Xbeads: A Platform for Flow Cytometry Sample QC and Harmonization

Summary Flow cytometry is a cornerstone of modern biomedical research and clinical diagnostics, offering unparalleled insights into the human immune system. Its capacity to rapidly analyze thousands of cells per second while simultaneously measuring dozens of parameters has made it indispensable for studying a wide range of biological processes. However, the complexity of flow cytometry assays introduces challenges in standardization and reproducibility. The variability in flow cytometry arises from several factors, including instrumentation and setup, reagent stability and quality, and experimental methods. These factors, especially in high-parameter experiments, can significantly impact assay results, hindering reliable data comparison across time, instruments, and testing sites. To address these challenges, we developed Xbeads, a novel internal assay quality control system. When added to samples prior to staining and processing, Xbeads can assess the performance of the entire cytometry workflow, including sample processing, data acquisition, and antibody reagent activity. By monitoring changes in Xbead signals, errors can be identified and normalized to ensure quantitative comparability across different experimental conditions. In our Phase I studies, we successfully demonstrated the utility of Xbeads in quantifying cellular antigen expression. We developed tightly-defined Xbeads for different antigens that were stable during storage and could be combined to create antigen cocktails. Xbeads effectively corrected for errors in instrument setup, reagent degradation, and staining methods. In this Phase II proposal, we plan to expand the Xbead system to include a broader range of validated antigens, encompassing both surface and intracellular targets. We will also conduct a mock flow cytometry clinical trial to assess the ability of Xbeads to normalize variation in a typical study using multiparameter staining panels. The successful completion of the Phase II studies will yield a robust commercial offering of Xbeads that has been rigorously validated to correct for instrument, reagent, and processing complexity. By addressing the challenges of assay variability, we aim to enhance the reproducibility and utility of flow cytometry in both research and clinical applications.