Mueller Claudius, Harpole Michael G, Espina Virginia
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle, MS1A9, Manassas, VA, 20110, USA.
Methods Mol Biol. 2017;1606:85-102. doi: 10.1007/978-1-4939-6990-6_6.
Bone metastasis from primary cancer sites creates diagnostic and therapeutic challenges. Calcified bone is difficult to biopsy due to tissue hardness and patient discomfort, thus limiting the frequency and availability of bone/bone marrow biopsy material for molecular profiling. In addition, bony tissue must be demineralized (decalcified) prior to histomorphologic analysis. Decalcification processes rely on three main principles: (a) solubility of calcium salts in an acid, such as formic or nitric acid; (b) calcium chelation with ethylenediaminetetraacetic acid (EDTA); or (c) ion-exchange resins in a weak acid. A major roadblock in molecular profiling of bony tissue has been the lack of a suitable demineralization process that preserves histomorphology of calcified and soft tissue elements while also preserving phosphoproteins and nucleic acids. In this chapter, we describe general issues relevant to specimen collection and preservation of osseous tissue for molecular profiling. We provide two protocols: (a) one-step preservation of tissue histomorphology and proteins and posttranslational modifications, with simultaneous decalcification of bony tissue, and (b) ethanol-based tissue processing for TheraLin-fixed bony tissue.
原发癌部位的骨转移会带来诊断和治疗方面的挑战。由于组织硬度和患者不适，钙化骨难以进行活检，这限制了用于分子分析的骨/骨髓活检材料的获取频率和可用性。此外，在进行组织形态学分析之前，骨组织必须进行脱矿质（脱钙）处理。脱钙过程主要基于三个原理：（a）钙盐在酸（如甲酸或硝酸）中的溶解性；（b）钙与乙二胺四乙酸（EDTA）的螯合作用；或（c）在弱酸中的离子交换树脂。骨组织分子分析的一个主要障碍是缺乏一种合适的脱矿质方法，既能保留钙化和软组织成分的组织形态学，又能保留磷酸化蛋白和核酸。在本章中，我们描述了与用于分子分析的骨组织标本采集和保存相关的一般问题。我们提供了两种方案：（a）一步法保存组织形态学、蛋白质及翻译后修饰，同时对骨组织进行脱钙；（b）针对TheraLin固定的骨组织的乙醇基组织处理方法。
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