Abstract 5502: Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types
Authors: Larkin Selena , George Tad , Kaldjian Eric 
Published: March 2024

Abstract
Background: Tissue consists of heterogenous cell types, each with diverse functions and functional states, where spatial organization can impact patient health status. Resolving this complexity at the subcellular level has historically been challenged by image resolution, the number of targets that can be simultaneously assessed, and throughput. Such barriers can be broken using high plex and whole slide biomarker imaging data, in one single cycle thus also mitigating issues that arise with multiple cyclic rounds.
Methods: A range of whole slide healthy and diseased tissue types were stained with 15 to 18-plex validated biomarker panels. The tissues’ inherent autofluorescence was isolated into discrete fluorescence channels for enhanced information about each tissue’s morphology. Whole slide spatial staining and imaging was conducted on the Orion™ spatial biology platform, and H&E staining was performed after immunofluorescence (IF) imaging on the same section and imaged by brightfield microscopy. The full protocol is fairly quick and simple, using standard histology tools: - Mount sections on glass slides- De-paraffinize and perform antigen retrieval- Quench autofluorescence- Stain slides with a panel of ArgoFluor™ conjugated antibodies- Coverslip with ArgoFluor Mounting Medium and cure overnight- Image whole slides at 20X magnification using the Orion instrument- Process to ome.TIFF and analyze- De-coverslip in aqueous solution- Perform H&E staining and scanning on same section.
Results: Imaging data within each tissue type presented here reveals important biological insights which can then be used for quantitative analysis. The little to no tissue degradation due to the one round staining and imaging process also enables H&E capabilities after IF, further enhancing biomarker quantitation. The combination of speed and resolution proved effective for comprehensive phenotypic profiling and characterization of tissue architecture, tumor heterogeneity, and the immune response.
Conclusions: There is a need to show tissue biology in a high quality, high resolution, subcellular fashion as it provides a more in-depth understanding of each biological sample and thus can lead to improved spatial research of various human diseases. The single-stain and scan method described here improves the acquisition of subcellular spatial context and greatly reduces/removes errors in multiplex image data compared to cyclic methods. A single stain and image cycle across a 12-18 plex sample provides accurate downstream biomarker quantitation, offering better prediction of patient outcomes.
Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5502.