Semi-Automated Prenatal Screening Using Maternal Blood

描述（由申请人提供）： 科学家们已经记录了母体血液中胎儿细胞的现象，并设想将其用于非侵入性产前筛查。一个关键的限制因素是母体循环中胎儿细胞的数量很少，这使得胎儿细胞分离困难，并限制了遗传分析的准确性。目前的重点是简单，实用和可重复的方法富集和胎儿细胞的基因检测。 该项目的目标是开发简单的半自动化方法，用于富集，检测和诊断母体血液中的胎儿细胞。该项目结合了胎儿祖细胞研究的最新进展，一种新的富集方法，以及用于产前遗传分析的快速自动化细胞检测。 在I期研究中，我们评估了使用透射光显微镜自动检测May-Giemsa染色的有核红细胞（NRBC）的可行性，然后通过荧光原位杂交（FISH）进行胎儿性别和/或非整倍体检测。我们的研究结果表明，NRBC在妊娠期间存在于母体血流中，并且可以有效地富集到有利于自动化细胞检测的比例。我们还观察到，这些细胞可能不是理想的靶细胞，因为这些细胞中的大多数处于分化的晚期阶段并正在经历凋亡。此外，我们发现NRBC用于FISH分析的适用性是高度可变的，因为它与其分化状态有关。因此，我们评估了胎儿祖细胞作为产前筛查的替代细胞类型的潜力。为了分离和富集，我们采用了一种更简单的分离方法，该方法允许检测一种以上的胎儿祖细胞类型。我们采用玫瑰花结法（StemCell Technologies，Inc）来实现从全血中选择性富集造血祖细胞类型，同时减少细胞损失。然后通过FISH成功地处理富集的样品以鉴定母体血液中的雄性胎儿细胞。 在II期研究中，我们将（i）基于临床环境中FISH信号的存在评估胎儿细胞检测效率的自动化分析，以及（ii）重新定位目标1中识别的胎儿细胞，并基于细胞形状、密度和大小记录胎儿细胞描述符。然后，这些信息将被用来进一步开发自动化参数的形态学鉴定的祖细胞胎儿细胞，和（iii）的基础上的目标1和2的结果，我们将实施最终的修改和/或优化，以完全开发一个半自动化的成像系统，用于检测和分析母体血液中的胎儿细胞，使用透射和荧光显微镜。 目前医学细胞遗传学研究的最终目标是使低成本、低风险的产前遗传筛查广泛可用。该项目将开发对实现这一目标至关重要的仪器。这里提出的创新技术方法有可能彻底改变产前诊断的未来。

DESCRIPTION (provided by applicant): Scientists have documented the phenomenon of fetal ceils in maternal blood, and envisioned using them for noninvasive prenatal screening. A key limiting factor is the small number of fetal cells in the maternal circulation, making fetal cell isolation difficult and limiting the accuracy of genetic analysis. Current emphasis is on simple, practical and reproducible methods for enrichment and genetic testing of fetal cells. The goal of this project is to develop simple semi-automated methods for enrichment, detection, and diagnosis of fetal cells in maternal blood. This project combines recent advances in fetal progenitor cell research, with a novel enrichment approach, and speedy automated cell detection for prenatal genetic analysis. In the Phase I study we evaluated the feasibility of automatically detecting May-Giemsa stained nucleated red blood cells (NRBCs) using transmitted light microscopy, followed by fetal gender and/or aneuploidy detection via fluorescent in-situ hybridization (FISH). Our results demonstrated that NRBCs are present in the maternal blood stream during pregnancy, and can be effectively enriched to proportions that are conducive to automated cell detection. We also observed that these cells may not be the ideal target cell, because most of these cells are at late stages of differentiation and undergoing apoptosis. Moreover, we found that the suitability of NRBCs for FISH analysis was highly variable as it is related to its state of differentiation. Consequently, we evaluated the potential of fetal progenitor cells as an alternative cell type for prenatal screening. For separation and enrichment, we employed a more simple isolation method that would allow for detection of more that one fetal progentior cell type. We adopted the Rosette Method (StemCell Technologies, Inc) to achieve the selective enrichment of hematopoietic progenitor cell types from whole blood with reduced cell loss. The enriched samples were then successfully processed via FISH to identify male fetal cells in maternal blood. In the Phase II study, we will (i) evaluate automated analysis for detection efficiency of fetal cells based on the presence of FISH signals in the clinical environment, and (ii) relocate fetal cells identified in aim 1 and record fetal cell descriptors based on cellular shape, density, and size. This information will then be used to further develop automated parameters for morphological identification of progenitor fetal cells, and (iii) based on the results of aim 1 and 2, we will implement the final modifications and/or optimizations to fully develop a semi-automated imaging system for the detection and analysis of fetal cells in maternal blood using transmitted and fluorescence microscopy. The ultimate goal of much of the current research in medical cytogenetics is to make low-cost, low-risk prenatal genetic screening widely available. This project will develop instrumentation that will be vital in the realization of this goal. The innovative technological approach presented here has the potential to revolutionize the future of prenatal diagnosis.