytometry platform utilizing smart tube fixation

Abstract The identification and quantification of biomarkers in clinical blood samples play a pivotal role in personalized medicine, enabling disease monitoring and therapeutic assessment. Protein phosphorylation, a key post-translational modification, reflects cellular signaling in immune or tumor cells. To exploit this, we developed a platform detecting phosphorylation states of key signaling proteins from clinical blood samples via flow cytometry. Our approach analyzes phosphorylated STAT5 (pSTAT5), ERK1/2 (pERK1/2), and p38 (p-p38), vital components in immune regulation and cancer progression. A major challenge in the analysis of phosphorylated proteins is the rapid degradation or change in phosphorylation state ex vivo, which requires immediate stabilization of samples after collection. To solve this problem, we use Smart Tubes, which enable rapid fixation of clinical blood samples and preserve the phosphorylation state upon collection, ensuring signal integrity and minimizing variability. Sample collection with Smart Tubes requires no technical equipment and can be easily implemented and standardized at any clinical trial site. Our test system employs flow cytometry, enabling the analysis of linage-specific markers (e.g., T/B cells, monocytes) and phospho-specific antibodies to quantitatively detect pSTAT5, pERK1/2, and p-p38 in peripheral blood mononuclear cell (PBMCs) subpopulations. Validation experiments with specific inhibitors demonstrate sensitivity and specificity in detecting these phosphorylated targets, even in heterogeneous cell populations within blood samples. The method allows for its application across a range of clinical conditions, from cancer immunotherapy monitoring to autoimmune disease characterization. By integrating rapid sample fixation with advanced analytical techniques, this platform addresses key challenges in analyzing phosphorylated biomarkers in clinical samples. The use of flow cytometry as the central analytical tool enables single-cell resolution, allowing detailed investigation of individual cell populations within complex samples by differential marker expression. This precision makes it possible to differentiate and study specific subpopulations of immune cells or circulating tumor cells, within a heterogeneous sample. The ability to preserve and accurately measure phosphorylation states directly in patient-derived samples provides invaluable insights into real-time cellular signaling and facilitates the optimization of therapeutic strategies targeting dysregulated signaling pathways. In summary, our system can detect phosphorylated proteins in clinical samples, with Smart Tubes ensuring sample stability and reliable biomarker analysis, advancing translational research and precision medicine. Citation Format: Valerie Oberhardt, Philipp Metzger, Carla N. Castro, Holger Weber. Detection of phosphorylated biomarkers in clinical blood samples: a flow cytometry platform utilizing smart tube fixation [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 4616.