Abstract Description Multicolor flow cytometry has advanced significantly with instruments capturing more lasers and detectors. Spectral flow cytometry, which records full emission spectra rather than peak emissions, allows fluorochromes with distinct spectral fingerprints to expand panels beyond the 40+ color limit of traditional methods. Agilent’s NovoCyte Opteon spectral flow cytometer, with its five-laser configuration (349nm, 405nm, 488nm, 561nm, 637nm) and 70 fluorescence detection channels, exemplifies this advancement. Using the OMIP-069 panel as a foundation, we expanded it to detect more biomarkers by adding dyes that either fill spectral gaps or possess unique spectral fingerprints despite similar emission maxima. The original OMIP-069 panel allowed characterization of PBMC subsets (e.g., CD4+, CD8+ T cells, B cells, NK cells, monocytes) and included activation/differentiation markers. Our modified 45-color panel introduced five biomarkers: CD31, CD45RO, CD69, CD33, and LAG-3. CD31 and CD45RO identify recent thymic emigrants (RTEs) among CD4+ T cells, CD69 tracks early activated T cells, LAG-3 assesses exhausted T cells alongside PD-1, and CD33 excludes residual monocytes. The results demonstrate that despite incorporating highly overlapping fluorochromes, immune subsets were accurately resolved, preserving dimensionality reduction and clustering outcomes. This highlights the NovoCyte Opteon’s excellent performance, enabling high-dimensional panel design and robust biomarker resolution. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Xiaoyu et al. (2025) studied this question.