Rapid Heart Attack Detection using an AC Electrokinetic Device

描述（由申请人提供）：尽管最近的诊断和治疗取得了进展，但急性心肌梗死（MI）和冠状动脉疾病（CAD）仍然是全球死亡和残疾的主要原因。此外，鉴于全球预期寿命增加、人口增长以及肥胖和糖尿病流行率迅速上升，预计这一趋势将急剧恶化。目前检测CAD的方法很容易通过常规负荷测试和冠状动脉造影术进行诊断，然而，尽管存在众所周知的风险因素，但MI等灾难仍然高度不可预测。循环内皮细胞（CEC）和内皮衍生微粒（CMP）作为急性心肌梗死的生物标志物具有相当大的前景。已经证明，CEC在处于急性心肌梗死高风险或已经患有急性心肌梗死的个体中以显著水平存在于血液中，但在患有稳定CAD的个体中没有。还据报道，CFC-DNA水平升高是急性MI的强预后标志。目前用于分离CFC-DNA和CMP的方法繁琐、破坏性和不可靠。健康患者的游离DNA量为12.5 - 60.1 ng/mL，MI患者为113 - 909 ng/mL。用于鉴定EMP的标记物和抗体染色剂包括CD 144、血管性血友病因子（vWF）和一种凝集素染色剂--荆豆凝集素（UEA）。一旦FDA批准，我们的原型设备将以合理的成本提供POC即时预后信息。该项目将专注于使用专有原型ACE设备从MI患者血液中分离EMP和CFC-DNA的临床前方面，并进行分析以揭示进一步的知识和理解。现在的目标是开发一种强大的即时MI诊断系统，该系统将直接从全血中分离CFC-HMW DNA和EMP，然后将其纯化，用于使用凝胶电泳、qRT-PCR和荧光分析进行进一步分析。该I期应用的具体目标是：1 -使用原型ACE装置来表征荧光珠和裂解血液的性能。2 -使用原型ACE装置来表征MI血浆的性能并与金标准（QIAamp试剂盒用于CFC-DNA，FACS用于EMP）进行比较。3 -使用原型ACE装置从10名对照和10名MI患者中分离EMP和CFC-DNA，并与Scripps Health为相同患者确定的CEC数据进行比较。SBIR第一阶段赠款将允许建造该设备，这将进一步实现快速生产和商业化。未来的第二阶段建议将根据需要修改原型，并将构建一个分析系统，将新鲜全血与先前冷冻的血浆相关联，并整合PCR扩增，通过荧光和基因分型识别内皮标志物 公共卫生相关性：该项目旨在开发一种即时心脏病发作诊断系统，该系统将为所有美国人提供快速，具有成本效益的筛查。该系统通过使用原型AC电动装置快速分离血液中的无细胞循环DNA和内皮微粒来实现这一点，这是一种具有越来越多共识的早期心脏病发作生物标志物。该设备和生物标志物的进一步开发将创造一种新的即时心脏病发作诊断设备，这将为所有美国人带来更好的医疗保健质量，并通过早期检测和后续治疗降低与心脏病发作相关的成本。

DESCRIPTION (provided by applicant): Acute myocardial infarction (MI) and coronary artery disease (CAD) continue to be the leading cause of death and disability in the world despite recent diagnostic and treatment advances. Moreover, this trend is expected to dramatically worsen given global life expectancy increases, population growth, and the rapidly escalating prevalence of obesity and diabetes. Current methods of detection for CAD is easily diagnosed through routine stress testing and coronary angiography, however catastrophes such as MI remain highly unpredictable despite well known risk factors. Circulating endothelial cells (CECs) and endothelial derived microparticles (CMPs) have considerable promise as a biomarker for acute Myocardial Infarction. It has demonstrated that CECs were found in the blood at significant levels in individuals at high risk for or those who already had an acute myocardial infarction, but not in individuals with stable CAD. It has also been reported that the elevated levels of CFC-DNA is a strong prognostic marker in acute MI. Current methods for isolated CFC-DNA and CMPs are cumbersome, destructive and unreliable. The amount of cell-free DNA in healthy patients varies from 12.5 to 60.1 ng/mL and in MI patients from 113 to 909 ng/mL. Markers and antibody stains for identifying EMPs include CD144, von Willebrand Factor (vWF) and Ulex Europaeus Agluttin (UEA), a lectin stain. Our prototype device will enable POC immediate prognostic information at a reasonable cost once the FDA approves it. This project will focus on the pre-clinical aspect of using a proprietary prototype ACE device to isolate EMPs and CFC-DNA from MI patient blood and analyzing to reveal further knowledge and understanding. The goal is to now develop a robust point-of-care MI diagnostic system, which will separate CFC-HMW DNA and EMPs directly from whole blood and then elute it for further analysis using gel electrophoresis, qRT-PCR and fluorescence analysis The specific aims for this Phase I application are: 1 - Use prototype ACE device to characterize performance for fluorescent beads and lysed blood. 2 - Use prototype ACE device to characterize performance for MI plasma and compare against gold standards (QIAamp kit for CFC-DNA, FACS for EMPs). 3 - Use prototype ACE device to isolate EMPs and CFC-DNA from 10 controls and 10 MI patients and compare to CEC data identified for the same patients by Scripps Health. The SBIR Phase I grant will allow the construction of this device, which will further enable rapid translation for manufacturing and commercialization. A future Phase II proposal will modify the prototype as needed, and will construct an Analytical System, correlate fresh whole blood vs. previously frozen plasma, and integrate PCR amplification for identification of endothelial markers through fluorescence and genotyping PUBLIC HEALTH RELEVANCE: This project is directed at the development of a point-of-care heart attack diagnostic system that will allow rapid, cost effective screening for all Americans. The system does this by rapidly isolating cell free circulating DNA and endothelial microparticles in blood, an early heart attack biomarker that has a growing consensus, using a prototype AC Electrokinetic device. Further development of this device and biomarker will create a new point-of-care heart attack diagnostic device that will lead to better quality healthcare for all Americans as well as lowering costs associated with heart attacks through early detection and subsequent treatment.