SCANNING CYTOMETRY TO SORT FETAL NRBCS IN MATERNAL BLOOD

罕见的胎儿有核红细胞（fnRBCS）在母体循环中的发现，导致了使用外周静脉穿刺进行产前遗传筛查的想法。开发这种用于获得用于基因检测的胎儿细胞的微创方法的动机是降低成本和风险，并增加程序的可用性。通过超声波、子宫颈穿刺术和绒毛取样检测遗传异常。虽然超声波是非侵入性的，但许多遗传异常并不导致可检测的形态异常。因此，超声波与胎盘穿刺术或慢性绒毛取样（取决于胎龄）相结合，用于筛查高危妊娠。这些技术涉及将针头插入子宫，并对胎儿造成风险，包括流产的可能性很小。一个简单的血液测试将代表一个低风险的技术，具有巨大的潜力，用于广泛的筛查。利用外周母血的挑战是定位估计的1/10，000，000 fnRBC。到目前为止，流式细胞术和其他分选方法未能达到这种罕见事件细胞选择所需的准确性和通量。此外，所有目前提出的方法都将富集步骤应用于含nRBC的血沉棕黄层。这些富集步骤进一步减少fnRBC的数量。富集中fnRBC损失的可接受水平未知，因为不可能直接测量母体外周血中的数量（和变化）。我们建议进一步开发基于图像的细胞分析技术。我们建议进一步开发基于图像的细胞分析技术（扫描细胞术）：1）直接测量母体血沉棕黄层中胎儿fnRBC的数量，2）评估该技术作为产前遗传筛查的常规细胞选择方法。两种策略扫描细胞计数的发展-一个较慢的，技术上的低风险的方法和一个更未经测试的高速连续扫描方法，提出了进一步发展的图像为基础的原位分选。拟议的用于大规模罕见事件检测的高性能扫描细胞术具有极其广泛的临床和研究用途的潜力。翻修（新字体）：主要批评和翻修是生物学方面的（工具部分获得了极好的评价）。医学遗传学和FISH专家Karen Arden博士被添加为研究者，领导我们先前完善的DAPI/抗HbF-FITC双重染色与双重X和Y染色体FISH染色相结合，用于4色荧光技术。还将对fnRBC与孕龄的分布进行全面统计分析，但是，拟定的工作将使用仪器首次直接测量fnRBC数量，该仪器在第一项研究的适当数量的患者中具有较高的超速率事件准确度。

The finding of rare fetal nucleated red blood cells (fnRBCS) in the maternal circulation has led to the idea of developing prenatal genetic screening using peripheral venipuncture. The motivations for developing this a minimally invasive method for obtaining fetal cells for genetic testing are to reduce cost and risk, and increase the availability of the procedure. Genetic anomalies are to be detected by ultrasound, amniocentesis and chorionic villus sampling. Although ultrasound is non- invasive, many genetic abnormalities do not result in detectable morphological abnormalities. Thus, ultrasound is combined with amniocentesis or chronic villus sampling (depending on gestational age) for screening at-risk pregnancies. These techniques involve inserting a needle into the uterus and entail risks to the fetus that include a small chance of miscarriage. A simple blood test would represent a low-risk technique with tremendous potential for use in widespread screening. The challenge in utilizing peripheral maternal blood is locating the estimated 1/10,000,000 fnRBCs. Thus far, flow cytometry and other sorting methods have failed to achieve the accuracy and throughput necessary for this magnitude of rare event cell selection. In addition, all currently proposed methods apply enrichment steps to the nRBC- containing buffy coat. These enrichment steps further reduce the number of fnRBCs. The acceptable level of fnRBC loss in enrichment is unknown because the direct measurements of the numbers (and variations) in maternal peripheral blood have not been possible. We propose to further develop image-based cell analysis technology. We propose to further develop image-based cell analysis technology (scanning cytometry) to: 1) measure directly the number of fetal fnRBCs residing in the maternal buffy coat, and 2) evaluate the technology as a routine cell selection method for prenatal genetic screening. Two strategies for scanning cytometry development- a slower, technologically low-risk method and a more untested high-speed continuous-scanning method-are proposed for further development of image-based in situ sorting. The proposed high-performance scanning cytometry for large-scale rare event detection has potential for extremely broad clinical and research use. Revisions (in new font): The primary critiques and revisions are biological (the instrumentation portion received excellent reviews). Dr. Karen Arden, an medical genetics and FISH expert, was added as an investigator to lead the combining of our previously perfected DAPI/anti- HbF-FITC dual stain with a dual X and Y chromosome FISH stain for a 4-color fluorescence technique. Full statistical analysis of the distribution of fnRBCs vs. gestation age will also be performed, but a proposed work will, however, provide the first direct measurement of the numbers of fnRBCs using an instrument with demonstrated high ultra-rate event accuracy on an appropriate number of patients for this first study.