Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease

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, paraffinembedded 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. (Hepatology Communications 2020;4:708-723).

Comparison of liver biopsies before and after direct-acting antiviral therapy for hepatitis C and correlation with clinical outcome

“…c Fibrosis staging was first evaluated using light microscopy and the Ishak criteria (scale of 0 to 6). Then, CPA was calculated for each liver biopsy using a previously published method 19 , 25 , 26 . The percentage of hepatocytes involved by macrovesicular steatosis was first estimated by a pathologist (H.L.S.)…”

Patients with advanced hepatic fibrosis before or after direct-acting antiviral therapy for chronic hepatitis C are at risk for development of liver cancer

“…1 and 4). The study patients did not have signi cant differences in the amount of brosis pre-and post-treatment regardless of whether this was determined by a pathologist using the Ishak brosis staging system [25] or by the calculated CPA [22,26] (Table 2). This may have been due to the short duration between DAA treatment completion and collection of their follow-up liver biopsies, which ranged between 6.0 and 28.4 months (Table 2).…”

“…Liver biopsies are still important for the diagnosis of several liver diseases including autoimmune hepatitis, primary biliary cholangitis, and in ltrative processes, such as amyloidosis. Liver biopsies also provide essential material for research studies of the hepatic microenvironment and responses to immunotherapy [26,44] . They are also useful when non-invasive assessment and serologic study results are discrepant in staging hepatic brosis [21] .…”

“…Each patient's pre-and post-DAA treatment liver biopsies (n = 10) were evaluated by a board-certi ed liver pathologist (H.S.L) who was blinded to the patient's serology, clinical history, and prior pathology reports. Biopsies were evaluated using established numerical scoring systems for histologic features of hepatitis in ammatory activity [24,25] , staging of brosis [25] and grading of steatosis [26] . The modi ed hepatitis index (MHAI) evaluates several parameters of hepatic in ammatory activity, including periportal/periseptal interface hepatitis, con uent necrosis, focal lytic necrosis, apoptosis/in ammatory activity, and portal in ammation [25] .…”

Highly multiplexed 2-dimensional imaging mass cytometry analysis of HBV-infected liver

“…For innate immune cells, CD45 + events were further gated for CD68 + , CD16 + , CD14 + , CD11b + and CD57 + cells. Notably, expression patterns for CD68, CD16 and CD14 were highly co-localized with similar characteristics in quantitative and phenotype analyses (50). Therefore, we simplified our language and data presentation by using CD68 expression to define hepatic Kupffer cells, with a separate description of CD11b + cells which were also enriched for CD68 and HLA DR. We further defined CD45 + CD3 -CD57 + cells as a subset of mature NK cells that can accumulate with aging and may be altered in various viral infections including chronic hepatitis B (40,51).…”