40‐Parameter/37‐Color Spectral Cytometry Panel for Robust Immunoprofiling of Human Lymphoid Subsets in Cancer Patients

ABSTRACT The analysis of immune cell compartments in cancer patients is crucial to predict treatment efficacy and relapse. We introduce a robust 40‐parameter, 37‐channel spectral cytometry panel designed to profile human lymphoid subsets and CAR‐T cell expansion, with the capability to assess exhaustion status by profiling immune checkpoints and activating receptors in cancer patients. Developed for the 5‐laser Cytek Aurora, the panel optimizes fluorophore selection and uses three pairs of mutually exclusive markers assigned to a single fluorescent parameter to simplify setup and ensure robust data, adopting a conservative design choice to keep similarity indices below 0.85; though higher overlaps can still yield high‐quality data when best practices are applied. The panel enables detailed analysis of well‐defined lymphoid subsets using a conventional gating strategy, as well as detection of unconventional subsets with variable expression patterns by unsupervised algorithm‐based analysis. The effectiveness of the panel is demonstrated through a dataset simulating the progression of multiple myeloma, from pre‐malignant disease to a highly aggressive stage.