High‐Dimensional Spectral Flow Cytometry Panel for Immunophenotypic Characterization of Fixed Whole Blood Samples

Peripheral blood mononuclear cells (PBMCs) are commonly used for immune profiling in clinical research, but their isolation and cryopreservation present logistical challenges, particularly in settings with limited access to research facilities. To address this, we implemented a whole blood fixation protocol to preserve surface protein epitopes and enable high-dimensional flow cytometry without the need for immediate sample processing. A 36-marker lymphoid and 35-marker myeloid spectral flow cytometry panels were developed and optimized to characterize diverse immune populations, including T cell subsets, B cells, NK cells, monocytes, dendritic cells, and granulocytes. Panel design focused on antibody clone compatibility with fixed antigens, minimizing non-specific binding, and reliably detecting chemokine receptors. Performance was evaluated by comparing fresh and fixed whole blood, revealing consistent identification of major immune lineages. Long-term stability was demonstrated by using cryopreserved fixed blood from a healthy donor over two and half years, with immune profiles remaining stable. These findings support the use of fixed whole blood for high-dimensional spectral cytometry and its utility in longitudinal clinical immune monitoring.