Semi-Automated Prenatal Screening Using Maternal Blood

描述(由申请人提供)： 科学家已经记录了母体血液中的胎儿细胞现象，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的胎儿细胞数量很少，这使得分离胎儿细胞变得困难，并限制了基因分析的准确性。目前的重点是简单、实用和可重复的胎儿细胞培养和基因测试方法。 该项目的目标是开发简单的半自动方法来浓缩、检测和诊断母血中的胎儿细胞。该项目结合了胎儿祖细胞研究的最新进展，以及一种新的浓缩方法，以及用于产前基因分析的快速自动细胞检测。 在第一阶段的研究中，我们评估了使用透射光学显微镜自动检测May-Giemsa染色的有核红细胞(NRBCs)，然后通过荧光原位杂交(FISH)检测胎儿性别和/或非整倍体的可行性。我们的结果表明，在怀孕期间，NRBCs存在于母亲的血液中，并可以有效地浓缩到有利于自动细胞检测的比例。我们还观察到，这些细胞可能不是理想的靶细胞，因为这些细胞大多处于分化的后期，正在经历凋亡。此外，我们发现NRBCs在FISH分析中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替代细胞类型的潜力。对于分离和浓缩，我们采用了一种更简单的分离方法，可以检测到不止一种胎儿孕激素细胞类型。我们采用了Rosette方法(Stemcell Technologies，Inc.)，实现了从全血中选择性地浓缩造血祖细胞类型，减少了细胞损失。然后，通过FISH成功地对浓缩的样本进行处理，以识别母亲血液中的男性胎儿细胞。 在第二阶段研究中，我们将(I)根据临床环境中FISH信号的存在来评估自动分析对胎儿细胞的检测效率，以及(Ii)重新定位Aim 1中确定的胎儿细胞，并根据细胞形状、密度和大小记录胎儿细胞描述符。这些信息将被用来进一步开发用于祖先胎儿细胞形态鉴定的自动化参数，以及(Iii)根据目标1和目标2的结果，我们将实施最终修改和/或优化，以完全开发一种半自动成像系统，用于使用透射式和荧光显微镜检测和分析母体血液中的胎儿细胞。 目前许多医学细胞遗传学研究的最终目标是使低成本、低风险的产前基因筛查得到广泛应用。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.