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目的探讨微核、染色体试验中用乙醇部分代替甲醇固定淋巴细胞的可行性及相关技术方法。方法采用周围血半微量全血常规培养法进行试验。试验按固定液中甲醇和乙醇的比例不同分为Ⅰ、Ⅱ、Ⅲ、Ⅳ组,4种方法固定液中甲醇、乙醇和冰醋酸的比例分别为3∶0∶1、2∶1∶1、1∶2∶1、0∶3∶1(V∶V∶V),其他条件和操作均相同。取周围静脉血适量接种于RPMI-1640培养基中,于37℃培养72 h(染色体培养48 h),用体积分数为10%吉姆萨染液染色。结果Ⅰ、Ⅱ和Ⅲ组淋巴细胞均分散良好、染色均匀适中,胞浆呈蓝灰色,胞核呈紫红色,胞核轮廓清楚、边界清晰,均适合微核观察计数;Ⅳ组细胞容易聚集成团、胞浆易于破裂,致使适合观察的淋巴细胞数太少,从而影响微核的观察计数。Ⅰ、Ⅱ和Ⅲ组之间淋巴细胞着色良好率比较,差异有统计学意义(P<0.05);其中Ⅱ组淋巴细胞着色良好率高于Ⅲ组(P<0.05)。Ⅰ、Ⅱ和Ⅲ组淋巴细胞胞浆完整率和淋巴细胞微核细胞率分别比较,差异均无统计学意义(P>0.05)。Ⅰ、Ⅱ、Ⅲ和Ⅳ组淋巴细胞细胞成团指数比较,差异有统计学意义(P<0.05);其中Ⅰ、Ⅱ和Ⅲ组细胞成团指数均低于Ⅳ组(P<0.05)。Ⅰ、Ⅱ和Ⅲ组淋巴细胞染色体呈紫红色、分散良好、大小适中、背景清楚,均适合染色体观察;Ⅳ组不适用于染色体观察。结论微核和染色体试验的固定液中加入一定比例的乙醇(乙醇与甲醇的体积比不超过2∶1)代替甲醇固定淋巴细胞,不影响微核和染色体的观察,并可降低甲醇中毒风险。
Objective To investigate the feasibility of micronuclei and chromosomal test in which part of ethanol is used instead of methanol to fix lymphocytes and related techniques. Methods Peripheral blood semi-micro-whole blood routine culture method for testing. The test was divided into Ⅰ, Ⅱ, Ⅲ and Ⅳ groups according to the ratio of methanol and ethanol in the fixed solution. The proportions of methanol, ethanol and glacial acetic acid in the fixing solution of 4 methods were 3: 0: 1, 2: 1: 1, 1: 2: 1, 0: 3: 1 (V: V: V). Other conditions and operations are the same. Appropriate amount of peripheral venous blood was inoculated into RPMI-1640 medium and cultured at 37 ℃ for 72 h (chromosome culture for 48 h), and stained with 10% Giemsa stain. Results The lymphocytes in group Ⅰ, Ⅱ and Ⅲ were well dispersed, and the cells were uniformly stained. The cytoplasm was blue-gray, the nucleus was purplish red, the outline of nucleus was clear, and the boundaries were clear. All the cells were suitable for micronucleus observation and counting. Mission, cytoplasm easily rupture, resulting in the number of lymphocytes suitable for observation is too small, thus affecting the observation count micronuclei. The good rate of lymphocyte coloration between groups Ⅰ, Ⅱ and Ⅲ was statistically significant (P <0.05). The staining rate of lymphocytes in group Ⅱ was higher than that of group Ⅲ (P <0.05). There was no significant difference in the rates of complete cytoplasm of lymphocytes and micronuclei of lymphocytes between groups Ⅰ, Ⅱ and Ⅲ (P> 0.05). Group Ⅰ, Ⅱ, Ⅲ and Ⅳ lymphocyte conglomeration index, the difference was statistically significant (P <0.05); Ⅰ, Ⅱ and Ⅲ group of cells agglomeration index were lower than the group Ⅳ (P <0.05). The chromosomes of lymphocytes in group Ⅰ, Ⅱ and Ⅲ were purple, well dispersed, moderate in size and clear in background, which were suitable for chromosomal observation. Group Ⅳ was not suitable for chromosome observation. Conclusion The fixation of micronucleus and chromosome test by adding a certain proportion of ethanol (ethanol and methanol volume ratio of no more than 2: 1) instead of methanol fixed lymphocytes, does not affect the micronuclei and chromosome observations, and reduce the risk of methanol poisoning.