Hennerbichler Simone, Kroisel Peter M, Zierler Hannelore, Pertl Barbara, Wintersteiger Reinhold, Dohr Gottfried, Sedlmayr Peter
Institute of Histology and Embryology, Karl-Franzens-University Graz, Austria.
Prenat Diagn. 2003 Sep;23(9):710-5. doi: 10.1002/pd.668.
The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry.
CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis.
In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC.
On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.
本研究旨在评估通过激光扫描细胞术分析母血中胎儿有核红细胞（NRBC）的可行性。
对孕妇外周血进行密度离心后，通过磁性细胞分选获得CD71阳性细胞。将血红蛋白γ链的免疫荧光与DNA荧光染色（TO-PRO-3）以及用Y染色体特异性探针进行荧光原位杂交（FISH）相结合。使用激光扫描细胞仪（LSC）在载玻片上对细胞进行扫描。对血红蛋白γ和TO-PRO-3双阳性的事件进行重新定位，并对其形态和FISH反应性进行视觉评估。将LSC检测男性胎儿性别的结果与羊膜穿刺术的结果进行比较。
在15例怀有男性胎儿的妊娠中有8例，在9例怀有女性胎儿的妊娠中有0例（除1例男性/女性双胎妊娠外）检测到Y染色体阳性的NRBC。在怀有女性胎儿的妊娠中，所有曾生育过男性婴儿的女性均发现了非NRBC的Y染色体阳性细胞，而既往无流产史且无男性婴儿的女性未出现Y染色体阳性的非NRBC。
基于对当前妊娠中曾经定义的胎儿来源细胞的自动重新定位，激光扫描细胞术可能有助于重复（多）FISH分析和单细胞PCR用于非侵入性产前诊断。
