Vona Giovanna, Béroud Christophe, Benachi Alexandra, Quenette Alice, Bonnefont Jean Paul, Romana Serge, Dumez Yves, Lacour Bernard, Paterlini-Bréchot Patrizia
Unité INSERM 370, Faculté de Médecine Necker, Paris, France.
Am J Pathol. 2002 Jan;160(1):51-8. doi: 10.1016/S0002-9440(10)64348-9.
Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.
孕妇外周血中循环的胎儿细胞是无创基因分析的潜在靶点。它们包括上皮（滋养层）细胞，这些细胞比外周血白细胞大。我们使用上皮肿瘤细胞大小分离法（ISET）富集循环滋养层细胞。在妊娠11至12周时采集外周血。通过ISET分离的细胞用苏木精和伊红染色或进行免疫组织化学染色。对大的上皮细胞进行显微切割，并通过短串联重复序列和/或Y特异性引物的聚合酶链反应进行胎儿细胞鉴定。通过仅分析2毫升血液，我们在所有6名怀有男性胎儿的母亲中发现了数量不等（n = 1至7）的Y阳性细胞（总共23个中有15个）。相比之下，从7名怀有女性胎儿的母亲中分离出的26个细胞均未检测为阳性。使用短串联重复序列特异性标记对11个细胞进行了分析：其中6个显示出胎儿图谱，5个显示出母亲图谱，与Y特异性结果一致。仅将单细胞DNA的五分之一用于胎儿细胞评估，剩下足够的材料用于进一步的基因检测。我们还表明，ISET方法可用于荧光原位杂交分析以及检测单个显微切割细胞中的DNA点突变。我们得出结论，这是一种富集循环胎儿细胞并证明其胎儿来源的有效方法，并且可能对遗传性疾病的无创产前诊断具有重要意义。
