Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging.
Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging.
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DOI：
10.1038/labinvest.2015.2
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Angelo M
中科院分区：
文献类型：
--
作者：
Levenson RM;Borowsky AD;Angelo M
免疫组织化学（IHC）在癌症管理中的作用已经扩大到提供改进的诊断分类，以及对疾病预后，治疗和复发的指导。这些新的任务需要评估越来越多的蛋白质靶点;然而，常规的多路复用，通常使用连续的组织切片染色的单一分析物每个载玻片，可以耗尽小活检标本，复杂的载玻片到载玻片的蛋白质表达相关性，并留下足够的材料用于额外的分子测定。一种新的方法，质谱免疫组织化学（MSIHC），兼容高水平的目标复用和适用于福尔马林固定，石蜡包埋的样品可以规避许多这些问题。该策略采用用元素质量标签（例如通常在生物样本中不存在的同位素纯镧系元素）标记的抗体，而不是用典型的荧光团或发色团标记的抗体。然后使用激光或离子束在组织中检测金属标记的抗体，以释放标签用于随后的质谱检测。在给定的多重IHC组内，选择金属标记物，使得它们各自的质量不重叠。超过30种抗体已同时成像，如果部署完整的可用质谱，可能一次检测到多达100种抗体。MSIHC与常规IHC技术相比具有许多优点。不存在自发荧光导致的背景，动态范围为105，分别超过免疫荧光和显色IHC的100倍和1000倍。标记的一抗的检测改善了显色和荧光IHC的测定线性。与组织同时孵育的多重质量标记抗体似乎不会交叉干扰，并且由于质量标记不会降解，因此样品无限期稳定。多路离子束成像的成像分辨率可以优于光学显微镜。通过适当的仪器，MSIHC有可能改变研究和临床病理学实践。
The role of immunohistochemistry (IHC) in the management of cancer has expanded to provide improved diagnostic classification, as well as guidance on disease prognosis, therapy, and relapse. These new tasks require evaluation of an increasing number of protein targets; however, conventional multiplexing, usually achieved using serial tissue sections stained for a single analyte per slide, can exhaust small biopsy specimens, complicate slide-to-slide protein expression correlation, and leave insufficient material for additional molecular assays. A new approach, mass spectrometry immunohistochemistry (MSIHC), compatible with high levels of target multiplexing and suitable for use on formalin-fixed, paraffin-embedded samples can circumvent many of these issues. The strategy employs antibodies that are labeled with elemental mass tags, such as isotopically pure lanthanides not typically found in biological specimens, rather than with typical fluorophores or chromogens. The metal-labeled antibodies are then detected in tissue using lasers or ion beams to liberate the tags for subsequent mass spectrometry detection. Within a given multiplexed IHC panel, the metal labels are selected so that their respective masses do not overlap. More than 30 antibodies have been imaged simultaneously, and up to 100 antibodies could potentially be detected at once if the full available mass spectrum is deployed. MSIHC has a number of advantages over conventional IHC techniques. Background due to autofluorescence is absent and the dynamic range is 105, exceeding immunofluorescence and chromogenic IHC by 100-fold and 1000-fold, respectively. Detection of labeled primary antibodies improves assay linearity over both chromogenic and fluorescent IHC. Multiplexed mass-tagged antibodies incubated simultaneously with tissue do not appear to cross-interfere, and because the mass tags do not degrade, samples are stable indefinitely. The imaging resolution of multiplexed ion-beam imaging can be better than light microscopy. With appropriate instrumentation, MSIHC has the potential to transform research and clinical pathology practice.
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