环保透明脱蜡液Van-Clear与传统试剂在组织处理后HE染色及FISH法检测乳腺癌HER2基因中的比较

Comparison of Tissue Dewaxing by Environment-friendly Transparent Dewaxing Solution Van-Clear and Conventional Reagents for HE Staining and Detection of HER2 Genes by FISH in Breast Cancer Tissues

目的比较环保透明脱蜡液Van-Clear和二甲苯透明脱蜡制作的组织切片苏木精-伊红(HE)染色优良率,对比荧光原位杂交(FISH)法检测2种透明脱蜡液制作的乳腺癌组织切片的人类表皮生长因子受体2(HER2)基因扩增阳性率,探讨Van-Clear替代二甲苯的可行性。方法收集中山市博爱医院2013年2月~2015年12月门诊及住院部送检的乳腺浸润性导管癌标本89例、乳腺增生标本125例、子宫平滑肌瘤标本193例、子宫平滑肌肉瘤标本12例、肺癌穿刺标本16例、绒毛标本139例,同一病变部位切取2个样本,用抽签法随机分为2组,命名为A、B组。A组采用二甲苯透明脱蜡制作切片574张;B采用Van-Clear透明脱蜡制作切片574张。依据切片染色情况判定切片等级,采用SPSS20.0软件比较A、B组切片HE染色优良率;A、B组中乳腺浸润性导管癌标本再各制作切片89张,采用SPSS 20.0软件比较FISH法检测的HER2基因扩增的差异。结果 (1)生物显微镜下,A、B两组切片HE染色结果细胞轮廓清晰、透明度好,细胞核与细胞质蓝红相映、色彩鲜艳,核质对比明显,核膜及核染色质颗粒清晰可见、分辨良好,分裂象染色体呈现黑蓝色,各种成分层次分明。(2)A、B两组切片HE染色优良片分别为570张、572张,中等、差片4张、2张。染色优良率分别为99.30%、99.65%,两组间染色优良率差异无统计学意义(χ2=0.50,P>0.05)。(3)荧光显微镜下,A、B两组切片乳腺浸润性导管癌HER2双探针FISH检测结果组织轮廓和背景均清晰,探针定位准确,可见耀眼的红/绿荧光信号。(4)A、B两组切片检测乳腺浸润性导管癌HER2基因,FISH阳性率分别为24.72%、26.97%,两组间阳性率差异无统计学意义(χ2=0.50,P>0.05),且FISH结果符合率为97.75%。结论环保透明脱蜡液Van-Clear有替代传统试剂二甲苯应用于组织切片HE染色及FISH法检测乳腺癌HER2基因的潜在可能。

Objective To compare HE staining after tissue dewaxing by environment-friendly transparent dewaxing solution Van-Clear and conventional reagents,and compare positive rate of human epidermal growth factor receptor-2（HER2）gene amplification by in situ hybridization（FISH）assay in breast cancer tissue sections after the two dewaxing methods,and further explore the feasibility of Van-Clear replacing xylene.Methods Totally,89 cases of infiltrating ductal breast carcinoma,125 cases of hyperplasia of mammary glands,193 cases of leiomyoma of uterus,12 cases of leiomyosarcoma of uterus,16 cases of lung cancer,and 139 cases of villus specimens submitted by the outpatient and inpatient departments in Zhongshan Bo＇ai Hosipital were all collected from February 2013 to December 2015.Two samples were taken from the same lesion site,and randomly assigned to group A and B.Group A used xylene dewaxing to make 574 slices,while group B used Van-Clear transparent dewa-xing to make 574 slices.The slices were graded according to staining levels.Good HE staining rate was compared between group A and B by using SPSS 20.0.Eighty-nine sections from breast infiltrating ductal carcinoma tissues in group A and B were prepared.Difference in HER2 gene amplification detected by FISH was compared between the two groups.Results（1）Under the biological microscope,the HE staining results of the sections in group A and B were as follows：The cells had clear outline and good transparency.Their nucleus and cytoplasm had bright colors such as blue and red.The nucleoplasm had obvious contrast.The nuclear membrane and nuclear staining granules were clearly visible and well resolved.The mitotic chromosome was dark blue with distinct components and gradations.（2）In group A and B,the number of the HE staining slides of different quality was as follows,high quality：468and 476,fine quality：102and 96,medium quality：4and 2,bad quality,0and 0,respectively.The good staining rate was 99.30% and 99.65%in group A and B,respectively（χ~2=0.50,P0.05）.（3）Under the fluorescence microscope,the detection results of HER2 genes in infiltrating ductal breast carcinoma specimens by using dual probe FISH technique were as follows：The tissue profile and the background were both clear;the probe was accurate;and the red/green fluorescence signals were visible.（4） HER2 genes in the infiltrating ductal breast carcinoma specimens were detected,and the positive rate of FISH was 24.72%and 26.97%,respectively（χ~2=0.50,P0.05）.Besides,the coincidence rate of FISH result was 97.75%.Conclusion The environment-friendly transparent dewaxing solution Van-Clear has the potential to replace the conventional reagent xylene in the application of the HE staining and the use of FISH technique to detect HER2 genes in the breast cancer after both tissue processed.