Abstract
Background/Objectives: Performing hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) on the same specimen slide provides advantages that include specimen conservation and the ability to combine the H&E context with biomarker expression at the individual cell level. We previously used invisible deposited chromogens and dual-camera imaging, including monochrome and color cameras, to implement simultaneous H&E and IHC. Using this approach, conventional H&E staining could be simultaneously viewed in color on a computer monitor alongside a monochrome video of the invisible IHC staining, while manually scanning the specimen. Methods: We have now simplified the microscope system to a single camera and increased the IHC multiplexing to four biomarkers using translational assays. The color camera used in this approach also enabled multispectral imaging, similar to monochrome cameras. Results: Application is made to several clinically relevant specimens, including breast cancer (HER2, ER, and PR), prostate cancer (PSMA, P504S, basal cell, and CD8), Hodgkin's lymphoma (CD15 and CD30), and melanoma (LAG3). Additionally, invisible chromogenic IHC was combined with conventional DAB IHC to present a multiplex IHC assay with unobscured DAB staining, suitable for visual interrogation. Conclusions: Simultaneous staining and detection, as described here, provides the pathologist a means to evaluate complex multiplexed assays, while seated at the microscope, with the added multispectral imaging capability to support digital pathology and artificial intelligence workflows of the future.