APAAP高敏感信号放大技术在乳腺癌HPV DNA原位杂交检测中的应用

目的　探讨原位杂交 APAAP信号放大技术检测福尔马林固定 石蜡包埋乳腺癌组织HPV感染的实用性。方法　利用HPV通用型及亚型特异性探针 ,设定 4种杂交检测程序对 73例进展期乳腺癌和 34例乳腺良性病变进行HPVDNA原位杂交 APAAP信号放大检测。结果　在适宜的杂交程序下 ,39 19%的乳腺癌病例检测到HPVDNA ;良性病变中仅 1例 (2 94 % )纤维腺瘤检测到HPVDNA ,明显低于乳腺癌组 (P <0 0 1)。乳腺的大汗腺样化生区有细胞核阴性 细胞浆着色的假阳性反应 ,假阳性反应不足以被 1mmol L左旋咪唑阻断 ,但能被 0 2mol L盐酸前处理所消除。APAAP信号检测各步骤间的不同缓冲液可影响杂交结果。结论　只要杂交条件得当 ,原位杂交 APAAP检测技术在乳腺组织HPV感染研究中可获得满意结果。乳腺大汗腺样化生区内源性碱性磷酸酶含量较高 ,低浓度盐酸前处理有消除内源性碱性磷酸酶作用。

Utilization of APAAP high sensitivity signal detection after HPV DNA in situ hybridization on breast carcinoma

Objective To study the possibility of detecting HPV infection on formalin-fixed, paraffin-embedded breast tissues by in situ hybridization-APAAP signal amplification system. Methods 73 cases of advanced breast cancer and 34 cases of benign breast lesions were detected by HPV common probe and type-specific probes according to 4 different hybridizing protocols. Results Under the proper hybridizing condition, 39.19% of breast cancer cases was positive for HPV DNA, the positive cases showed dark blue stain on their nuclei; whereas, only 1 case(2.94%) of fibroadenoma out of benign lesion showed positive by HPV DNA In situ hybridization, the positive rate in benign lesion group was obviously lower than breast cancer group(P<0.01).The apocrine metaplasia areas of breast disclosed pseudo-positive stain in their cytoplasm even omitting the HPV probes during the protocol. The pseudo-positive reaction was removed by 0.2N HCl pretreatment but not by 1mM levamisole hydrochloride. Conclusion A satisfactory result can be obtained at HPV infection study on paraffin embedded breast tissues by In situ hybridization-APAAP detection technique. Since the apocrine metaplasia areas of breast showed obviously endogenous alkaline phosphatase activity, if the target molecule localized in cytoplasm, it is better to do blocking endogenous alkaline phosphatase pretreatment by low concentration HCl.