APIII - Advancing Practice, Instruction & Innovation Through Informatics

Multispectral imaging and pathology: H & E & Beyond

http://www.cri-inc.com

Richard Levenson, MD (rlevenson@cri-inc.com) 1, Michael Feldman, MD, PhD 2, Emma Furth, MD 2, Jonathan Hogan 1, Cliff C. Hoyt 1, Kirk W. Gossage, PhD 1 .1CRI, Woburn, MA; 2Dept. of Pathology, University of Pennsylvania, Philadelphia, PA.

Context: Hematoxylin and eosin-based stains and immunohistochemical procedures together form the backbone of clinical histopathology. Novel imaging and analyitical methods can extract additional qualitative and quantitative information from these relatively venerable procedures. In particular, multispectral imaging (MSI) approaches appear promising. In immunohistochemistry, for example, they can be used to: 1) enable detection of target co-localization; 2) elucidate the spatial relationships between IHC-identified cell types; and 3) detect multiple signal transduction events. Moreover, MSI-enabled multiplexing can reduce sample depletion by allowing multiple studies to be performed on individual sections. Multiplexing is accomplished by measuring absorbance spectra at every pixel of an image—appropriate algorithms then automatically resolve absorption patterns of overlapping chromogens to generate quantitative images of individual analytes. Examples will be shown of ER and PR-expression in breast cancer, as well as of double-immunophenotyping in hematopathology. With standard H&E-stained slides, spatial and spectral characteristics of the samples can be used to generate reasonably accurate mimics of some special stains, particularly those highlighting fibrosis.

Design: Samples of breast, prostate and other tissues were double-immunostained for a variety of targets, including ER and PR, using red and brown chromogens and a hematoxylin counterstain. Conventional H&E stains of liver and other tissues were also prepared.

Technology: The Nuance™ multispectral imaging system (CRi, Woburn, MA), which can be attached to any microscope, was used to acquire spectral datasets. Automated software tools were developed to quantitate nuclear percent-positivity and degree of colocalization of dual nuclear markers, as well as the assessment of immunophenotype for multiple membrane markers.

Results: Acquisition and analysis of individual fields could be accomplished in less than 10 seconds. Spectral analysis of double-IHC-stained specimens generated simultaneous ER and PR positivity as well as co-localization statistics. When MSI was performed on standard hematoxylin and eosin-stained specimens of liver involved with a variety of fibrotic processes, multispectral and additional imaging processing tools were used to highlight the fibrotic regions, which were pseudocolored to recapitulate typical Masson trichrome coloration. The resulting images were compared to authentic trichrome staining performed on adjacent sections. The H&E "trichrome" results generated results substantially equivalent to those obtained by actual trichrome stains.

Conclusion: Multispectral imaging promises to greatly increase the utility and information content of multiple simultaneous immunostains, enabling per-cell multiparameter measurements. In the case of H&E-stained slides, it appears that MSI can also extract additional information and reduce the use of special stains and their associated costs and delays.