Velociraptor Machine Learning Quantifies Similarity to Known Cell Types and Matches Cells Across Flow and Imaging Cytometry Platforms

ABSTRACT Suspension flow cytometry enables high‐throughput cellular profiling at the single cell level, but these data lack positional information. Conversely, tissue‐based imaging cytometry techniques reveal a cell's location within the tissue architecture and can provide insight into cell biology. It would be especially valuable if data analysis tools could incorporate data from imaging and flow cytometry platforms to gain complementary strengths when quantifying features of cells and populations. We hypothesized that per‐cell Marker Enrichment Modeling (MEM) might provide a way to register cells between flow and imaging cytometry analysis. Here, we developed the Velociraptor machine learning workflow for cross‐platform cytometry analysis. Velociraptor begins with a graph‐based implementation of MEM to calculate per‐cell quantitative phenotype labels. With this information, Velociraptor can then quickly calculate similarity between each cell's phenotype and search terms describing established cell types, cells of interest, or cells observed in other samples. Velociraptor was effective in registering cells within and between cytometry platforms. Integrated identification of cell populations was tested in several challenges, including comparisons of high dimensional datasets from cancer and immunology. Tested instrument types included imaging mass cytometry (IMC), cyclic immunohistochemistry (cycIHC), suspension mass cytometry (CyTOF), and suspension spectral flow cytometry (SFC). Between IMC and CyTOF, a comparison across imaging and flow cytometry platforms that use the same mass tag probes, Velociraptor accurately identified and registered immune cell types (median F1‐measure of 0.81). Between SFC and CyTOF, a comparison of two fundamentally different probe types—fluorophores and metal tags—in suspension flow cytometry, Velociraptor was even more accurate at identifying and registering cells (concordance correlation coefficient of 0.99). Velociraptor was especially useful in heterogeneous samples where individual cells diverged in phenotype from the bulk population. In IMC imaging of human breast cancer, a previously unappreciated tumor cell subset was revealed by Velociraptor, characterized as CD15 + , and validated as spatially segregated to the tumor core. In both cycIHC (8‐dimensional imaging) and IMC imaging (40‐dimensional imaging), Velociraptor accurately identified macrophages using a single search label as input. Notably, Velociraptor worked effectively with both extremely rare and highly abundant cell types and with cell search labels calculated from data and theoretical labels based on literature and expertise. The Velociraptor algorithm is freely available at https://github.com/cytolab .