High Throughput Radiation Biodosimetry

高通量辐射生物剂量测定

• 批准号: 7326167
• 负责人: STEPHEN D DERTINGER
• 金额: $ 12.61万
• 依托单位: LITRON LABORATORIES, LTD.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7326167
• 关键词: Accidents; Address; Affect; Apoptotic; Area; Biological; Biological Assay; Bleomycin; Blood; Blood specimen; Cell Fraction; Cells; Chromatin; Chromosomes; Clinical; Clinical Management; Clinical assessments; Communities; DNA Double Strand Break; Data; Development; Dicentric chromosome; Disasters; Dose; Dyes; End Point; Event; Exhibits; Flow Cytometry; Frequencies; Future; Goals; Gold; Health; Health Personnel; Hospitals; Human; Human Resources; Individual; Investigation; Ionizing radiation; Laboratories; Laboratory Study; Lymphocyte; Mammalian Cell; Managed Care; Measurement; Measures; Medical; Methodology; Methods; Microscopic; Microscopy; Modeling; Monitor; Mus; Necrosis; Nuclear; Numbers; Patient Care; Patients; Peripheral Blood Lymphocyte; Phase; Phase II Clinical Trials; Population; Positioning Attribute; Procedures; Process; Purpose; Radiation; Radiation therapy; Rate; Readiness; Reliance; Reporting; Research; Resource Allocation; Risk Assessment; Scheme; Scientist; Score; Security; Shipping; Ships; Site; Staging; Staining method; Stains; Standards of Weights and Measures; Stream; System; Techniques; Testing; Training; Triage; United States Food and Drug Administration; United States National Institutes of Health; Validation; Visual; Whole Blood; Work; base; biodosimetry; commercialization; data acquisition; design; dosimetry; instrumentation; lymphoblastoid cell line; micronucleus; portability; research study; tool

DESCRIPTION (provided by applicant): In instances of accidental radiation exposure or radiological terror events, incomplete or missing physical dosimetry will be the norm. Biodosimetry is therefore needed to make rational clinical management decisions, and for long-term carcinogenic risk assessments. The "gold-standard" biodosimetry techniques measure chromosome damage in circulating lymphocytes (dicentrics or micronuclei), and have proven valuable when the number of victims is small. Unfortunately, these assays are too cumbersome to monitor large populations. The goal of this project is to supply the Nation with a high throughput radiation biodosimetry tool that overcomes this limitation. The assay that will be optimized and validated is a flow cytometry-based method that provides rapid measurements of chromosomal damage. The endpoint that we will utilize as a chromosome damage indicator is micronucleus formation in circulating lymphocytes. The research proposed herein is an extension of recent advances made by Litron scientists regarding the scoring of micronuclei in cultures of immortalized mammalian cells. To date, this proof-of-principle work has focused on the mouse lymphoblastoid cell line L5178Y, and has demonstrated the importance of differentially staining MN from the chromatin of apoptotic and necrotic cells. Results have lent strong support to our premise that accurate flow cytometric enumeration of mammalian cell MN may be achieved with a two dye, sequential staining procedure. Even so, further work with this scoring system is needed to investigate its compatibility with primary lymphocytes, and to develop methods that overcome likely obstacles to the analysis of primary lymphocytes for the purpose of radiation biodosimetry. Phase I experiments have thus been designed to 1) evaluate the compatibility of a flow cytometric process for enumerating micronucleus frequencies in primary lymphocytes, 2) devise a simple method for normalizing flow cytometry-based micronucleus frequencies for the fraction of cells that have undergone division, and 3) determine the degree of correspondence between micronucleus measurements accomplished by microscopy versus an optimized flow cytometric method. Ultimately, this line of investigation seeks to enhance the Nation's state of preparedness for a large-scale radiological disaster by rapidly supplying health care providers with dose-estimates. Radiation dose will be unknown for most exposures that occur in the context of an accident or a terrorist event. Traditionally, certain changes in blood are used to estimate dose, and thereby make rational treatment decisions. Unfortunately, the low throughput capacity of traditional radiation biodosimetry assays cannot address a large scale disaster. Thus, there is a need for new methodologies with higher throughput. We propose to develop such a radiation biodosimetry assay based on the extremely high analysis rates that are possible with flow cytometry instrumentation.

描述（由申请人提供）：在意外辐射暴露或放射性恐怖事件的情况下，不完整或缺失的物理剂量测定将是常态。因此，需要生物剂量测定来做出合理的临床管理决策以及长期致癌风险评估。 “黄金标准”生物剂量测定技术可测量循环淋巴细胞（双着丝粒或微核）中的染色体损伤，并且在受害者数量较少时已被证明是有价值的。不幸的是，这些测定太麻烦，无法监测大量人群。该项目的目标是为国家提供克服这一限制的高通量辐射生物剂量测定工具。将要优化和验证的测定是一种基于流式细胞术的方法，可快速测量染色体损伤。我们将用作染色体损伤指标的终点是循环淋巴细胞中微核的形成。本文提出的研究是 Litron 科学家在永生化哺乳动物细胞培养物中微核评分方面取得的最新进展的延伸。迄今为止，这项原理验证工作主要集中在小鼠类淋巴母细胞系 L5178Y 上，并证明了从凋亡细胞和坏死细胞的染色质中对 MN 进行差异染色的重要性。结果有力地支持了我们的前提，即通过两种染料的连续染色程序可以实现哺乳动物细胞 MN 的准确流式细胞术计数。即便如此，仍需要对该评分系统进行进一步研究，以研究其与原代淋巴细胞的兼容性，并开发方法来克服为辐射生物剂量测定目的而分析原代淋巴细胞可能遇到的障碍。因此，I 期实验的目的是 1) 评估流式细胞术过程的兼容性，用于计算原代淋巴细胞中的微核频率，2) 设计一种简单的方法，用于标准化已经历分裂的细胞部分的基于流式细胞术的微核频率，以及 3) 确定通过显微镜完成的微核测量与优化流程之间的对应程度 细胞计数法。最终，这一调查旨在通过快速向医疗保健提供者提供剂量估计来提高国家对大规模放射性灾难的准备状态。对于事故或恐怖事件中发生的大多数照射，辐射剂量是未知的。传统上，血液的某些变化被用来估计剂量，从而做出合理的治疗决策。不幸的是，传统辐射生物剂量测定的低通量能力无法解决大规模灾难。因此，需要具有更高吞吐量的新方法。我们建议基于流式细胞仪仪器可能实现的极高分析率来开发这种辐射生物剂量测定法。