利用胎儿血红蛋白表型分选、检测母血中胎儿有核红细胞

Sorting and detection of fetal nucleated erythrocytes in maternal blood using fetal specific haemoglobin.

目的:建立高纯度、高得率、高特异性的分选技术,富集纯化母血中胎儿有核红细胞(FNRBC).方法:70例孕妇外周血,经密度梯度离心初步富集后行血红蛋白γ链免疫组化染色,以显微操作术挑取阳性细胞.采用单细胞PEP-PCR技术检测SRY基因,并进行荧光原位杂交分析.结果:胎儿NRBC具有鲜明的形态学、免疫学特征.每例母血FNRBC数5～27个,平均0.82±0.37/ml.PEP-PCR检测的灵敏度、特异度分别为95.35%、100%.4例FISH结果均与实际核型相符.结论:以γ-血红蛋白为分选标志,结合显微操作技术,可以有效地获取高纯度、高浓度研究模板,保证后继分析的准确性,为无创性产前诊断发展奠定基础.

Objective：To establish an effective sorting method for fetal nucleated red blood cells （FNRBC） from maternal peripheral blood to obtain highly concentrated and purified target cells. Methods ：70 cases of pregnant women were taken as the study group. After preliminary enrichment using density gradient centrifugation. FNRBCs were identified by immunohistochemistry stain with anti-γ-globin monoclonal antibody and collected by micromanipulation. Primer extension preamplification （PEP） and ploymerase chain reaction （PCR） based on single cell were adopted to detect SRY gene. Fluorescence in situ hybridization was also applied. Results：Fetal NRBCs show unique morphological characteristics and haemoglobin phenotype. The number of FNRBCs in each case was 5 - 27, with a mean of 0.82 ± 0.37/ml. The sensitivity and specificity of PEP-PCR was 95.35% and 100% respectively. In 4 cases receiving FISH, the results were all in coincidence with actual karyotype. Conclusion：The sorting method associating γ-haemoglobin immunohistochemistry stain and micromanipulation can guarantee the quantity and quality of research template, which make accurate genetic analysis using FNRBC in maternal blood more possible.