SCANNING CYTOMETRY TO SORT FETAL NRBCS IN MATERNAL BLOOD

扫描细胞术对母血中的胎儿 NRBCS 进行分类

• 批准号: 6043638
• 负责人: JEFFREY H PRICE
• 金额: $ 25.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6043638
• 关键词: bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical automation; biomedical equipment development; clinical research; erythrocytes; female; flow cytometry; human pregnant subject; image processing; imaging /visualization /scanning; pregnancy circulation

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.

在母体循环中发现罕见的胎儿有核红细胞（fnRBCS）引发了利用外周静脉穿刺进行产前基因筛查的想法。开发这种获取胎儿细胞进行基因检测的微创方法的动机是降低成本和风险，并提高该程序的可用性。通过超声波、羊膜穿刺术和绒毛膜绒毛取样来检测遗传异常。尽管超声是非侵入性的，但许多遗传异常不会导致可检测到的形态学异常。因此，超声波与羊膜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。这些技术涉及将针插入子宫，会给胎儿带来风险，其中包括流产的可能性很小。简单的血液测试代表了一种低风险技术，具有广泛筛查的巨大潜力。利用母体外周血的挑战是定位估计的 1/10,000,000 个 fnRBC。迄今为止，流式细胞术和其他分选方法未能达到如此大规模的罕见事件细胞选择所需的准确性和通量。此外，目前提出的所有方法都对含有 nRBC 的血沉棕黄层应用富集步骤。这些富集步骤进一步减少了 fnRBC 的数量。富集过程中 fnRBC 损失的可接受水平尚不清楚，因为无法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），以：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。为进一步开发基于图像的原位分选，提出了两种扫描细胞术开发策略——一种速度较慢、技术上风险较低的方法和一种未经测试的高速连续扫描方法。所提出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。修订（以新字体）：主要的批评和修订是生物学的（仪器部分获得了极好的评价）。医学遗传学和 FISH 专家 Karen Arden 博士被任命为研究人员，负责领导将我们之前完善的 DAPI/抗 HbF-FITC 双染色与 X 和 Y 染色体双 FISH 染色相结合，以实现 4 色荧光技术。还将对 fnRBC 的分布与孕龄进行全面统计分析，但拟议的工作将使用在适当数量上具有高超率事件准确性的仪器，首次直接测量 fnRBC 的数量第一项研究的患者。