不同温度及染色方法对细胞DNA倍体诊断的影响  

Effects of different temperatures and staining methods on DNA ploidy diagnosis

作　　者：黄荣祥 HUANG Rong-xiang;Xiamen Motic Medical Laboratory;

机构地区：[1]厦门麦克奥迪医学检验所,福建厦门361006

出　　处：《生物医学工程与临床》2018年第6期686-691,共6页Biomedical Engineering and Clinical Medicine

摘　　要：目的通过介绍3种Feulgen染色法,以经典的标准Feulgen染色法作为对照,评估温度及染色方法对细胞DNA倍体诊断的影响。方法根据细胞病理学TBS诊断标准,将收集的标本划分为未见上皮内病变/恶性肿瘤(NILM)、高度鳞状上皮内病变(HSIL)两组,采用标准Feulgen染色法(25℃)、改良Feulgen染色法(40℃)、快速Feulgen染色法(55℃)对这两组涂片分别染色,用MotiCytometer全自动细胞图像分析系统测定各倍体[二倍体(2c)、四倍体(4c)、多倍体(> 4c)]细胞核积分光密度(IOD)及其变异系数(CV)值,绘制酸解曲线图,并对测量结果进行统计分析。结果 3种染色法细胞核染色深,核轮廓清晰,胞浆及玻片背景干净。标准Feulgen染色法最佳水解时间为40~70 min。改良Feulgen染色法最佳水解时间为15~30 min。快速Feulgen染色法反应迅速,平台期短,在0.5~3.0 min IOD差异存在显著统计学意义(F=12.40,P <0.01),而1.0~2.0 min IOD差异无统计学意义(F=0.34,P> 0.05),故1.0~2.0 min可作为最佳水解时间。3种染色法2c细胞核IOD最大值分别为127、128、127,差异无统计学意义(F=0.00,P> 0.05),4c及> 4c细胞3种染色法反应最大值差异均无统计学意义(P> 0.05)。同一温度下正常细胞、病变细胞最佳反应时间一致。结论不同温度下的Feulgen染色方法各具优劣,病理诊断工作者在选择染色温度时,应综合考虑水解、染色之间的关系,根据实验室的需求选用适宜的染色方法。Objective To evaluate the effects of different temperatures and staining methods on DNA ploidy diagnosis by introducing 3 different Feulgen staining methods, with standard Feulgen staining method as control. Methods According to cytopathology The Bethesda System（TBS） diagnosis standard, the specimens were divided into negative for intraepithelial lesion or malignancy（NILM） group and high-grade squamous intraepithelial lesion（HSIL） group. The standard Feulgen staining method（25 ℃）, improved Feulgen staining method（40 ℃） and rapid Feulgen staining method（55 ℃） was used to stain and MotiCytometer ICM to test cell nuclear integral optical density（IOD） and coefficient of variation（CV）. The hydrolysis curve was drawn and measurement results was analyzed. Results The nuclei by 3 staining methods were deep stained with clear nuclear outline, clean cytoplasm and glass background. The optimal hydrolysis time in standard Fenlgen staining method was 40 - 70 minutes, improved Feulgen staining method was 15 - 30 minutes. Rapid Feulgen staining method was rapid with short platform period, the difference of IOD in 0.5 - 3.0 minutes was statistically significant（F = 12.40, P 〈 0.01）, while that in 1.0 - 2.0 minutes was no statistically significant（F = 0.34, P 〉 0.05）, so 1.0 - 2.0 minutes was considered optimal hydro].ysis time. The maximum IOD values of 2c cell nucleus in 3 staining methods were 127, 128 and 127, respectively, and the differences were no statistically significant（F = 0.00, P 〉 0.05）, and the maximum response values of 3 staining methods of 4c and 〉 4c cells showed no statistically significant difference（P 〉 0.05）. Normal cells and pathological cells showed same optimal reaction time at the same temperature. Conclusion It is demonstrated that Feulgen staining methods have their own advantages and disadvantages at different temperatures. The relationship between hydrolysis and staining method should be considered when choosing the staining temperature

关 键 词：病理学 Feulgen 图像分析 积分光密度 酸解 DNA 

分 类 号：R361.3[医药卫生—病理学]