IntroductionMonitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations.MethodsWe evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique.ResultsOverall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson’s ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5%vs.68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 minvs.159 min) and lower recovery rates (median 53.1%vs.26.8%) associated with SFCvs.MC.DiscussionAltogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents.