Immune Cell and Cell Cluster Phenotyping, Quantitation, and Visualization Using In Silico Multiplexed Images and Tissue Cytometry.

Phenotyping immune cells and cell clusters in situ, including their activation state and function, can aid in interpretation of spatial relationships within the tissue microenvironment. Immune cell phenotypes require multiple biomarkers. However, conventional microscopy setups can only image up to four biomarkers at one time. In this report, we describe and give an example of a workflow to phenotype, quantitate, and visualize greater than four biomarkers in silico utilizing multiplexed fluorescence histology and the TissueFAXS quantitative imaging system with a conventional microscopy setup. Biomarkers were conjugated to Cy3 or Cy5. Multiplexed staining was performed on formalin-fixed paraffin-embedded tissue sections. We imaged the slides, inactivated the dyes, and repeated the process until all biomarkers were stained. Phenotype profiles were built based on in silico combinations of the biomarkers. We used algorithms that aligned all images to create a composite image, isolated each cell in the image, and identified biomarker positive cells in the image. The in silico phenotypes were quantitated and displayed through flow cytometry-like histograms and dot scatterplots in addition to backgating into the tissue images. The advantage of our workflow is that it provides visual verification of cell isolation and identification as well as highlight characteristics of cells and cell clusters.