FETAL ERYTHROID CELLS IN MATERNAL CIRCULATION

分子遗传学的最新进展使考虑 利用少量胎儿红系对胎儿进行基因检测 通过胎盘迁移到母体循环的细胞 在怀孕期间。这样的孕妇血液检测最终可能会 补充，甚至可能取代像这样的侵入性程序 羊膜穿刺术和绒毛取样。这对美国的 这些分子技术的发展，以首次明确识别 区分胎儿细胞和母体细胞，然后确定 胎儿红系细胞发生的孕期变异性， 包括有核红细胞，在母体循环中。我们有 发展了一种明确而直接的流式细胞术方法 从母体中鉴定、量化和分离胎儿细胞 外周血样。我们的技术利用了这样一个事实 细胞表面红系标记物血糖素A也是 多态MN血型的遗传决定因素，并利用 针对M型和N型的等位基因特异性的单抗 血糖素A蛋白。通过用这两种抗体进行双重标记 使用双色流式细胞术，我们可以毫不含糊地区分胎儿 母体血液中细胞的比例为50% 和孩子在这个基因座上是不一致的。在试点研究中 我们已经确定有可检测的胎儿红系细胞 绒毛取样前的母体循环(9-12周 怀孕)和在足月简单的分娩之后，有 胎儿细胞频率在这段时间内的增加。我们建议申请 这些技术适用于100对信息丰富的母亲/胎儿 在正常送货后确认，样品存放在整个仓库中 怀孕了。然后将这些技术应用于事件 母亲/胎儿配对有各种不良后果，包括前 子痫。这项研究也将为开发和 胎儿细胞扩增和直接分选技术的应用 碘化丙啶阳性胎儿细胞(网织红细胞和有核细胞) 红细胞)。可能的胎儿细胞将作为一个群体被捕获 在试管中，或铺设在幻灯片栅格上，用于直接显微镜 有核细胞的分析。SORTED的胎儿派生 网织红细胞和有核细胞将使用等位基因特异性进行验证 通过聚合酶链式反应和/或FISH分析血糖蛋白A基因的表达 Y染色体在男性胚胎中存在的Y染色体

Recent advances in molecular genetics make it feasible to consider genetic testing of the fetus via the small numbers of fetal erythroid cells that migrate across the placenta into the maternal circulation during pregnancy. Such a maternal blood test could ultimately complement, or perhaps even replace such invasive procedures as amniocentesis and chorionic villus sampling. It is critical to the development of these molecular techniques to first definitively identify and distinguish fetal cells from maternal cells, and second determine the gestational variability in the occurrence of fetal erythroid cells, including nucleated erythroblasts, in the maternal circulation. We have developed a flow cytometric method of unambiguously and directly identifying, quantifying and isolating fetal cells from maternal peripheral blood samples. Our technique takes advantage of the fact that the cell surface erythroid lineage marker glycophorin A is also the genetic determinant of the polymorphic MN blood group, and utilizes allele-specific monoclonal antibodies to the M and N forms of the glycophorin A protein. By double labeling with these two antibodies and using two-color flow cytometry, we can unambiguously distinguish fetal cells in maternal blood in those 50 percent of cases where the mother and child are genetically discordant at this locus. In pilot studies we have determined that there are detectable fetal erythroid cells in the maternal circulation prior to chorionic villus sampling (9-12 weeks gestation) and after full-term uncomplicated birth, and that there is an increase in fetal cell frequency over this time. We propose to apply these techniques to a population of 100 informative maternal/fetal pairs identified following normal delivery with samples banked throughout gestation. These techniques will then be applied to incident maternal/fetal pairs with a variety of adverse outcomes, including pre- eclampsia. This study will also provide samples for the development and application of techniques for fetal cell enrichment and direct sorting of propidium iodide-positive fetal cells (reticulocytes and nucleated erythroblasts). Putative fetal cells will be captured as a population in a test tube, or laid out on a slide grid for direct microscopic analysis for nucleated cells. The fetal derivation of sorted reticulocytes and nucleated cells will be verified using allele-specific PCR for glycophorin A mRNA and by PCR and/or FISH analysis for the presence of a Y chromosome in male conceptuses.