High Throughput Radiation Biodosimetry

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，不完整或丢失的物理剂量测量将是标准。因此，需要生物剂量学来做出合理的临床管理决策，并进行长期致癌风险评估。“金标准”生物剂量学技术测量循环淋巴细胞(双着丝粒或微核)的染色体损伤，并已被证明在受害者数量较少时有价值。不幸的是，这些检测过于繁琐，无法监测大量人口。该项目的目标是向国家提供一种克服这一限制的高通量辐射生物剂量测定工具。将被优化和验证的检测方法是一种基于流式细胞术的方法，它提供了对染色体损伤的快速测量。我们将用来作为染色体损伤指标的终点是循环淋巴细胞中微核的形成。这里提出的研究是Litron科学家在永生化哺乳动物细胞培养中微核评分方面最新进展的延伸。到目前为止，这项原理验证工作主要集中在小鼠淋巴母细胞系L5178Y上，并证明了将MN与凋亡和坏死细胞的染色质进行区别染色的重要性。结果有力地支持了我们的假设，即用两种染料的顺序染色程序可以实现对哺乳动物细胞MN的准确的流式细胞术计数。即便如此，还需要进一步研究这一评分系统，以研究其与初级淋巴细胞的兼容性，并开发出克服初级淋巴细胞分析的可能障碍的方法，用于辐射生物剂量学目的。因此，设计了第一阶段实验，以1)评估流式细胞术方法计数初级淋巴细胞微核率的兼容性，2)设计一种简单的方法，以归一化基于流式细胞术的微核率，用于已经历分裂的细胞部分，以及3)确定显微镜与优化的流式细胞术方法所完成的微核测量之间的对应程度。最终，这项调查旨在通过迅速向卫生保健提供者提供剂量估计来加强国家对大规模辐射灾难的准备状态。对于发生在事故或恐怖事件背景下的大多数暴露，辐射剂量将是未知的。传统上，血液中的某些变化被用来估计剂量，从而做出合理的治疗决定。不幸的是，传统的辐射生物剂量测定的低处理能力不能应对大规模灾难。因此，需要具有更高吞吐量的新方法。我们建议开发这样一种基于流式细胞仪的极高分析率的辐射生物剂量测定方法。

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.