Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

Abstract

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 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. 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.

Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells

“…This has led to an exponential growth of the amount of data generated from a single aliquot of a relatively small volume of a sample (e.g., blood). Such technological advances have thereby contributed to an increase in our knowledge about immune cells in both homeostatic and disease conditions, due to the enhanced capability of measuring millions of cells stained with dozens of markers simultaneously ( 33 – 36 ). However, new challenges in flow cytometry data analysis have also emerged.…”

An autologous ex vivo model for exploring patient-specific responses to viro-immunotherapy in glioblastoma

“…Samples were processed and stained according to the standardized EuroFlow protocols www.euroflow.org/protocols . 35 All incubations were performed in the dark. Briefly, the cells of the co-culture were stained with the antibody panel for 30 min at room temperature, washed with PBS and incubated with a viability marker (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation, Thermo Fisher Scientific) for 30 min at 4°C.…”

Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

“…Despite offering more reliable results, the cost of disposables remains a hindrance to the widespread adoption of these systems (15). Mass cytometry cannot provide information about the cell size, internal complexity and autofluorescence profile which can be counted as a drawback depending on what the researchers are looking for (128).…”

“…Another beneficial feature is the automated subtraction of cellular autofluorescence. In studies comparing mass cytometry and spectral cytometry results, researchers underscore the significance of selecting an efficient panel (128,132).…”