Spectral flow cytometry is an upcoming technique that allows for extensive multicolor panels, enabling simultaneous investigation of a large number of cellular parameters in a single experiment. To fully explore the resulting high-dimensional single cell datasets, high-dimensional analysis is needed, as opposed to the common practice of manual gating in conventional flow cytometry. However, preparing spectral flow cytometry data for high-dimensional analysis can be challenging, because of several technical aspects. In this article, we will give insight into the pitfalls of handling spectral flow cytometry datasets. Moreover, we will describe a workflow to properly prepare spectral flow cytometry data for high dimensional analysis and tools for integrating new data at later time points. Using healthy control data as example, we will go through the concepts of quality control, data cleaning, transformation, correcting for batch effects, subsampling, clustering and data integration. This methods article provides an R-based pipeline based on previously published packages, that are readily available to use. Application of our workflow will aid spectral flow cytometry users to obtain valid and reproducible results.
