Advancing Immune Profiling and Cell and Gene Therapy Monitoring in Clinical Trials with High-Dimensional Spectral Flow Cytometry 9327

Abstract Description Objective This study developed and qualified spectral flow cytometry assays for high-dimensional immune phenotyping in clinical trials, overcoming challenges in panel design, marker resolution, and assay reproducibility. Methods Markers were selected based on expression patterns, and fluorophores were assigned strategically to minimize spectral overlap, especially for co-expressed markers. Antibody titrations optimized stain index, balancing resolution and spectral interference. Single stain controls using PBMCs and beads determined unmixing strategies, with autofluorescence correction enhancing resolution. Fully stained samples were analyzed using FlowJo, and assays were qualified through precision testing across various conditions. Results Assays resolved 17–18 markers with robust T-cell subset profiling. Strategic fluorophore assignment and titration minimized resolution loss, while PBMCs and bead controls achieved superior unmixing. All metrics met stringent precision criteria, confirming assay reproducibility and robustness. Conclusion These assays demonstrate the transformative potential of spectral flow cytometry for immune monitoring in cell and gene therapies, enabling precise immune landscape characterization and advancing translational research. We developed assays capable of accurately monitoring immune responses in CGT such as CAR T cells. This advancement enhances immune profiling capabilities, CGT monitoring, and broader adoption of spectral flow cytometry. Funding Sources PPD, part of Thermo Fisher Scientific, inc. Topic Categories Technological Innovations in Immunology (TECH)