Brightfield multiplex immunohistochemistry with multispectral imaging

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.