Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC). However, as our understanding of the complex tumor microenvironment grows, there is increasing demand for multiplexed biomarker detection. Currently, multiplexed IHC assays are almost exclusively based on immunofluorescence because brightfield techniques are limited by the broad spectral absorption of chromogens and a reliance on conventional 3-channel color cameras. In this work, we overcome these limitations by combining new chromogens possessing narrow absorbance bands with matched illumination channels and monochrome imaging. Multiplex IHC was performed using four or five covalently deposited chromogens and hematoxylin nuclear stain to preserve morphological context and detail. Brightfield illumination was provided with a tungsten lamp/filter wheel combination or filtered light emitting diodes to provide up to 12 illumination wavelengths. In addition, an automated rapid imaging system was developed, using a synchronized 12-LED illuminator, that could capture images at all wavelengths in under 1 s. In one example, a four-biomarker multiplex assay was designed and used to distinguish regions of adenocarcinoma and squamous cell carcinoma in non-small cell lung cancer. The technology was also validated with a five-biomarker assay in prostate cancer. Spectrally unmixed images of each biomarker demonstrated concordant expression patterns with DAB single stain on serial sections, indicating faithful identification of each biomarker. In each assay, all chromogens were well resolved by spectral unmixing to remove spectral crosstalk. While further characterization and refinement of the assay, and improvements in automation and user interface are necessary for pathologist acceptance, this approach to multiplex IHC and multispectral imaging has the potential to accelerate adoption of multiplexing by combining the medical value of high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy.
Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.
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.
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