应用磁珠细胞分离仪从母血中分离胎儿细胞的研究

Using magnetically activated cell sorting to isolate fetal cells from maternal blood

目的 研究利用免疫磁珠细胞分离仪分离孕妇血中胎儿细胞的效率。方法 孕妇血采自孕龄为 2 1～40周 ,初孕的孕男性单胎正常孕妇 16例。脐血来自胎龄 38～ 40周的男性新生儿 10例。标本根据CD71抗体的效价标记磁珠后放在磁场中进行磁珠细胞分离。分离后的脐血标本在流式细胞仪上检测分离效率 ,孕妇血标本经套式PCR方法鉴定男性SRY基因片段。根据CD71-FITC抗体效价标记脐血中的单个核细胞。同时利用流式细胞仪检测和分离脐血中的胎儿细胞。结果 利用磁珠细胞分离仪分离孕妇血中的胎儿有核红细胞 ,经套式PCR技术寻找男性胎儿SRY基因片段。经第一次扩增有效率 87 5 % ,第二次扩增有效率 10 0 0 %。脐血中胎儿有核红细胞在分离前占 5 3 2 %～ 74 9% ;分离后 ,流式细胞仪富集为 99 2 %～ 99 8% ,免疫磁珠细胞分离仪富集为 97 3%～ 99.4%。结论 免疫磁珠细胞分离仪可富集到 98 3%的胎儿有核红细胞 ,经套式PCR技术在分离后的孕妇血中可确定 10 0 %的男性胎儿SRY基因片段。该项技术简单 ,价廉 。

Objective To study the efficiency of using magnetically activated cell sorting (MACS) to isolate fetal cells in maternal circulation.Methods 20mL anticoagulant maternal blood in 16 cases were gained. These normal primigravidas with male fetus were in 21～40 pregnant weeks. 5ml anticoagulant cord blood in 10 cases of male neonates (after 38～40 pregnant weeks) were gained.①MACS isolation protocol:We used antibody-CD71 to mark mononuclear cells from the maternal blood and the cord blood respectively to get fetal erythrocytoblasts according to the antibody's efficiency.After indirectly labeling magnetic beads,these samples from maternal blood and cord blood were laid in the magnetic field for the isolation of the fetal cells respectively. The isolated cells from the cord blood were analyzed in Flow Cytometry to detect the efficiency of cell separation with MACS.At the same time,we used nest-PCR technique to look for SRY gene sequences in the sorted cells from the maternal blood to detect the male fetal sexes.②Flow Cytometry isolation protocol:We used CD71-FITC to label the mononuclear cells from the cord blood . In Flow Cytometry the samples were analyzed and sorted to get fetal erythrocytoblasts.Results ① Before sorting the cells,there were NRBCs about 53.2%～74.9% in the cord blood.After using MACS and Flow Cytometry to sort the fetal cells respectively,the isolation rates of the fetal cells were 97.3%～99.4% and 99.2%～99.8% respectively in these two methods.②After using MACS to isolate the fetal cells from the maternal blood,we used nest-PCR to look for SRY gene sequences.The efficiency was 87.5% when using the first couple primer to proliferate DNA.The efficiency was 100% when using the second couple primer to proliferate DNA again.Conclusion The MACS technique is a simple and inexpensive method.It has a high potency for sorting fetal cells from maternal blood.In the future the approach for noninvasive prenatal diagnosis will be used generally in clinic.