Abstract 3767: A novel method to minimize HIER-induced alterations on H&E staining in an integrated mIF-H&E workflow
Authors: Hwang Kevin , Vezeau Grace , Olejnik Edyta , Wood Douglas , Cardenes Ruben , Duro Lauren , Chatterjee Gourab , Lee Je H. 
Published: March 2024

Abstract
Background: Hematoxylin and eosin staining (H&E) is widely used as an anatomical assay for clinical diagnosis. Researchers and clinicians also rely on molecular in situ techniques, such as multiplexed immunofluorescence (mIF), to gain deeper insights on cellular phenotypes and tissue microenvironment. As a result, there has been significant interest in combining anatomical stains with molecular imaging techniques, most commonly by using a terminal H&E stain after mIF staining. This allows a combination of the two assays but has been observed to show alterations in the H&E staining pattern. In this work we identify heat-induced epitope retrieval (HIER) as the root cause of alterations of the terminal H&E stain after mIF. We further demonstrate a new workflow combining an initial H&E stain with subsequent InSituPlex assay, to avoid these alterations and thus enable co-registration of highly sensitive multiplexed immunofluorescence with an unaltered H&E stain.
Methods: FFPE tissue slides were stained using a standard HIER protocol or a full mIF assay, followed by a standard H&E stain. Slides were imaged using a Zeiss Axioscan.Z1 scanner. Additional H&E-stained slides were prepared, and the H&E stain removed using a destaining protocol before carrying out InSituPlex® mIF staining for multiple markers. Fluorescence and brightfield images of the same slide were overlaid using UltiStacker® to assess qualitative differences between pre- and post-mIF H&E, and UltiAnalyzer.AI® was used to quantify cell densities and signal intensities between mIF image pre- and post-H&E.
Results: Alterations in H&E staining were observed between slides directly stained with H&E and slides stained with a terminal H&E after HIER alone or a full mIF assay. Calculated cell counts and tissue area were consistent throughout, but some tissue microstructures could be difficult to resolve after either HIER or full mIF. Our destaining protocol was successful in removing hematoxylin and eosin from directly-stained H&E slides and allowing subsequent mIF staining. For most biomarkers, qualitative and quantitative differences in InSituPlex mIF staining were minimal. Brightfield and fluorescent images were co-registered with sub-micron accuracy using UltiStacker, enabling molecularly defined cellular phenotyping within a traditional H&E image.
Conclusions: While the mIF to H&E workflow is widely used and is capable of allowing combined anatomical and cellular phenotyping, alterations in the H&E stain exist due to the use of HIER in most mIF protocols. An alternative method is possible in which H&E-stained slides are destained and then restained for mIF or other spatial assays, providing the same valuable combination of assay information but without the HIER-induced alterations in H&E staining pattern.
Citation Format: Kevin Hwang, Grace Vezeau, Edyta Olejnik, Douglas Wood, Ruben Cardenes, Lauren Duro, Gourab Chatterjee, Je H. Lee. A novel method to minimize HIER-induced alterations on H&E staining in an integrated mIF-H&E workflow [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 3767.