免疫组织化学双染技术在唾液腺淋巴上皮性病变中的应用评价

Application of double stained immunohistochemistry technology in lymphoepithelial lesions of salivary gland

目的:根据抗体阳性表达的不同部位(细胞核、细胞质和细胞膜)、染色顺序及匹配不同的显色系统,探讨免疫组织化学双染技术在唾液腺淋巴上皮性病变诊断中的最佳染色方法。方法:挑选良性淋巴上皮病(benign lymphoepithelial lesion,BLEL)、淋巴上皮癌(lymphoepithelial carcinoma,LEC)和黏膜相关淋巴组织结外边缘区B细胞淋巴瘤(extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue,MALT淋巴瘤)各20例,分别进行AE1/AE3、Ki-67、CD20cy抗体的免疫组织化学单染和两两组合的双染检测。每个病例依据不同抗体的表达部位(细胞核、细胞质及细胞膜)、不同的抗体染色顺序和显色剂,分别采用3种双染方法检测,即①先Ki-67(DAB显色),再AE1/AE3或CD20cy(AEC显色);②先AE1/AE3或CD20cy(DAB显色),再Ki-67(AEC显色);③先Ki-67(AEC显色),再AE1/AE3或CD20cy(BCIP/NBT显色)。所得结果均与单染相比较,采用SPSS 17.0软件包对数据进行χ^2检验和配对t检验,分析染色强度和阳性比率有无差异。结果:所有双染方法中抗体定位均准确,但方法 1中各抗体染色强度(P=0.765)和阳性比率(P>0.05)均无显著差异,而方法 2和方法 3中抗体的阳性比率和染色强度均有不同程度下降(P<0.05)。结论:免疫组织化学双染技术在唾液腺淋巴上皮性病变中的最佳染色方法为先进行阳性定位于细胞核如Ki-67的染色,配合使用DAB显色剂,后进行阳性定位于细胞膜或细胞质如AE1/AE3或CD20cy的染色,配合使用AEC显色剂。

PURPOSE: Depending on the sites of positive antibody expression(nucleus, cytoplasm and membrane), this study was aimed to investigate the best method of applying double stained immunohistochemistry technology in lymphoepithelial lesions of salivary gland. METHODS: The expression of AE1/AE3,Ki-67 and CD20 cy was examined by single stained immunohistochemistry and double stained immunohistochemistry respectively in 20 benign lymphoepithelial lesions(BLEL), 20 lymphoepithelial carcinomas(LEC) and 20 extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue(MALT). Depending on the different sites of antibody expression(nucleus, cytoplasm and membrane), the sequence of staining and chromogenic system, three methods of double stained immunohistochemistry were performed:① Ki-67(DAB chromogen),AE1/AE3 or CD20cy(AEC chromogen);② AE1/AE3 or CD20cy(DAB chromogen),Ki-67(AEC chromogen);③ Ki-67(AEC chromogen),AE1/AE3 or CD20cy(BCIP/NBT chromogen). The results of double stained immunohistochemistry were compared with single stained immunohistochemistry. The data were analyzed by using SPSS17.0 software package. RESULTS: The results of immunohistochemistry demonstrated specificity and accuracy of the immunostaining procedures in all three methods, no significant difference of the positive strength(P=0.765) and positive ratio of antibody(P>0.05) was found in method 1. Otherwise, there were significant differences of the positive strength and positive ratio of antibody in method 2 and 3(P <0.05). CONCLUSIONS: The best method is that nucleus positive antibody should be chosen first(Ki-67), followed by DAB chromogen, cytoplasm and membrane positive antibody should be chosen second(AE1/AE3 or CD20cy), combined with AEC chromogen for double stained immunohistochemistry in lymphoepithelial lesions of salivary gland.