CN107179406A - 癌栓多标免疫组化染色试剂盒 - Google Patents
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Abstract
本发明属于免疫组织化学技术领域，涉及一种显示血管和淋巴管内癌栓的癌栓多标免疫组化染色试剂盒，用于解决HE染色片中脉管内癌栓与间质中浸润癌巢不易明确区分的问题。这种免疫组化染色试剂盒由A、B、C三种抗体的混合液组成；抗体A为CD34或CD31，抗体B为Podoplanin Protein或D2‑40，抗体C为Cytokeratin (Pan)；用于在同一张切片上显示血管内皮细胞、淋巴管内皮细胞和癌细胞，可以清晰地显示脉管内的癌细胞，尤其微小癌栓，避免因人为判读差异造成的脉管内癌栓的过度诊断或诊断不足。
Description
技术领域
本发明属于免疫组织化学技术领域，具体地，本发明涉及一种显示血管和淋巴管内癌栓的癌栓多标免疫组化染色试剂盒。
技术背景
癌肿不同阶段的治疗策略不同，因此其病理分期对临床治疗和预后具有重要意义。癌肿病理分期主要方法是，通过显微镜下观察癌细胞在组织器官中浸润范围，有无脉管内癌栓，有无淋巴结转移，并结合临床影像学判断有无远处器官转移，最终确定癌肿的病理分期，因此脉管内癌栓是病理学观察的一项重要指标。然而在HE染色切片中毛细血管、小静脉和淋巴管内早期癌栓的判断比较困难。一是由于早期癌栓微小而不易被发现；二是浸润癌巢收缩与周围间质形成空隙，这种形态很难与脉管内癌栓区别。
我们设计了CD34（或CD31）、Podoplanin Protein（或D2-40）和Cytokeratin (Pan)混合型第一抗体的癌栓多标免疫组化染色试剂盒，其中CD34和CD31作为血管内皮细胞标志物，Podoplanin Protein（或D2-40）作为淋巴管内皮细胞标志物，Cytokeratin (Pan)作为癌细胞标记物。
CD34（或CD31）、Podoplanin Protein（或D2-40）和Cytokeratin (Pan)混合型第一抗体配方，用于在同一张切片上显示血管内皮细胞、淋巴管内皮细胞和癌细胞，可以清晰地显示脉管内的癌细胞，尤其微小癌栓，避免因人为判读差异造成的脉管内癌栓的过度诊断或诊断不足。
发明内容
本发明的目的在于，为避免病理医生对癌栓的过度诊断或诊断不足，提供一种用于显示血管和淋巴管内癌栓的癌栓多标免疫组化染色试剂盒。该试剂盒染色过程简便快捷，可以在同一张切片显示丰富且对比性强的病理图像信息，使癌栓（尤其微小癌栓）的诊断更为容易且确定。
为实现上述目的，本发明采用如下技术手段：
一种癌栓多标免疫组化染色试剂盒，所述试剂盒中的第一抗体为多种抗体混合液。
其中，所述混合型第一抗由A、B、C三种抗体的混合液组成；其中，抗体A为CD34或CD31，抗体B为Podoplanin Protein或D2-40，抗体C为Cytokeratin (Pan)；其中A、B两种抗体为同一种属来源，抗体C为另一种属来源的抗体。
采用的检测放大系统为不同种属的HRP和AP酶标的混合液。
采用的显色剂为DAB、AP-Red组合或AEC、BCIP/NBT组合。
所述混合型第一抗体在同一切片同一时间同时反应。
所述检测方法采用与混合型第一抗体对应的HRP和AP酶标的混合型第二抗体。
本发明的另一目的在于提供一种上述癌栓多标免疫组化染色试剂盒的应用，该试剂盒可应用于显示血管和淋巴管内的癌栓。
癌栓多标免疫组化染色步骤如下：
⑴组织切片放入67℃恒温箱中干烤2h。
⑵常规二甲苯脱蜡3次，每次6分钟，100%、100%、95%、85%梯度乙醇中水化，每次3分钟，最后自来水冲洗。
⑶抗原热修复，自然冷却至室温，自来水冲洗，PBS 冲洗3×3分钟。
⑷在3%过氧化氢中室温孵育10分钟，阻断内源性过氧化物酶，PBS冲洗 3×3分钟。
⑸用正常动物血清封闭10分钟，甩去多余血清，不洗，滴加混合型第一抗体，室温孵育1小时，PBS 冲洗3×3分钟。
⑹滴加混合型第二抗体，室温孵育15分钟，PBS 冲洗3×3分钟。
⑺分步显色，显微镜镜下监测显色过程，适时终止反应。
⑻苏木素复染，水性封固剂封固，等水性封固剂完全干燥后，用中性树胶加盖玻片封固。
本发明的显著优点
本发明癌栓多标免疫组化染色试剂盒首次将混合型第一抗体和多种属检测放大系统，应用于血管和淋巴管内癌栓的诊断和鉴别诊断，不但保持了混合型抗体中各个抗体与单染时同样的敏感性和特异性外，还具有染色步骤少，实验时间短，稳定性高，实验结果直观对比，所带来的信息量远比传统单染高等优点。
附图说明
图1. 早期浸润性尿路上皮癌的癌栓多标免疫组化染色（×200），混合型第一抗体为CD34、D2-40和Cytokeratin (Pan)的抗体混合液，淋巴管内皮细胞和血管内皮细胞显示红色（黑白图中显示浅色），癌细胞显示棕色（黑白图中显示深色）。癌细胞位于衬附红色内皮细胞的脉管内，属于脉管内癌栓；组织中还散在一些周围有空隙的微小癌巢，但空隙的内表面没有红色内皮细胞衬附，因此不属于脉管内癌栓，而属于癌细胞浸润间质。
