杰享第03期：肿瘤免疫治疗时代的多重免疫组织化学、免疫荧光技术综述

（一）目前的多重检测技术主要是检测显色染料、金属同位素、荧光基团等发出的信号来达到检测标志物的目的。各个mIHC/IF平台的工作原理图（Diagram showing mechanism of each of the mIHC/IF platform）如图1所示：

图1A.DISCOVERY ULTRA system: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. The HRP is reacted with an appropriate substrate bound to a chromogenic dye, leading to the precipitation of insoluble, coloured precipitates at the site where the antigens are found.

图1B.Metal-based IHC techniques such as IMC and MIBI:a primary antibody bound to the target antigen is tagged with a metal isotope of known molecular mass. Analysis is carried out using mass spectrometry in MIBI and laser ablation coupled to mass cytometry in IMC.

图1C.Vectra: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. A fluorophore-conjugated tyramide molecule serves as the substrate for HRP, resulting in an antigen-associated fluorescence signal.

图1D.Nanostring’s DSP: the target antigen will bind the primary antibody which is coupled to a photo cleavable oligonucleotide tag. UV light is used to cleave the oligonucleotide tags and is collected using a micro capillary tube and stored in a microplate well. The oligonucleotide tags will bind to the reporter probe via the target-specific capture probe. Reporter probes are imaged and counted by the nCounter analysis system.

（二）现有的多重检测技术可同时检测5-40+标志物，不同成像平台的比较（Overview and comparison of the different imaging modalities）如表1所示：

（三）Vectra, Chipcytometry, or DISCOVERY ULTRA平台mIHC/IF图像示例（Representative mIHC/IF images captured through the Vectra, Chipcytometry, or DISCOVERY ULTRA imaging system）：

图2. Representative mIHC/IF images captured through the Vectra, Chipcytometry, or DISCOVERY ULTRA imaging system.

1. mIHC/IF of pancreatic adenocarcinoma FFPE sections labelled with DAPI (blue), CD73 (green), CD8 (yellow), CD68 (red), FoxP3 (cyan), CD3(magenta) and CK (orange) were scanned using the Vectra imaging system.
2. Mouse pancreas FFPE sections labelled with CD45 (brown), CD274(green), CD3e (purple), CD4 (cyan), CD8a (pink), CD11b (yellow), CD31 (darkbrown), CD326/EpCAM (red), B220 (orange), F4/80 (blue), NK1.1 (purple), Pan-CK(maroon), Hoechst 33342 (dark blue) were scanned using the Chip cytometry imaging system.
3. Cholangiocarcinoma FFPE sections labelled with CD20 (blue), CD8(red), CD68 (turquoise), CD3 (yellow) were scanned using the DISCOVERY ULTRA imaging system.
   
   （四）IMC技术最大的优势在于可同时检测40+标志物，图3为人体组织切片IMC图像示例（Representative IMC images of human tissue sections）：

1. T cell panel (A; CD45RO depicted ingreen, CK in cyan, collagen in yellow, CD8 in red, CD4 in magenta, and Ki67 in white)
2. Basic lineage panel (B; CD68 in green, CD20 in cyan, PD-L1 in yellow, VISTA in red, CD3 in magenta, and CD45 in white)
3. Function panel (C; OX40 in green, CD38 in cyan, Ki67 in yellow, ecadherin in red, collagen in magenta, and granzymeB in white).

（五）mIHC/IF图像分析仍然面临很多挑战，现有的解决方案各有优劣势，其中HistoCAT、QuPath属于开源的分析软件，常用的mIHC/IF 图像分析平台（Some of the commonly adopted analytic platforms for mIHC/IF）优劣势比较如下：

（六）Ultivue’s InSituPlex可使用普通的荧光显微镜成像，不需要配置昂贵的显微镜，因此可在大部分实验室开展检测。图4为人体组织样本Ultivue’s InSituPlex图像示例（Representative Ultivue’s InSituPlex images of human tissue samples labelled with CD8 (green), CD68 (yellow), PD-L1 (red) and CK/Sox10 (cyan)）：

图4. Representative Ultivue’s InSituPlex images of human tissue samples labelled with CD8 (green), CD68 (yellow), PD-L1 (red) and CK/Sox10(cyan). Whole slide imaging of tonsil section (A), high magnification view ofHCC (B), and radioembolization-treated HCC (C, Y-90 visible as microspheres).

（七）目前已研究过多种mIHC/IF Panel，通过同时分析多个标志物来预测患者接受PD-1/PD-L1抑制剂治疗的应答反应。Meta分析中使用mIHC/IF技术的文献清单（List of papers using mIHC/IF in the meta-analysis）如下：

杰论

综上所述，mIHC/IF在肿瘤免疫治疗领域具有广阔的应用前景。与传统的IHC只能检测一个标志物不同，mIHC/IF能够在单个组织切片中检测多个标志物，同时提供有关细胞组成和空间排列的全面信息，使我们能够更深入地了解癌症的发病机制和对免疫治疗的反应。同时mIHC/IF技术处理的组织样本可以长期保存，供进一步研究使用。但是，这类技术的检测成本及实用性方面，仍然是一个值得关注的问题。

迈杰转化医学作为国内精准诊断整体解决方案的领导者，致力于解决精准医疗药物研发及患者用药痛点，围绕生物标志物研究、伴随诊断开发，建立了完善的核酸组学、蛋白组学、细胞组学技术平台。我们拥有国内领先的IHC检测平台，配备有Leica Bond Max、Ventana BenchMark、Dako Autostainer Link48三大进口自动化平台，以及用于mIHC检测的Leica Bond RX、PerkinElmerVectra3 System平台，在mIHC检测方法学开发及验证方面积累了丰富的经验，如三标四色的Panel(CD4, CD8, PD-L1 )，及五标六色的Panel(CD4, CD8, CD163, PD-L1, Pan-Keratin)等，如有mIHC检测方法开发及服务需求，请联系迈杰转化医学商务部（邮箱：MARKETING@MEDxTMC.com）。

文献链接

Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy - Tan - 2020 - Cancer Communications - Wiley Online Library（https://onlinelibrary.wiley.com/doi/10.1002/cac2.12023

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