Abstract 4569: Intra- and inter-run assessment of reproducibility and quantification of UltiMapper™ I/O APC and T-act kits for tissue multiplexing
Authors: Phillips Bonnie , Patel Katir , Hebert Courtney , Sharma Aditi , Buell Jamie , Downing Sean 
Published: August 2019

Abstract
Background: The field of immuno-oncology research has enthusiastically adopted multiplex immunohistochemistry (IHC) techniques to establish the spatial relationships between various immune cell populations in a tumor biology in context. Multiplexing enables researchers to gain a deeper understanding and insight into the tumor microenvironment. Unfortunately, many multiplexing technologies face several challenges, specifically in generating highly robust, reproducible, and easily quantifiable data sets. Ultivue’s UltiMapper I/O APC and T-act kits utilize InSituPlex® technology, a new method of multiplexed IHC that utilizes a streamlined workflow with single antigen retrieval, staining, amplification, and detection steps, allowing for the completion of the assay in 5 hours. Here we assess these kits for intra- and inter-run reproducibility and quantification.
Methods: Intra-run reproducibility and quantification was accomplished by manually staining 5 serial sections from three tissue types (deidentified samples of tonsil, melanoma, NSCLC) with one set for each of the UltiMapper I/O APC (CD11c, CD20, CD68/CD163, and MHC Class II) and T-act (CD3, Granzyme B, Ki67, and pan-Cytokeratin/SOX10) kits. Inter-run assessment was determined by staining each slide from a set of five serial sections of each tissue type independently. Images were acquired using the ZEISS® Axio Scan.Z1, without the need for linear unmixing allowing for direct whole slide imaging. Analysis was accomplished using Indica Labs HALO® software. Coefficients of variation (CVs) were calculated based on resulting data.
Results: Analysis of intra-run serial section images revealed that cell counts from section to section had CVs of &lt;10% across all markers, in all tissues, for both the UltiMapper I/O APC and T-act kits. This included total cell counts and average signal intensity. Similar results were seen for inter-run comparisons for both kits (&lt;10% CV).
Conclusions: The results presented here indicate that InSituPlex technology is potentially much more reproducible than other tissue multiplexing techniques currently available. Histological standards for coefficients of variation in IHC based assays are typically &lt;15%. Data presented here falls well within that standard indicating the potential for future translational applications. In conclusion, InSituPlex is a highly reproducible and quantifiable multiplexing technology that is able to produce these results across a variety of tissue types and markers.
Note: This abstract was not presented at the meeting.
Citation Format: Bonnie Phillips, Katir Patel, Courtney Hebert, Aditi Sharma, Jamie Buell, Sean Downing. Intra- and inter-run assessment of reproducibility and quantification of UltiMapper™ I/O APC and T-act kits for tissue multiplexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4569.