(a) Flowchart for the use of SPADE on a simulated 3D flow cytometry data set. (b) 3D scatterplot of a simulated data set, which is constructed from four abundant subpopulations. (c) Density down-sampled version of b showing the same structure but with fewer cells. (d) Agglomerative hierarchical clustering on the down-sampled cells to generate cell clusters. (e) SPADE tree connecting all cell clusters. (f) SPADE tree color-coded according to the median expression intensities of markers of cells within each node, where each node is a cell cluster from d; each of the three trees represents the distribution of the different marker expression across all the cell clusters.