35-color CytoFLEX mosaic panel profiling B cells, myeloid cells, and diagnostic markers in chronic lymphocytic leukemia (CLL) 2306495

Introduction Advances in spectral flow cytometry have enabled high-resolution analysis of immune cell diversity, particularly within the B cell compartment. Building on previous work, we refined and validated a high-dimensional spectral B cell panel using PBMC samples from CLL patients. The panel was optimized for the CytoFLEX mosaic 88 Spectral Detection Module integrated with the CytoFLEX LX platform with UV laser capability. Methods The panel incorporates refined fluorophore selection and marker combinations to resolve major and minor B cell subsets, including transitional, naïve, double-negative, switched and unswitched memory, and antibody-secreting cells, while maintaining robust spectral unmixing and minimizing overlap. It supports simultaneous assessment of surface immunoglobulins (IgA, IgG), light chains, activation markers, and co-receptors, enabling broad phenotypic coverage in a single tube. Additionally, the high multiplexing capacity allows inclusion of markers for progenitors and other immune subsets such as dendritic cells, monocytes, and NK cells. Results Leveraging the multiplexing capacity of spectral technology, the panel design also accommodates markers for progenitor cells and additional immune subsets, including dendritic cells, monocytes, and NK cells. Conclusion Applying this panel to CLL samples can reveal disease-associated immunophenotypic patterns, highlighting its potential for translational research and future clinical applications in B-cell malignancies.