Martel-Petit V, Petit C, Marchand M, Fleurentin A, Fontaine B, Miton A, Lemarie P, Philippe C, Jonveaux P
Laboratoire de Génétique Médicale, CHU Nancy, France.
Prenat Diagn. 2001 Feb;21(2):106-11. doi: 10.1002/1097-0223(200102)21:2<106::aid-pd983>3.0.co;2-#.
Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.
母体循环中存在的胎儿有核红细胞非整倍体的无创产前诊断受到细胞数量极少且来源不确定的阻碍（孕期循环中的成红细胞70%源自母体）。因此，在成红细胞富集步骤之后，一种能够在母体细胞中筛选出胎儿细胞的方法必不可少。在本研究中，先通过菲可梯度法进行成红细胞富集，然后用抗CD71或抗GPA抗体进行阳性免疫磁选，在此之后，开发了一种从经典的克勒豪尔试验改编而来的快速、简单且直接的化学染色方法来筛选胎儿细胞。胎儿和成红细胞之间的精确区分基于胎儿血红蛋白（Hb）和成人血红蛋白在构成上的差异。胎儿细胞呈现出强烈的粉红色细胞质染色，而含有成人血红蛋白的母体细胞则无色。细胞质完整性的保留使得能够区分形态特征，并通过荧光原位杂交（FISH）同时可视化核杂交信号。使用双色X和Y特异性DNA探针，通过FISH分析对15名孕妇的血样进行了检测，并将结果与细胞遗传学或超声性别测定结果进行了比较。对于12例妊娠成功确定了胎儿性别（5例XY/7例XX），2例原位杂交失败，1例在克勒豪尔试验后未观察到胎儿成红细胞。该筛选方法应用于一名有囊性纤维化（CF）风险的孕妇。经过克勒豪尔试验后，通过显微切割收集胎儿成红细胞，通过引物延伸预扩增（PEP）扩增全基因组DNA，随后进行巢式CF PCR。胎儿基因型通过传统产前诊断成功鉴定并得到证实。
