These include the speed at which cells are analyzed, the high accuracy and resolution of the technology, and the low operating costs per sample. This technology emerged as a powerful tool for detailed analysis of complex populations and several other factors have contributed to the success and widespread use of flow cytometry. Examples of applications of automated tools that have successfully improved the characterization of different cell populations in vaccination studies are also presented.įlow cytometry allows to simultaneously quantify expression of extracellular and intracellular molecules targeted by dyes or monoclonal antibodies, as well as to measure multiple characteristics of a single cell such as size and granularity. Analysis based on computational tools might overcame both the subjectivity of manual gating and the operator-biased exploration of expected populations. In this review, an overview of the automated analysis pipeline is provided, spanning from the pre-processing phase to the automated population analysis. The potentialities of automated analyses range from the improvement of the data quality in the pre-processing steps up to the unbiased, data-driven examination of complex datasets using a variety of algorithms based on different approaches. Filling the gap between algorithms developers and final users is fundamental for exploiting the advantages of computational tools in the analysis of cytometry data. However, the use of these tools has been preferentially restricted to bioinformaticians, while few of them are routinely employed by the biomedical community. In recent years, the development of many tools for the automated analysis of multiparametric cytometry data has been reported, with an increasing record of publications starting from 2014. When many parameters are investigated simultaneously, it is not feasible to analyze all the possible bi-dimensional combinations of marker expression with classical manual analysis and the adoption of advanced automated tools to process and analyze high-dimensional data sets becomes necessary. Multiparametric flow cytometry is particularly suitable for deep analysis of immune responses after vaccination, as it allows to measure the frequency, the phenotype, and the functional features of antigen-specific cells. Flow and mass cytometry are used to quantify the expression of multiple extracellular or intracellular molecules on single cells, allowing the phenotypic and functional characterization of complex cell populations.
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