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Published on: March 24, 2015
Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease
Omar A Saldarriaga1, Benjamin Freiberg2, Santhoshi Krishnan3,4
1Department of Pathology University of Texas Medical Branch Galveston TX.
Abstract:
Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t-distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.
Insights
Spectral imaging microscopy effectively analyzes intrahepatic macrophages in liver biopsies. This technique identified specific macrophage populations associated with advanced fibrosis in chronic liver diseases, paving the way for personalized therapies.
Area of Science:
- Immunology
- Hepatology
- Biomedical Imaging
Background:
- Intrahepatic macrophages play a role in liver injury and fibrosis, but their specific contribution is not fully understood.
- Stellate cells are known contributors to fibrosis, highlighting a knowledge gap in macrophage involvement.
- Multispectral imaging offers advanced capabilities for in situ analysis of human tissues.
Purpose of the Study:
- To investigate the role of intrahepatic macrophages in the development of liver fibrosis.
- To establish and validate a multiplex spectral imaging protocol for analyzing macrophage populations in human liver biopsies.
- To identify specific macrophage phenotypes associated with advanced fibrosis in chronic liver diseases.
Main Methods:
- Developed and optimized a multiplex spectral imaging protocol using five key macrophage markers (CD68, MAC387, CD163, CD14, CD16).
- Applied the protocol to human liver biopsies from patients with chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis.
- Utilized advanced imaging modalities including cell phenotyping, tissue segmentation, and t-distributed stochastic neighbor embedding for data analysis.
Main Results:
- Identified significant differences in macrophage populations between patients with advanced fibrosis and controls.
- Found increased prevalence of CD163+CD16+, CD68+, and CD68+MAC387+ macrophage populations in patients with advanced fibrosis.
- Demonstrated the feasibility of batch analysis for high-throughput screening of liver biopsy samples.
Conclusions:
- Spectral imaging microscopy is a powerful tool for in situ analysis of cellular profiles in human liver biopsies.
- Specific macrophage populations are significantly associated with advanced liver fibrosis.
- This technology holds potential for developing personalized therapeutic strategies for liver diseases.
