de Graaf I M, Jakobs M E, Leschot N J, Ravkin I, Goldbard S, Hoovers J M
Department of Clinical Genetics, Academic Medical Centre, Amsterdam, The Netherlands.
Prenat Diagn. 1999 Jul;19(7):648-52. doi: 10.1002/(sici)1097-0223(199907)19:7<648::aid-pd600>3.0.co;2-x.
In this study we evaluated the performance of a system for the enrichment, identification and analysis of fetal cells in maternal peripheral blood. Blood samples were collected from women after chorionic villus sampling and enriched for the presence of nucleated erythrocytes using a three-step procedure, namely: (a) centrifugation to separate nucleated red blood cells (NRBCs) from the majority of red blood cells (RBCs) and white blood cells (WBCs); (b) selective lysis of the remaining maternal RBCs; (c) separating the NRBCs from the remaining WBCs in a three-layer density gradient. Fetal cells were identified by using a monoclonal antibody against the gamma-chain of fetal haemoglobin (anti-HbF) and a nuclear stain (DAPI). Additionally, to further increase the specificity of the identification, and to eliminate some of the undesired staining by maternal leukocytes, a fluorescent antibody (CD45) was added. The sex chromosome complement of the cells was determined by fluorescence in situ hybridization (FISH) with X and Y-specific probes and the results were compared with the karyotypes obtained after analysis of chorionic villi. Using the described method, in all cases where the woman was carrying a male fetus (n=18) at least one XY cell was found, while no male cells were found in women carrying a female fetus. However, in the majority of cases with a male fetus (n=11) female HbF positive cells were found indicating the presence of maternal nucleated erythrocytes. The study demonstrates that the combination of anti-HbF and CD45 is a useful, but not fully specific, marker for fetal NRBCs and that additional markers are needed.
在本研究中，我们评估了一个用于富集、鉴定和分析母体外周血中胎儿细胞的系统的性能。在绒毛取样后从女性身上采集血样，并使用三步程序富集有核红细胞，具体步骤如下：(a) 离心以将有核红细胞（NRBCs）与大多数红细胞（RBCs）和白细胞（WBCs）分离；(b) 选择性裂解剩余的母体RBCs；(c) 在三层密度梯度中从剩余的WBCs中分离出NRBCs。通过使用针对胎儿血红蛋白γ链的单克隆抗体（抗-HbF）和核染色剂（DAPI）来鉴定胎儿细胞。此外，为了进一步提高鉴定的特异性，并消除母体白细胞的一些不必要染色，添加了一种荧光抗体（CD45）。通过使用X和Y特异性探针的荧光原位杂交（FISH）确定细胞的性染色体组成，并将结果与绒毛分析后获得的核型进行比较。使用所述方法，在所有怀有男性胎儿的病例（n = 18）中，均发现至少一个XY细胞，而在怀有女性胎儿的女性中未发现男性细胞。然而，在大多数怀有男性胎儿的病例（n = 11）中，发现了女性HbF阳性细胞，表明存在母体有核红细胞。该研究表明，抗-HbF和CD45的组合是胎儿NRBCs的一种有用但并非完全特异性的标志物，还需要其他标志物。
