Abstract 1600: Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes
Authors: Roberts Esteban , Day William , Kelly Brian D. , Polaske Nathan W. , Ashworth-Sharpe Julia , Johnson Donald , Lefever Mark , Kosmeder Jerry , Zhang Hongjun , Zhang Jian , Tsao Tsu-Shuen , Farrell Mike , Bredno Joerg , Ochs Robert , Morrison Larry 
Published: August 2018

Abstract
Introduction
Immunohistochemistry (IHC) is a valuable means to examine the distribution of protein expression at the subcellular level throughout human tissues while retaining morphology of the cells and tissue. Given the large and growing number of established protein biomarkers and the often very limited amount of tissue provided by tumor biopsy, the ability to evaluate multiple biomarkers on a single-slide mounted specimen via multiplexed IHC is of growing importance. However, due to the limited number of conventional chromogens, their broad absorbance spectra, and deposition chemistry based on only two enzymes, brightfield multiplexing has been typically limited to only two protein targets.
Methods
To expand multiplexing capability, our project objective was to reduce the antibody incubation, detection, and enzyme deactivation steps to produce high-level multiplexed chromogenic assays with short run times and minimal exposure to potentially destructive enzyme deactivation reagents. We utilized unmodified and tagged primary antibodies with secondary anti-primary and anti-tag antibodies conjugated to several enzymes employing orthogonal enzymatic detection systems. Peroxidase-catalyzed tyramide signal amplification (TSA) and quinone methide signal amplification (QMSA) chemistry adapted to two different hydrolase enzymes provided several compatible chromogens with relatively narrow absorbance bands and good spectral separation. All assays were fully automated and performed on the Benchmark XT platform.
Results
Multiplex IHC staining was demonstrated using combinations of TSA and QMSA, with the former catalyzed by horseradish peroxidase (HRP) and the latter established to work successfully with two different hydrolase enzymes: alkaline phosphatase (AP) and neuraminidase. Three- and four-chromogen multiplex assays were demonstrated on a variety of targets including breast cancer (Her2, ER, PR, Ki-67) and immune cell targets (CD3, CD8, cytokeratin, FOXP3, PD-L1) on a variety of tumor and tonsil tissues. Use of both tagged and untagged primary antibodies and up to three enzymes allow reduction of antibody removal and enzyme deactivation steps, significantly reducing assay time. Quantitative comparisons of multiplex staining to ‘gold standard' DAB staining on serial sections verified the multiplex assay performance.
Conclusion
We have expanded on the current HRP and AP brightfield IHC detection technologies by utilizing TSA- and QMSA-based chromogens and introducing the new neuraminidase detection chemistry. These detection chemistries combined with tagged and untagged primary antibodies provided up to four-chromogen brightfield multiplex IHC with verified assay performance.
Citation Format: Esteban Roberts, William Day, Brian D. Kelly, Nathan W. Polaske, Julia Ashworth-Sharpe, Donald Johnson, Mark Lefever, Jerry Kosmeder, Hongjun Zhang, Jian Zhang, Tsu-Shuen Tsao, Mike Farrell, Joerg Bredno, Robert Ochs, Larry Morrison. Expanded multiplexing capability in brightfield immunohistochemistry utilizing multiple chromogen chemistries and detection enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1600.