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

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（0.95×10⁷/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（0.81×10⁶/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）；③DGC+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1.99×10⁶/mL，较DGC+MACS阴性筛选（CD45^-）（2.56×10⁶/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）；④DGC+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。

Abstract:

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 4 groups：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 ①Compared with the other three groups，DGC method had the largest total cell number（0.95×10⁷/mL），the largest FNRBC number（39.87/mL） and the lowest proportion of FNRBC（0.004‰），with statistically significant differences（P=0.000）；②DGC+MACS negative / positive screening（CD45^-/CD71⁺） method got the least total cells number（0.81×10⁶/mL）and the least FNRBC number（7.47/mL），with statistically significant differences（P=0.000）；③In the DGC+MACS single antibody screening method，the total number of cells（1.99×10⁶ /mL）obtained by DGC+MACS positive screening（CD71⁺） was lower than that by DGC+MACS negative screening（CD45^-）（2.56×10⁶/mL）. 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）；④There 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+MACS positive screening（CD71⁺） is the best without reducing the number of target cells.