Multiplex Immunofluorescence Profiling of Tumor Biopsies Using UltraPlex mxIF Technology

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An unmet need in tissue diagnostics is the ability to simultaneously detect three or more markers on a single tissue specimen, i.e. multiplexing. Unlike other immunoassays, i.e. flow cytometry, ELISA and bead assays, which solely quantify the biomarker in a sample, a multiplex immunoassay quantifies the biomarker, identifies the location of the cell in the tissue and identifies the spatial relationship of the biomarkers with respect to each other. Being able to identify the contextual relationships of immune cell phenotypes and other biomarkers in a tumor is believed to be critical to understanding the state of the immune system before and after immunotherapy. Multiplex immunostaining can also be described as image cytometry or flow cytometry in situ. Detecting multiple biomarkers on a single slide presents many challenges: correct selection of biomarkers, sourcing and qualifying antibodies against the biomarkers, detection methods, optimization of the staining-detection protocol and how to visualize, record and interpret the resulting complex staining pattern, all without generating artefact from cross-talk at each step. Today we shall demonstrate a complete solution for standardization of immunofluorescent staining and detection, digitization and analysis for tumor biopsy material. We have demonstrated that this protocol can be carried out on the Leica Biosystems BOND RX auto-stainer and the stained tissue can be imaged on the Aperio system that allows the user to easily digitize the whole slide at high-resolution, manage the resulting huge image files and provide accurate quantitative interpretation in the form of mark-up images and numerical data at the per cell level providing detailed phenotype information.