Abstract 2459: Membrane stain-enhanced quantification of HCR™ Pro RNA-ISH using HALO image analysis software

Abstract Accurate quantification of RNA in situ hybridization (RNA-ISH) signals is critical for understanding spatial gene expression, particularly at the single-cell level. Traditional methods using Hematoxylin or DAPI staining often fall short due to their inability to clearly define cell boundaries, leading to inaccurate dot counting. In the context of digital pathology, this challenge can hinder precise interpretation of spatial gene expression data. Here, we demonstrate how the incorporation of membrane stains enhances RNA-ISH signal quantification by delineating cell borders, enabling precise dot counting with HALO image analysis software. Membrane stains were applied across both chromogenic and fluorescent HCR™ Pro RNA-ISH assays, facilitating robust signal detection in brightfield and fluorescent imaging modalities. This approach also enabled the integration of cell-type specific markers (e.g., CD3 and CD4) to provide insights into gene expression dynamics within distinct cell subpopulations relevant to pathology workflows. Comparative analysis revealed superior accuracy in dot quantification when using membrane-enhanced staining compared to traditional Hematoxylin/DAPI-expanded methods. These findings underscore the importance of combining membrane stains with advanced imaging analysis tools like HALO in digital pathology for precise and reproducible quantification of RNA-ISH signals. This advancement paves the way for more detailed studies of differential gene expression and cellular heterogeneity within clinical and research pathology settings. Citation Format: Randy Chen, Anne Hellebust, Aneesh Acharya, Harry Choi. Membrane stain-enhanced quantification of HCR™ Pro RNA-ISH using HALO image analysis software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2459.