基于免疫磁珠法的不同筛选方案对胎儿有核红细胞富集效果的比较  

The comparison of enrichment effects of different screening schemes for fetal nucleated red blood cells from maternal blood by magnetic-activated cell sorting

作　　者：唐苗苗 宾雅棣 赵蓝波 李奇灵 孙超 Tang Miaomiao;Bin Yadi;Zhao Lanbo;Li Qiling;Sun Chao(Department of Obstetrics and Gynecology,The First Affiliated Hospital of Xi’an Jiaotong University;Health Science Center,Xi'an Jiaotong University)

机构地区：[1]西安交通大学第一附属医院妇产科,西安710000 [2]西安交通大学医学部,西安710000

出　　处：《重庆医科大学学报》2023年第2期155-161,共7页Journal of Chongqing Medical University

基　　金：陕西省科技厅基础研究面上资助项目(编号:2020JQ-530)。

摘　　要：目的:将密度梯度离心(density gradient centrifugation,DGC)与免疫磁珠法(magnetic-activated cell sorting,MACS)相结合,应用4种不同的筛选方案,对脐血中的胎儿有核红细胞(fetal nucleated red blood cells,FNRBC)进行分离,评估不同策略富集FNRBC的效果。方法:对10例脐血标本(12 mL/例)按4种不同方案进行筛选:DGC;DGC+MACS阴性筛选(CD45^(-));DGC+MACS阳性筛选(CD71^(+));DGC+MACS阴性/阳性筛选(CD45^(-)/CD71^(+))。通过细胞计数板计数,K-B染色后显微镜下人工FNRBC计数,从获得总细胞量、FNRBC量和FNRBC比例经t检验后评估不同方案富集FNRBC的效果。结果:(1)与其他3组相比,DGC方案可获得的总细胞量(0.95×10^(7)/mL)最多,FNRBC数量(39.87/mL)最多,FNRBC比例最低(0.004‰),差异均具有统计学意义(均P=0.000);(2)与其他3组相比,DGC+MACS阴性/阳性筛选(CD45^(-)/CD71^(+))组合法所获得的总细胞量最少(0.81×10^(6)/mL),获得FNRBC数量也最少(7.47/mL),差异均具有统计学意义(均P=0.000);(3)DGC+MACS单种抗体筛选方案中,DGC+MACS阳性筛选(CD71^(+))所获得总细胞量为1.99×10^(6)/mL,较DGC+MACS阴性筛选(CD45^(-))(2.56×10^(6)/mL)低,差异有统计学意义(P=0.000),而两方案所得FNRBC数量之间差异无统计学意义(P=0.910);(4)DGC+MACS阴性筛选(CD45^(-)),DGC+MACS阳性筛选(CD71^(+)),DGC+MACS阴性/阳性筛选(CD45^(-)/CD71^(+))所得FNRBC比例之间差异无统计学意义(P=0.030、0.050、0.210)。结论:DGC方案获得FNRBC数量最多;DGC+MACS阴性/阳性筛选(CD45^(-)/CD71^(+))方案去除混杂细胞能力最强。在不降低获取目标细胞数量基础上,DGC+MACS阳性筛选(CD71^(+))方案富集FNRBC效果最好。Objective:To isolate fetal nucleated red blood cells(FNRBC)from maternal umbilical cord blood through density gradient centrifugation(DGC)and magnetic-activated cell sorting(MACS) by four different screening schemes,and to evaluate the screening effect of different methods.Methods:Ten samples of umbilical cord blood(12 mL/case)were collected.Each sample was divided into 4groups:Group 1:DGC;Group 2:DGC+MACS negative screening(CD45^(-));Group 3:DGC+MACS positive screening(CD71^(+));Group 4:DGC+MACS negative/positive screening(CD45^(-)/CD71^(+)).After screening,the number of cells was counted,and the t-test was carried out to evaluate the enrichment efficiency of each method.K-B staining was used to identify FNRBC count,and then the number and the proportion of FNRBC were calculated.Results:(1)Compared with the other three groups,DGC method had the largest total cell number(0.95×10^(7)/mL),the largest FNRBC number(39.87/mL) and the lowest proportion of FNRBC(0.004‰),with statistically significant differences(P=0.000);(2)DGC+MACS negative/positive screening(CD45^(-)/CD71^(+)) method got the least total cells number(0.81×10^(6)/mL)and the least FNRBC number(7.47/mL),with statistically significant differences(P=0.000);(3)In the DGC+MACS single antibody screening method,the total number of cells(1.99×10^(6)/mL)obtained by DGC+MACS positive screening(CD71^(+)) was lower than that by DGC+MACS negative screening(CD45^(-))(2.56×10^(6)/m L).The difference was statistically significant(P=0.000).However,there was no significant difference in the number of FNRBC between the two methods(P=0.910);(4)There was no significant difference in the proportion of FNRBC between DGC+MACS negative screening(CD45^(-)),DGC+MACS positive screening(CD71^(+)) and DGC+MACS negative/positive screening(CD45^(-)/CD71^(+))(P=0.030,0.050,0.210).Conclusion:The number of FNRBC obtained by DGC is the largest.DGC+MACS negative/positive screening(CD45^(-)/CD71^(+))has the strongest ability to remove mixed cells.The effect of enriching FNRBC by DGC+MA

关 键 词：胎儿有核红细胞 密度梯度离心 免疫磁珠筛选 

分 类 号：R714[医药卫生—妇产科学]