Optimization of a 40‐Color Spectral Flow Cytometry Panel for Human Blood Immunophenotyping: Impact of Blood Volume, Sample Preservation, and Overnight Staining

Spectral flow cytometry and high-parameter panels provide a powerful tool to meet the growing demands for deep immunophenotyping. This study aimed to adapt a published 40-color spectral flow cytometry panel for whole blood analysis, evaluate the impact of cryopreservation, and explore the potential benefits of overnight staining on data quality and cost-effectiveness. The optimization process revealed several artifacts, including loss of IgG signal and spectral profile changes in PerCP-eFluor710, which were addressed through protocol and reagent modifications. Cryopreservation resulted in significant alterations to granulocyte morphology and viability, limiting direct comparisons with fresh blood samples. Preservation also led to altered distributions in 30% of gated populations, particularly affecting T cell, dendritic cell, and monocyte subsets. Overnight staining with reduced antibody concentrations enhanced resolution for key markers while allowing for 5- to 20-fold reductions in antibody usage, substantially lowering panel costs. Contrary, extended incubation times also decreased the detection of markers critical for identification of NKT-like and regulatory T cell subsets, including CD2 and CD39. This underscores the importance of fluorochrome interference, preservation effects, and extended incubation times when optimizing high-dimensional panels, suggesting opportunities to improve staining protocols in clinical and epidemiological research, particularly where resolution or cost-effectiveness are primary concerns.