Center for Reproductive Medicine, Anhui Medical University Affiliated Provincial Hospital, Hefei, PR China.
Fetal Diagn Ther. 2012;32(3):194-200. doi: 10.1159/000338124. Epub 2012 Jun 1.
We tested the feasibility of the in vitro culture of fetal nucleated erythroblasts from maternal blood for noninvasive prenatal screening as a substitute for culturing fetal nucleated erythroblasts from fetal villi.
Nucleated blood cells separated via Percoll from 52 samples of fetal villi and maternal peripheral blood were cultured with or without magnetic-activated cell sorting glycophorin A (MACS-GPA+), and detected by an anti-hemoglobin-epsilon (FITC) antibody. Gender of the epsilon-positive cells were identified by FISH and further confirmed by PCR of the villi karyotype. Developmental stages of nucleated erythroblasts from villi and blood with MACS-GPA+ were analyzed by Wright-Giemsa staining.
In the maternal blood, epsilon-positive cells were found in 4 and 24 cultured samples with and without MACS-GPA+, respectively. Also, Y-signals were visualized in 3 out of 4 and in 15 out of 24 cases in the epsilon-positive cells. Although epsilon-positive cells were found in all villus samples irrespective of MACS-GPA+ sorting, Y-signals were visualized in 31 out of 52 cases. Proerythroblasts and basophilic erythroblasts occupied 7 and 1% in fetal and maternal samples (with MACS-GPA+), respectively.
The in vitro culturing of fetal nucleated erythroblasts from maternal blood is not feasible with the current techniques for prenatal diagnosis, because the fetal nucleated erythroblast is not well developed in vitro. This may be attributed to the low proportion of these erythroblasts at an early stage in the fetal circulation and the low permeability of these cells to the maternal blood.
我们测试了从母亲外周血中体外培养胎儿有核红细胞进行无创性产前筛查的可行性，以替代从胎儿绒毛中培养胎儿有核红细胞。
通过 Percoll 从 52 例胎儿绒毛和母亲外周血样本中分离有核血细胞，分别进行和不进行磁性激活细胞分选糖蛋白 A（MACS-GPA+）培养，并通过抗血红蛋白-ε（FITC）抗体进行检测。通过荧光原位杂交（FISH）鉴定ε阳性细胞的性别，并通过对绒毛核型的 PCR 进一步确认。通过 Wright-Giemsa 染色分析 MACS-GPA+有核红细胞的发育阶段。
在母亲外周血中，分别有 4 例和 24 例培养样本中存在 MACS-GPA+和无 MACS-GPA+的ε阳性细胞。在这 4 例中的 3 例和 24 例中的 15 例ε阳性细胞中观察到 Y 信号。尽管所有绒毛样本均存在ε阳性细胞，无论是否进行 MACS-GPA+分选，但在 52 例中有 31 例观察到 Y 信号。原红细胞和嗜碱性红细胞分别占胎儿和母亲样本（带 MACS-GPA+）的 7%和 1%。
目前的产前诊断技术不能实现从母亲外周血中体外培养胎儿有核红细胞，因为胎儿有核红细胞在体外发育不良。这可能归因于这些红细胞在胎儿循环早期的比例较低，以及这些细胞对母体血液的通透性较低。
