Conventional histological and cytological staining with simultaneous immunohistochemistry enabled by invisible chromogens

Conventional histological stains, such as hematoxylin plus eosin (H&E), and immunohistochemistry (IHC) are mainstays of histology that provide complementary diagnostic information. H&E and IHC currently require separate slides, because the stains would otherwise obscure one another. This consumes small specimen, limiting the total amount of testing. Additionally, performing H&E and IHC on different slides does not permit comparison of staining at the single cell level, since the same cells are not present on each slide, and alignment of tissue features can be problematic due to changes in tissue landscape with sectioning. We have solved these problems by performing conventional staining and IHC on the same slide using invisible IHC chromogens, such that the chromogens are not visible when viewing the conventional stain and the conventional stain is excluded from images of the IHC. Covalently deposited chromogens provided a convenient route to invisible chromogen design and are stable to reagents used in conventional staining. A dual-camera brightfield microscope system was developed that permits simultaneous viewing of both visible conventional stains and invisible IHC chromogens. Simultaneous staining was demonstrated on several formalin-fixed paraffin-embedded tissue specimens using single and duplex IHC, with chromogens that absorb ultraviolet and near infrared light, followed by H&E staining. The concept was extended to other conventional stains, including mucicarmine special stain and Papanicoulou stain, and further extended to cytology specimens. In addition to interactive video review, images were recorded using multispectral imaging and image processing to provide flexible production of color composite images and enable quantitative analysis.