Label-Free Identification of Human Eosinophils Using 808 nm Side Scatter

Accurate identification and quantification of eosinophils is critical for the diagnosis and monitoring of eosinophil-associated disorders. While flow cytometry remains a powerful tool for leukocyte characterization, conventional instruments equipped with 405 nm or 488 nm side scatter (SSC) detectors offer limited resolution for eosinophil discrimination overlap in scatter with neutrophils. Using a spectral flow cytometer equipped with six distinct SSC detectors, we report a novel, label-free approach for eosinophil detection leveraging high 808 nm near-infrared SSC (IRSSC) uniquely observed in human eosinophils. This optical signature is independent of antibody labeling, activation fixation, or permeabilization, and shows strong concordance with conventional CD66b/CD16 gating strategies (R = 0.997). Notably, the high 808 nm SSC is absent in murine eosinophils, suggesting a species-specific structural feature such as in human eosinophils. These findings establish IRSSC as a robust, reagent-free biomarker for eosinophil detection, with broad implications for both clinical diagnostics and translational immunology.