Abstract 2040: Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis
Authors: Morrison Larry E. , Lefever Mark R. , Behman Lauren J. , Kapadia Monesh J. , Bauer Daniel R. 
Published: June 2022

Abstract
Anatomical pathology relies upon visual evaluation of clinical specimens using brightfield microscopy. This necessarily utilizes histological stains and immunohistochemical (IHC) chromogens that absorb visible light, between 430 and 690 nm. To complement the visible stains, we developed chromogens for use in IHC that absorb light outside the visible range, and imaging methods for detecting dyes absorbing from the ultraviolet (UV) through the near infrared (NIR). Invisible chromogens partnered with conventional visible stains on the same slide provides complementary information, enabling pathologists to gain deeper insights from a patient’s tissue.
Methods: Invisible IHC was performed using conventional antibodies and antibody-enzyme conjugates utilizing covalently deposited chromogens absorbing in the UV and NIR. This was combined with IHC using visible chromogens or histological stains. Resulting stained specimens were evaluated interactively in real-time using a dual-camera microscope system comprising color and monochrome cameras, or by recording single microscope fields using multispectral imaging and the monochrome camera.
Results: Multiplex IHC was performed with different combinations of visible and invisible chromogens, with the highest level of multiplexing - 7 biomarkers + hematoxylin counterstain (5 visible IHC chromogens plus a UV and an NIR chromogen) - demonstrated on a formalin-fixed paraffin-embedded (FFPE) prostate tumor. Images of each stain were recorded using multispectral imaging, and staining patterns were concordant with single stain DAB analysis on serial sections. In addition to increased IHC multiplexing capacity, multiplexing of IHC with histological stain was demonstrated by combining an NIR absorbing chromogen, identifying MART-1 expression, with H&E staining on FFPE melanoma specimens. The dual-camera microscope presented video of the visible H&E staining (color) next to the NIR IHC staining (monochrome) on the computer monitor as the specimen was manually scanned. Archiving was afforded by multispectral imaging which also enabled quantitative analysis. An additional advantage of the NIR chromogen was the suppression of melanin pigment, the absorbance of which was significantly reduced in the NIR. Other conventional stains multiplexed with invisible IHC on colon, lung, pancreas, and tonsil FFPE tissues also showed good separation of conventional and IHC staining.
Conclusion: Taking advantage of invisible chromogens, IHC multiplexing capacity can be increased and conventional histological stains can be combined with IHC on a single slide. Clinical benefits include saving precious specimen, enabling differentiation of cell populations by simultaneous expression of multiple biomarkers, and providing biomarker expression within the cellular and morphological context of conventional stains.
Citation Format: Larry E. Morrison, Mark R. Lefever, Lauren J. Behman, Monesh J. Kapadia, Daniel R. Bauer. Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2040.