Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. pearance. In a group of 20 primigravidas sampled on more than one occasion postpartum, quinacrine fluorescent signals ("Y body") were seen at a frequency of 0.01-0.1% of lymphocytes. These frequencies were noted to remain "practically unchanged" up until 1 year after delivery, when the study ended. In a related study, Ciaranfi et al. (5) (4) to include samples from women 5-7 years postpartum (5). In a series of 62 women who delivered a male infant, more than half had detectable male lymphocytes 2 years after birth. In some cases, metaphases with a Y chromosome were visualized 5 years postpartum. These studies, although intriguing, were performed in the 1970s using techniques less sensitive and less accurate than those available today.During this decade, investigators using PCR amplification of Y chromosome-specific sequences to identify fetal cells in maternal blood noted "false positive" results-i.e., detection of male DNA when the fetus was female (6, 7). Although laboratory contamination as a source of true false positives can be a concern (8), it appears that, in some cases, the male DNA was a real finding, possibly originating from a prior pregnancy (9). Other investigators studied postpartum maternal samples without using cell separation techniques to enrich for specific subpopulations of fetal cells and concluded that fetal cells did not persist postpartum (10).The study reported here occurred in two parts. Initially, we used a monoclonal antibody to cluster differentiation antigen (CD) 34 to isolate fetal hematopoietic stem cells from the blood of pregnant women. The surprising results, described below, suggested that the gender of the circulating CD34+ cells did not always correlate with the fetal gender of the current pregnancy. This led to the hypothesis that fetal CD34+ cells could persist from a prior pregnancy. We tested this hypothesis by fluorescence-activated cell sorting of four specific subpopulations of mononuclear cells from women who were not presently pregnant but who had previously given birth to a male infant. Although we were in...

Fetal cells were isolated from the peripheral blood of a pregnant woman at 19 weeks of gestation whose fetus had Down syndrome. An amniocentesis had been performed 2 weeks earlier because of abnormalities detected on an antenatal sonogram. Fetal cells were separated by fluorescence-activated cell sorting using monoclonal antibody to the transferrin receptor (TfR). Fluorescence in situ hybridization studies with probes for chromosomes Y and 21 revealed a small number of 47,XY,+21 cells in the TfR+ sorted fraction. Although preliminary, the results of this study suggest the possibility that one day, fetal chromosome aneuploidy will be routinely diagnosed from maternal venous blood samples.

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.

No abstract