Abstract 4250: Multispectral imaging of brightfield multiplex immunohistochemistry
Authors: Bauer Daniel R. , Lefever Mark , Leibold Torsten , Behman Lauren , Roberts Esteban , Ashworth-Sharpe Julia , Morrison Larry 
Published: September 2020

Abstract
Introduction. Brightfield microscopy is the current gold standard for tissue-based cancer diagnoses and has long been the choice of pathologists. Accordingly, the clinical use of brightfield-based immunohistochemistry (IHC) far exceeds immunofluorescence (IF), except in multiplexing applications for which IF is predominately used. Brightfield multiplex imaging is currently limited by 1) the broad spectral absorbance of conventional chromogens and 2) color cameras that only have three broad and overlapping color channels. In this work, we combine narrowband covalently deposited chromogens (CDCs; Day et al., Lab Invest 2017 vol.97 p.104) with matched narrow illumination channels and monochrome imaging to demonstrate that high-order multiplexing is possible in brightfield.
Material and Methods. Multiplex IHC was performed using 4 or 5 CDCs and hematoxylin (htx) nuclear staining on formalin-fixed paraffin-embedded (FFPE) tissue sections. Spectral widths of 5 CDC absorbance bands ranged from 69 to 85 nm (FWHM), and 150 nm for a 6th broader CDC. Illumination was provided with a tungsten lamp/filter wheel combination (average bandwidth = 28nm) or filtered light emitting diodes (LEDs; average bandwidth = 23nm). Images were recorded on microscopes fitted with either illumination source and monochrome CCD or CMOS cameras. Furthermore, a custom-designed 12 wavelength LED illuminator facilitated high-speed multispectral imaging with short exposure times (average Δt=10ms). On-slide spectra of all chromogens were measured. Spectral unmixing was used to calculate protein abundances, which were pseudo-colored to render a visualization.
Results. Multiple assays were tested on FFPE sections of lung, prostate, and colon tumors with up to 5 IHC markers (5 CDCs + htx). Spectral cross-talk between chromogen signals was significantly reduced with spectral unmixing (SNR≈30). Biomarker concentrations were pseudo-colored to produce customizable visualizations of each assay. Brightfield renditions of each assay were nearly identical to a traditional visualization. Multiplex staining results were concordant with single stain DAB IHC of serial sections. Automated control and image acquisition using the 12 LED illuminator permitted acquisition of all 12 wavelengths in 700 ms per 1 mm2 of tissue.
Conclusion. Feasibility of brightfield IHC multiplexing was achieved by matching narrowband CDCs with specific light channels to enable detection of up to six analytes (5 CDCs plus nuclear stain), approaching the capabilities of IF. Our 12-color system is expandable to even higher-order multiplexing and boasts imaging speed roughly comparable to that of a clinical brightfield scanner. This system presents an attractive alternative to IF by combining high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy.
Citation Format: Daniel R. Bauer, Mark Lefever, Torsten Leibold, Lauren Behman, Esteban Roberts, Julia Ashworth-Sharpe, Larry Morrison. Multispectral imaging of brightfield multiplex immunohistochemistry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4250.