Saldarriaga Omar A, Freiberg Benjamin, Krishnan Santhoshi, Rao Arvind, Burks Jared, Booth Adam L, Dye Bradley, Utay Netanya, Ferguson Monique, Akil Abdellah, Yi Minkyung, Beretta Laura, Stevenson Heather L
Department of Pathology University of Texas Medical Branch Galveston TX.
Visiopharm Hoersholm Denmark.
Hepatol Commun. 2020 Mar 3;4(5):708-723. doi: 10.1002/hep4.1494. eCollection 2020 May.
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 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+. Spectral imaging microscopy is a powerful tool that enables analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.
肝内巨噬细胞会影响微环境的组成、宿主对肝损伤的免疫反应以及纤维化的发展。与星状细胞相比，巨噬细胞在纤维化发展中的作用仍不明确。多光谱成像能够检测人福尔马林固定、石蜡包埋组织中的多种标志物。这项前沿技术非常适合分析人类肝脏组织，因为它可以对荧光团信号进行光谱解混、扣除自发荧光并保留肝脏结构。我们分析了在巨噬细胞群体中常见的五种不同抗体（CD68、MAC387、CD163、CD14和CD16）。在优化单重染色并建立光谱库后，我们将所有抗体组合成多重方案，并用于对从患有慢性肝病的代表性患者（包括慢性丙型肝炎、非酒精性脂肪性肝炎和自身免疫性肝炎）采集的活检组织进行染色。测试了各种成像方式，包括细胞表型分析、组织分割、t分布随机邻域嵌入图以及有助于比较和可视化已识别的巨噬细胞和其他细胞图谱的表型矩阵。然后，我们使用批量分析算法测试了该平台分析每组多名患者肝脏活检多个感兴趣区域的可行性。与对照组相比，丙型肝炎病毒阳性且伴有晚期纤维化的患者中有五个群体存在显著差异。与对照组相比，其中三个群体在晚期纤维化患者中显著增加，包括CD163 + CD16 +、CD68 +和CD68 + MAC387 +。光谱成像显微镜是一种强大的工具，能够分析人类肝脏活检中的巨噬细胞和其他细胞，未来可能会带来更个性化的治疗方法。
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