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

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 imag e, 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 p rovides visual verification of cell isolation and identification as well as highlight characteristics of cells and cell clusters. © 2018 International Society for Advancement of Cytometry
Source: Cytometry Part A - Category: Molecular Biology Authors: Tags: Technical Note Source Type: research