Abstract 3832: Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining

Introduction: Recent insights into the tumor microenvironment have fueled the need for additional information beyond the one or two phenotypes provided by traditional immunohistochemistry (IHC). As a response, manual and automated fluorescent multiplex immunohistochemistry (fIHC) techniques have been recently developed and accepted by the immuno-oncology space. Fluorescent multiplex immunohistochemistry (fIHC) assays are designed to simultaneously measure multiple biomarkers in tissue sections with visual context that is lost in other methods, such as flow cytometry. Here we describe a novel optimization strategy to achieve quantitative, robust, and specific multiplex fIHC staining results with both manual and automated multiple-color Opal procedures. Methods: Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents both manually and on a fully automated Leica BOND RX™ stainer. The impact of different reagent concentrations and quantities were analyzed in respect to signal specificity and robustness, stripping efficiency, signal co-localization, signal to noise, and color separation. Images were acquired on a Vectra 3.0® automated imaging system, and analyzed with inForm® software. Results: The goals of this study were two-fold: -to understand the impact of Opal reagent concentrations/quantities on fluorescent signal intensities acquired from cells within the context of tissue. -to develop an Opal reagent optimization method that yields more consistent, quantitative results from separated and co-localized fluorescent signals. We’ve applied this novel fIHC experimental approach and optimization strategy to Opal monoplex and multiplex assays and explored staining robustness, contextual specificity, staining order independence, and co-localized signal separation. Using the novel optimization strategy, we have achieved optimal staining patterns with improved confidence in the quantitative characteristics of the assay. Tissue sections stained after optimization have exhibited staining order independence and closely align with traditional IHC patterns. Conclusion: This novel optimization strategy, developed for Opal fIHC assays, is more quantitative, improves staining results, and minimizes interference between co-localized biomarkers. Citation Format: Yi zheng, Carla Coltharp, Ryan Dilworth, Linying Liu, Darryn Unfricht, Cliff Hoyt, Milind Rajopadhye, Peter Miller. Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining [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 3832. doi:10.1158/1538-7445.AM2017-3832