外周血染色体吉姆萨显带制备条件优化研究

目的 对外周血染色体吉姆萨显带制备条件进行优化，以提高染色体制备成功率。方法 选取2021 年4 月～ 2022年4 月因不孕不育就诊于梧州市工人医院生殖医学中心的116 例成年患者的外周静脉血为研究对象，其中单因子梯度实验15 例，正交实验设计1 例，优化前组50 例和优化后组50 例。在现有染色体制备体系基础上，通过单因子梯度实验对影响外周血染色体制备成功率的秋水仙素作用浓度、作用时间及低渗处理时间进行优化，根据单因子梯度实验结果进行3 因素3 水平正交实验设计，以染色体制备成功率最高的条件组合作为最适的外周血染色体制备体系。并进一步对优化前、后两组＜ 320 带，320 ～ 400 带和400 ～ 550 带的分裂相所占比例进行比较分析。结果 单因子梯度实验结果表明，染色体制备成功率随着秋水仙素作用浓度、作用时间及低渗处理时间的增加均呈先升高后下降趋势。在秋水仙素不同作用浓度实验中，当秋水仙素作用浓度为0.24 μg/ml 时染色体制备成功率最高，达67.67%；在秋水仙素不同作用时间实验中，当秋水仙素作用时间为30 min 时染色体制备成功率最高，达62.33%；在不同低渗处理时间实验中，当低渗处理时间为35 min 时染色体制备成功率最高，为64.17%。根据单因子梯度实验结果，秋水仙素作用浓度设置0.20，0.24，0.28μg/ml 三个水平，秋水仙素作用时间设置25，30 和35 min 三个水平，低渗处理时间设置30，35，40min 三个水平，进行正交实验设计。结果表明秋水仙素作用浓度为0.24 μg/ml，秋水仙素作用时间30 min，低渗处理时间 40 min 是外周血染色体制备最适体系。优化前组吉姆萨显带 <320 带的分裂相占比(64.30%)显著高于优化后组(43.78%)，差异具有统计学意义(χ2=508.443，P ＜ 0.001)；优化后组320 ～ 400 带，400 ～ 550 带分裂相占比(40.30%，15.29%)均显著高于优化前组(33.12%，2.58%)，差异具有统计学意义(χ2=66.629，635.346，均P ＜ 0.001)。结论 经优化后确定的染色体制备体系可提高染色体制备成功率，值得临床应用与推广。

Study on the Optimization of Preparation Conditions for Chromosome Giemsa Banding in Peripheral Blood

Objective To optimize the preparation conditions of peripheral blood chromosome Giemsa banding to improve the success rate of chromosome preparation. Methods The peripheral venous blood of 116 adult patients who were admitted to the Reproductive Medicine Center of Wuzhou Gongren Hospital due to infertility from April 2021 to April 2022 were selected as the research objects. Among them,15 cases were used in the single-factor gradient experiment, 1 case was used in the orthogonal design experiment, and there were 50 cases in the pre-optimization group, 50 cases in the post-optimization group. On the basis of the existing chromosome preparation system, the action concentration, action time of Colchicine and hypotonic treatment time, which affect the success rate of chromosome preparation in peripheral blood, were optimized by single-factor gradient experiment. Then according to the results of the single-factor gradient experiment, a 3-factor and 3-level orthogonal design experiment was carried out, and the combination of conditions with the highest success rate of chromosome preparation was used as the most suitable peripheral blood chromosome preparation system. Furthermore, the proportion of split phase in ＜ 320 band，320 ～ 400 band and 400 ～ 550 band in the pre-optimization group and the post-optimization group was compared and analyzed. Results The results of the single-factor gradient experiment showed that the success rate of chromosome preparation increased first and then decreased with the increase of action concentration, action time of Colchicine and hypotonic treatment time. In the experiments with different action concentrations of Colchicine, the success rate of chromosome preparation was the highest when the action concentration of Colchicine was 0.24 μg/ml, which was 67.67%. In the experiments with different action time of Colchicine，when the action time of Colchicine was 30 min，the success rate of chromosome preparation was the highest，which was 62.33%. In the experiments with different hypotonic treatment time，when the hypotonic treatment time was 35 min，the success rate of chromosome preparation was the highest，which was 64.17%. According to the results of single factor gradient experiment，the action concentration of Colchicine was set at three levels 0.20, 0.24 and 0.28 μg/ml，the action time of Colchicine was set at three levels 25, 30 and 35 min，and the hypotonic treatment time was set at three levels 30, 35 and 40 min. Orthogonal design experiment was conducted. The results showed that the action concentration of Colchicine was 0.24 μg/ml，the action time of Colchicine was 30 min，and the hypotonic treatment time was 40 min，which was the optimal system for the preparation of peripheral blood chromosomes. The proportion of the split phase of the Giemsa band <320 band in the pre-optimization group (64.30%) was significantly higher than that in the post-optimization group (43.78%),and the difference was statistically significant (χ2=508.443, P＜0.001).The proportion of the split phase of the Giemsa band 320 ～ 400 band and 400 ～ 550 band in the post-optimization group (40.30%，15.29%) was significantly higher than that in the pre-optimization group (33.12%，2.58%)，and the difference was statistically significant (χ2=66.629, 635.346, all P ＜ 0.001). Conclusion The optimized chromosome preparation system could improve the success rate of chromosome preparation and was worthy of clinical application and promotion.