图2. 浸润性乳腺癌的癌栓多标免疫组化染色（×200），混合型第一抗体为CD31、D2-40和Cytokeratin (Pan)的抗体混合液，淋巴管内皮细胞和血管内皮细胞显示红色（黑白图中显示浅色），癌细胞显示棕色（黑白图中显示深色）。图上方静脉内见小癌栓，图下方的淋巴管内见大癌栓。
具体实施方式
下面结合具体实施例对本发明进行详细说明；以下实施例是为了进一步说明本发明，但不应视为限制本发明。本专利中所述的各种试剂，除特别指出外，均可从商业途径上获得。
实施例1：
研究对象为甲醛固定-石蜡包埋的早期浸润性尿路上皮癌，癌栓多标免疫组化染色步骤如下：
⑴组织切片放入67℃恒温箱中干烤2h。
⑵常规二甲苯脱蜡3次，每次6分钟，100%、100%、95%、85%梯度乙醇中水化，每次3分钟，最后自来水冲洗。
⑶在pH6.0的柠檬酸抗原修复液中高压修复1.5-2分钟，自然冷却至室温，自来水冲洗，PBS 冲洗3×3分钟。
⑷在3%过氧化氢中室温孵育10分钟，阻断内源性过氧化物酶，PBS冲洗 3×3分钟。
⑸用正常动物血清封闭10分钟，甩去多余血清，不洗，滴加CD34、D2-40和Cytokeratin (Pan)混合型第一抗体，室温孵育1小时，PBS 冲洗3×3分钟。
⑹滴加与第一抗体对应的HRP、AP酶标的混合型第二抗体，室温孵育15分钟，PBS冲洗3×3分钟。
⑺滴加AP-Red显色液，室温下孵育15-30min，显微镜镜下监测显色过程，适时终止反应；滴加DAB显色液，室温下孵育5-10min，显微镜镜下监测显色过程，适时终止反应。
⑻苏木素复染，水性封固剂封固，等水性封固剂完全干燥后，用中性树胶加盖玻片封固。
早期浸润性尿路上皮癌的癌栓多标免疫组化染色（×200）结果见图1所示。其中，淋巴管内皮细胞和血管内皮细胞显示红色，癌细胞显示棕色。A处为癌细胞位于衬附红色内皮细胞的脉管内，属于脉管内癌栓；B处为组织中还散在一些周围有空隙的微小癌巢，但空隙的内表面没有红色内皮细胞衬附，因此不属于脉管内癌栓，而属于癌细胞浸润间质。
实施例2：
研究对象为甲醛固定-石蜡包埋的浸润性乳腺癌组织，癌栓多标免疫组化染色步骤如下：
⑴组织切片放入67℃恒温箱中干烤2h。
⑵常规二甲苯脱蜡3次，每次6分钟，100%、100%、95%、85%梯度乙醇中水化，每次3分钟，最后自来水冲洗。
⑶在pH6.0的柠檬酸抗原修复液中高压修复1.5-2分钟，自然冷却至室温，自来水冲洗，PBS 冲洗3×3分钟。
⑷在3%过氧化氢中室温孵育10分钟，阻断内源性过氧化物酶，PBS冲洗 3×3分钟。
⑸用正常动物血清封闭10分钟，甩去多余血清，不洗，滴加CD31、D2-40和Cytokeratin (Pan)混合型第一抗体，室温孵育1小时，PBS 冲洗3×3分钟。
⑹滴加与第一抗体对应的HRP、AP酶标的混合型第二抗体，室温孵育15分钟，PBS冲洗3×3分钟。
⑺滴加AP-Red显色液，室温下孵育15-30min，显微镜镜下监测显色过程，适时终止反应；滴加DAB显色液，室温下孵育5-10min，显微镜镜下监测显色过程，适时终止反应。
⑻苏木素复染，水性封固剂封固，等水性封固剂完全干燥后，用中性树胶加盖玻片封固。
浸润性乳腺癌的癌栓多标免疫组化染色（×200）结果见图2所示。淋巴管内皮细胞和血管内皮细胞显示红色，癌细胞显示棕色。图上方静脉内见小癌栓，图下方的淋巴管内见大癌栓。
Claims (6)
1.一种癌栓多标免疫组化染色试剂盒，其特征在于：所述试剂盒中第一抗体为多种抗体混合液。
2.根据权利要求1所述的癌栓多标免疫组化染色试剂盒，其特征在于：所述混合型第一抗由A、B、C三种抗体的混合液组成；其中，抗体A为CD34或CD31，抗体B为PodoplaninProtein或D2-40，抗体C为Cytokeratin (Pan)；其中A、B两种抗体为同一种属来源，抗体C为另一种属来源的抗体。
3.根据权利要求1所述的癌栓多标免疫组化染色试剂盒，其特征在于：采用的显色剂为DAB、AP-Red组合或AEC、BCIP/NBT组合。
4.根据权利要求1所述的癌栓多标免疫组化染色试剂盒，其特征在于：所述多种抗体混合型的第一抗体在同一切片同一时间同时反应。
5.根据权利要求1所述的癌栓多标免疫组化染色试剂盒，其特征在于：所述检测方法采用与混合型第一抗体对应的HRP和AP酶标的混合二抗。
6.一种权利要求1所述的癌栓多标免疫组化染色试剂盒的应用方法，其特征在于：所述试剂盒应用于显示血管和淋巴管内的癌栓。
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