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目的比较环保固定液聚羟基丙烯酸、环保透明脱蜡液Van-clear单独或联合替代传统固定液4%中性缓冲甲醛、传统透明脱蜡液二甲苯应用于实时荧光定量PCR(qRT-PCR)法,检测非小细胞肺癌(NSCLC)表皮生长因子受体(EGFR)基因突变率的敏感性、特异性及符合率的差异,评估qRT-PCR法及其环保技术平台的临床应用价值。方法选取中山市博爱医院、中山市中医院2013年5月至2016年3月期间手术切除的原发性NSCLC标本91例,同一肿瘤病变部位切取5个样本,采用随机数字表法分为A、B、C、D、E。A组使用4%中性缓冲甲醛固定、二甲苯透明脱蜡制作切片、直接测序法检测EGFR基因突变;B组使用4%中性缓冲甲醛固定、二甲苯透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变;C组使用聚羟基丙烯酸固定、二甲苯透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变;D组使用4%中性缓冲甲醛固定、Van-clear透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变;E组使用聚羟基丙烯酸固定、Van-clear透明脱蜡制作切片、qRT-PCR法检测EGFR基因突变。分别测定5组NSCLC中EGFR基因第18、19、20、21号外显子的突变情况。结果 (1)B、C、D、E组与A组比较,EGFR基因发生突变人数百分率、基因靶位点突变率的差异均无统计学意义(P>0.05);(2)B、C、D、E组与A组比较,EGFR靶位点突变检测结果的灵敏度好、特异度强、符合率高。结论聚羟基丙烯酸、环保透明脱蜡液Van-clear单独或联合替代传统试剂,应用于qRT-PCR法检测NSCLC中EGFR基因突变,与以传统试剂应用于直接测序法的检测结果一致性好,有在临床推广应用的意义和价值。
Objective To compare the environmental protection liquid polyhydroxy acrylic acid and the transparent transparent dewaxing liquid Van-clear to replace the traditional fixed liquid 4% neutral buffered formalin alone or in combination with the traditional transparent dewaxing liquid xylene for real-time quantitative PCR (qRT-PCR) , To detect the sensitivity, specificity and coincidence rate of EGFR mutation rate in non-small cell lung cancer (NSCLC) and to evaluate the clinical value of qRT-PCR and its platform of environmental technology. Methods 91 patients with primary NSCLC resected from May 2013 to March 2016 in Pok Oi Hospital of Zhongshan City and Zhongshan Hospital of Traditional Chinese Medicine were selected. Five samples of the same tumor lesion were selected and divided into A, B, C, D, E. Group A was fixed with 4% neutral buffered formalin, transparent deparaffinization of xylene was made, and direct sequencing was used to detect EGFR gene mutation. Group B was fixed with 4% neutral buffered formalin and transparent deparaffinized with xylene for qRT-PCR The EGFR gene mutation was detected in group C, the sections were fixed with polyhydroxyl acrylic acid and transparent dewaxed with xylene, and the EGFR gene mutation was detected by qRT-PCR. The cells in group D were fixed with 4% neutral buffered formalin and cut with Van-clear transparent dewaxing. The EGFR gene mutation was detected by qRT-PCR. Group E was fixed with polyhydroxyl acrylic acid and sliced with Van-clear transparent dewaxing. EGFR gene mutation was detected by qRT-PCR. The mutations of exon 18, 19, 20, and 21 of EGFR gene in 5 NSCLC patients were respectively determined. Results (1) Compared with group A, there was no significant difference in the percentage of mutation of EGFR gene and mutation rate of gene target site in group B, C, D and E (P> 0.05); (2) D, E group compared with A group, EGFR target site mutation detection sensitivity, specificity, coincidence rate. Conclusion Polyhydroxy acrylic acid and transparent transparent dewaxing solution Van-clear, alone or in combination, can be used in qRT-PCR to detect EGFR gene mutation in NSCLC, which is in good agreement with the traditional sequencing method. Significance and value in clinical application.