Immunophenotyping is an approach to measuring the abundance and functional state of immune cells as a means to understand mechanisms of homeostasis, identify biomarkers of disease, and measure therapeutic and adverse responses to drugs such as immune checkpoint inhibitors. Such studies are conventionally performed using flow cytometry together with combinations of immunomarkers that allow for identification of cell type, maturation state, and activation status. Here we describe a method for profiling circulating human leukocytes with highly-multiplexed immunofluorescence microscopy. The method has distinct advantages over flow cytometry in that it has greater sensitivity for detecting rare cell populations, allows for repeat analysis and long-term storage of precious biological samples, and obviates the requirement for spectral deconvolution. Using an open-source multiplexed immunofluorescence protocol referred to as cyclic immunofluorescence (CycIF), and commercially available reagents and instruments (AccuCyte and CyteFinder; RareCyte Inc.), we show that imaging can provide 16-plex, single-cell intensity data in addition to information on cellular morphology. Analytic technologies borrowed from the field of mass cytometry, such as ViSNE and Phenograph, allow for downstream phenotypic analysis and high-dimensional biomarker discovery. Current efforts now aim to expand the number of validated immune targets and combine imaging with single-cell picking and sequencing technologies.

Citation Format: Jia-Ren Lin, Lance U'Ren, Gregory J. Baker, Joshua J. Nordberg, Zoltan Maliga, Jackie L. Stilwell, Eric P. Kaldjian, Peter K. Sorger. Slide-based multi-parametric immunophenotyping of human blood samples by cyclic immunofluorescence using the Accucyte-CyteFinder system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2017-1705