Steele Keith E, Brown Charles
MedImmune LLC, Gaithersburg, MD, USA.
Methods Mol Biol. 2018;1845:87-98. doi: 10.1007/978-1-4939-8709-2_6.
Multiplex immunohistochemistry allows the demonstration of multiple protein antigens in individual histological sections of formalin-fixed paraffin-embedded tumors or other types of tissue. Carefully designed and optimized immunohistochemistry (IHC) assays not only maximize the information available from limited tissues, but also enable a higher level interpretation of that information by demonstrating the histo-anatomical relationships among key cell types which express the included biomarkers. Programmable automated IHC instruments support the development and application of complicated multiplex IHC protocols, help save time and effort, and enhance immunostaining quality and reproducibility. Simple data can be extracted from immunostained tissues to include qualitative (descriptive) findings and semiquantitative analysis. The value of multiplex IHC can be increased further by the utilization of image analysis software either to better visualize multiple markers or by applying suitable digital scoring solutions to capture data (automated pathology).Here, we describe a five-marker multiplex based on application of two individual assays to serial sections of non-small cell lung carcinoma (NSCLC). We use this assay to label PD1, PD-L1, CD3, CD68, and cytokeratins in relation to tertiary lymphoid structures (TLS) and other regions of the tumor microenvironment. We illustrate how visualization of the immunostaining results can be used to understand TLS organization and other aspects of the tumor microenvironment, and briefly consider means to further yield additional information.
多重免疫组化能够在福尔马林固定石蜡包埋肿瘤或其他类型组织的单个组织切片中显示多种蛋白质抗原。精心设计和优化的免疫组化（IHC）检测不仅能最大限度地利用有限组织中获取的信息，还能通过展示表达所包含生物标志物的关键细胞类型之间的组织解剖关系，对这些信息进行更高层次的解读。可编程自动化免疫组化仪器支持复杂多重免疫组化方案的开发和应用，有助于节省时间和精力，并提高免疫染色质量和可重复性。可以从免疫染色组织中提取简单数据，包括定性（描述性）结果和半定量分析。通过利用图像分析软件更好地可视化多种标志物，或应用合适的数字评分解决方案来捕获数据（自动化病理学），多重免疫组化的价值可以进一步提高。在此，我们描述一种基于将两种单独检测应用于非小细胞肺癌（NSCLC）连续切片的五标志物多重检测方法。我们使用该检测方法标记与三级淋巴结构（TLS）及肿瘤微环境其他区域相关的PD1、PD-L1、CD3、CD68和细胞角蛋白。我们阐述了如何利用免疫染色结果的可视化来理解TLS组织及肿瘤微环境的其他方面，并简要考虑进一步获取更多信息的方法。
