High Throughput Radiation Biodosimetry

描述（由申请人提供）：在意外辐射照射或辐射恐怖事件的情况下，不完整或缺失的物理剂量测定将是常态。因此，需要生物剂量学来做出合理的临床管理决策，并进行长期致癌风险评估。“黄金标准”生物剂量测定技术测量循环淋巴细胞中的染色体损伤（双着丝粒细胞或微核），并已证明在受害者人数较少时很有价值。不幸的是，这些检测方法太麻烦，无法监测大量人群。该项目的目标是为国家提供一个高通量的辐射生物剂量测定工具，克服这一限制。将进行优化和验证的检测是一种基于流式细胞术的方法，可快速测量染色体损伤。我们将利用循环淋巴细胞中微核形成作为染色体损伤指标的终点。本文提出的研究是Litron科学家关于永生化哺乳动物细胞培养物中微核评分的最新进展的延伸。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样细胞系L5178Y上，并证明了从凋亡和坏死细胞的染色质中区别染色MN的重要性。结果提供了强有力的支持，我们的前提是，准确的流式细胞计数的哺乳动物细胞MN可以实现与两种染料，连续染色程序。即便如此，这个评分系统的进一步工作，需要调查其与初级淋巴细胞的兼容性，并开发方法，克服可能的障碍，分析初级淋巴细胞的辐射生物剂量学的目的。因此，设计了I期实验，以1）评价流式细胞术方法用于计数原代淋巴细胞微核频率的兼容性，2）设计一种简单的方法，用于标准化基于流式细胞术的已分裂细胞部分的微核频率，3）确定显微镜与优化流式细胞术方法完成的微核测量之间的对应程度。最终，这条调查线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害的准备状态。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法的低通量能力不能解决大规模的灾难。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辐射生物剂量测定法的基础上的极高的分析率，是可能与流式细胞仪。

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.