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ROCHESTER, NY, United States

A method for the enumeration of micronucleated erythrocyte populations while distinguishing platelet and platelet-associated aggregates involves the use of a first fluorescent labeled antibody having binding specificity for a surface marker for reticulocytes, a second fluorescent labeled antibody having binding specificity for a surface marker for platelets, and a nucleic acid staining dye that stains DNA (micronuclei) in erythrocyte populations. Because the fluorescent emission spectra of the first and second fluorescent labeled antibodies do not substantially overlap with one another or with the emission spectra of the nucleic acid staining dye, upon excitation of the labels and dye it is possible to detect the fluorescent emission and light scatter produced by the erythrocyte populations and platelets, and count the number of cells from one or more erythrocyte populations in said sample. In particular, the use of the second antibody prevents interference by platelet-associated aggregates in the scoring procedures.


The present invention relates a method for the enumeration of mammalian cell micronuclei, while distinguishing micronuclei from the chromatin of dead and dying cells. The method utilizes differential staining of chromatin from dead and dying cells, to distinguish the chromatin from micronuclei and nuclei that can be detected based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of micronuclei events relative to the number of nuclei can be used to assess the DNA-damaging potential of a chemical agent, the DNA-damaging potential of a physical agent, the effects of an agent which can modify endogenously-induced DNA damage, and the effects of an agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 481.62K | Year: 2012

DESCRIPTION (provided by applicant): Large-scale disaster situations put considerable demands on existing medical infrastructure, as well as local governing and safety agencies. When the additional factor of ionizing radiation and exposure of humans is part of the emergency, the lack of effective assessment tools and management strategies can easily become overwhelming. As part of efforts to devise methods and technologies that will address this critical need, this proposal is designed to develop and evaluate two radiation- sensitive endpoints that can be obtained from a small volume blood sample. The goal is to create an analytical platform that could be integrated into mobile response units that can quickly survey the thousands of subjects expected in certain emergency triage situations. The key to delivering useful dose/exposure estimation with a sufficiently rapid turnaround is the combination of cell enrichment via immunomagnetic technologies with high throughput analytical platforms that employ multi-well plates, e.g. 384 or 1536 well. An ultra-high throughput method that enumerates circulating lymphocytes will serve as an initial screen that can rapidly survey and flag subjects for further analysis. Using the same blood sample, the confirmatory assay will employ flow cytometric assessment of micronucleated reticulocytes as a marker of radiation-induced chromosome damage. This two-stage approach will enable the first screen to quickly dismiss the majority of individuals that have received minimal-to-no exposure and the subsequent assay will focus on definitive identification of subjects that will require further medical care. By quickly and efficiently sending the worried-well home, more effective allocation of resources and care can be directed where it is ultimately needed. Such an approach is critical to ensuring an immediate and sufficient response to large-scale emergencies such as those presented by exposure of human populations to ionizing radiation. PUBLIC HEALTH RELEVANCE: Accidental or intentional exposure of human populations to ionizing radiation will not only directly impact public health, but will also affect economics, politics and societies worldwide. The development of tools and strategies to assess and mitigate these impacts are of critical national and global importance. This proposal seeks to generate a rapid high throughput approach using two established biomarkers of radiation damage to determine exposure of individual humans in point-of-care triage settings.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.95K | Year: 2014

DESCRIPTION (provided by applicant): It is well recognized that current batteries of genetic toxicology assays exhibit relatively high sensitivity, meaning they effectively identify genotoxic carcinogens. However, a critical deficiency with current approaches exists-namely, the specificity of the in vitro mammalian cell genotoxicity tests is low, as they yield a high incidenc of positive results that do not have in vivo relevance (so-called misleading or irrelevant positives). This high incidence of irrelevant in vitro positive results leads to extensive and costy additional testing, often with whole animal models, or else abandonment of potentially valuable products. We will address this major problem with current in vitro mammalian cell genetic toxicity assays by developing commercial kits that enable an automated testing strategy that exhibits both high sensitivity and specificity. This system will categorize positive results according to the predominant mode of genotoxic activity, and importantly


A method for the enumeration of micronucleated erythrocyte populations while distinguishing platelet and platelet-associated aggregates involves the use of a first fluorescent labeled antibody having binding specificity for a surface marker for reticulocytes, a second fluorescent labeled antibody having binding specificity for a surface marker for platelets, and a nucleic acid staining dye that stains DNA (micronuclei) in erythrocyte populations. Because the fluorescent emission spectra of the first and second fluorescent labeled antibodies do not substantially overlap with one another or with the emission spectra of the nucleic acid staining dye, upon excitation of the labels and dye it is possible to detect the fluorescent emission and light scatter produced by the erythrocyte populations and platelets, and count the number of cells from one or more erythrocyte populations in said sample. In particular, the use of the second antibody prevents interference by platelet-associated aggregates in the scoring procedures.

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