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. ? ? ?
