ROCHESTER, NY, United States

Litron Laboratories, Ltd.

www.LITRONLABS.COM
ROCHESTER, NY, United States

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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.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 976.47K | Year: 2015

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 andquot misleadingandquot or andquot irrelevantandquot 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 will reliably identify irrelevant mode s of action that are likely to be nonoperational in vivo A secondary objective of the proposed work is the development of methods for characterizing clastogens That is we will develop tools that elucidate whether clastogenic activity is the result of DNA reactivity or whether it is mediated by an indirect mechanism PUBLIC HEALTH RELEVANCE Sub lethal DNA damage that cannot be faithfully repaired results in gene mutation and or chromosomal aberrations and these effects are known to contribute to carcinogenesis There is also emerging evidence that DNA damage contributes to germline genetic disorders and other disease sequelae for instance atherosclerosis Thus there is an important need for sensitive and specific assays to evaluate chemicals for genotoxic potential Furthermore in cases when genotoxic potential has been determined more efficient means of elucidating genotoxic mode of action would be useful for understanding human health risks


The present invention relates a simple method for evaluating free eukaryotic cell nuclei for biomarkers of DNA damage and/or transcription factor activation, activity, or expression levels and/or epigenetic modifications to chromatin or chromatin-associated factors. The invention also teaches useful strategies for combining nuclear biomarkers into a matrix of endpoints that are capable of elucidating genotoxicants primary mode of DNA-damaging activity. Kits for conducting methods according to the invention are also described.


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 eukaryotic cell micronuclei, while simultaneously acquiring cytotoxicity and mode of action information. The method utilizes differential labeling of chromatin from dead and dying cells to distinguish the chromatin from micronuclei, nuclei, and metaphase chromosomes, and differential labeling of metaphase events to provide additional information regarding cytotoxicity and genotoxic modes of action. Counting of micronuclei events relative to the number of nuclei and quantifying perturbations to the proportion of metaphase events 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, the effects of an agent which can modify exogenously-induced DNA damage, and genotoxic mode of action.


The present invention relates a simple method for evaluating free eukaryotic cell nuclei for biomarkers of DNA damage and/or transcription factor activation, activity, or expression levels and/or epigenetic modifications to chromatin or chromatin-associated factors. The invention also teaches useful strategies for combining nuclear biomarkers into a matrix of endpoints that are capable of elucidating genotoxicants primary mode of DNA-damaging activity. Kits for conducting methods according to the invention are also described.


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: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 172.59K | Year: 2016

DESCRIPTION provided by applicant Assessment of chemicalsandapos potential to cause chromosomal damage is an established and important part of preclinical genotoxicity safety testing for many consumer products industrial chemicals and all pharmaceutical agents Currently the mammalian erythrocyte micronucleus test is the most commonly employed assay for in vivo assessment of chromosomal damage but this assay reports specifically on genotoxicity that occurs in the bone marrow In order to obtain a more comprehensive understanding of potential genotoxicity testing guidanceandapos s recommend evaluation of a second tissue The liver the site of metabolism and in many cases activation of genotoxicants is usually regarded as the preferred second tissue Even so there is a lack of efficient and effective tools for studying liver genotoxicity The Comet assay and transgenic rodent mutation models can be employed to study the liver but these assays suffer from methodological and cost issues that limit their utility Another important consideration is that these assays are not highly amenable to integration with on going toxicology studies meaning additional animals are required for the liver genotoxicity assessment One alternative approach is to examine liver hepatocytes for the formation of micronuclei an established indicator of chromosomal damage However existing methods for examining liver micronuclei are still emerging and currently based on a multi step sample processing scheme followed by manual scoring by microscopy This approach is subjective and labor intensive and results in too few cells being scored for reliable enumeration of micronucleated hepatocytes a situation that diminishes the ability of the test to detect weakly genotoxic agents We will overcome these deficiencies by combining simple rapid tissue processing and staining with high speed flow cytometric analysis to greatly improve the execution of liver micronucleus scoring Furthermore we will multiplex several cytotoxicity measurements into the liver micronucleus assay thereby providing information that we predict will be important for interpreting the genotoxicity results The methodology will be reduced to practice in the form of commercially available kits and will contribute to the reduction and refinement of animal testing as it will make it feasible to integrate a liver genotoxicity assay ito ongoing toxicology studies Overall this project will meet a critical need in the practice of genetic toxicology by improving chemical safety assessments in several meaningful ways PUBLIC HEALTH RELEVANCE The assessment of chemical induced chromosomal damage in the liver is an important component of genetic toxicology safety testing Current methods are tedious costly and not amenable to integration with on going toxicology studies We propose to develop an easy efficient and automated method for processing and analyzing liver tissue for the frequency of micronucleated hepatocyte an indicator of chromosomal damage This method will substantially improve existing approaches for evaluating drugs and other chemicals for their ability to cause DNA damage to the liver Besides genetic toxicity our assay will provide concurrent assessments of overt toxicity information that is critical for interpreting DNA damage results The methodology will be made available through commercial kits and will contribute to the reduction and refinement of animal testing as it will make it feasible to integrate a liver genotoxicity assay into ongoing toxicology studies


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


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.

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