Abstract: 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.

Abstract: The present invention relates a method for the enumeration of in vivo gene mutation. The method utilizes differential staining of GPI-anchor deficient erythrocyte populations to distinguish between wild-type and pig-a gene mutants. Quantitative analyses can be conducted on erythrocytes and/or reticulocytes, and is based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of mutant erythrocytes or reticulocytes relative to the number of total erythrocytes or reticulocytes can be used to assess the DNA-damaging potential of an exogenous chemical agent, the DNA-damaging potential of an exogenous physical agent, the effects of an exogenous agent which can modify endogenously-induced DNA damage, and the effects of an exogenous agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

Abstract: The present invention relates a method for assessing in vivo hematotoxicity. The method utilizes differential staining of nucleated and non-nucleated blood cells, and also differential labeling of cells with functional versus dysfunctional mitochondrial membrane potential. Quantitative analyses can be conducted on stained whole blood specimens, and is based on blood cells' fluorescent emission and light scatter properties following exposure to an excitatory light source. The ratio of certain cell populations can be readily measured. Furthermore, it is also possible to express cell population values in terms of number per unit volume. This invention can be used to evaluate the hematotoxicity of drugs, chemicals, radiation, and other exogenous agents, or the effects that a suspected protective agent may have on induced hematotoxicity. Furthermore, the matrix of measurements provided by this invention is useful in estimating radiation dose, i.e., retrospectively.

Abstract: 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.

Abstract: The present invention relates a method for the enumeration of in vivo gene mutation. The method utilizes differential staining of GPI-anchor deficient erythrocyte populations to distinguish between wild-type and pig-a gene mutants. Quantitative analyses can be conducted on erythrocytes and/or reticulocytes, and is based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of mutant erythrocytes or reticulocytes relative to the number of total erythrocytes or reticulocytes can be used to assess the DNA-damaging potential of an exogenous chemical agent, the DNA-damaging potential of an exogenous physical agent, the effects of an exogenous agent which can modify endogenously-induced DNA damage, and the effects of an exogenous agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

Abstract: 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.

Abstract: The present invention relates a method for the enumeration of in vivo gene mutation. The method utilizes differential staining of GPI-anchor deficient erythrocyte populations to distinguish between wild-type and pig-a gene mutants. Quantitative analyses can be conducted on erythrocytes and/or reticulocytes, and is based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of mutant erythrocytes or reticulcoytes relative to the number of total erythrocytes or reticulocytes can be used to assess the DNA-damaging potential of an exogenous chemical agent, the DNA-damaging potential of an exogenous physical agent, the effects of an exogenous agent which can modify endogenously-induced DNA damage, and the effects of an exogenous agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

Abstract: 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.

Abstract: 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.

Abstract: The invention relates to methods and kits for the quantitative analysis of in vivo mutation frequencies of the Pig-A gene in individuals exposed to a genotoxicant, particularly using peripheral blood samples of vertebrates.

Abstract: 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.

Abstract: The present invention relates a method for assessing in vivo hematotoxicity. The method utilizes differential staining of nucleated and non-nucleated blood cells, and also differential labeling of cells with functional versus dysfunctional mitochondrial membrane potential. Quantitative analyses can be conducted on stained whole blood specimens, and is based on blood cells' fluorescent emission and light scatter properties following exposure to an excitatory light source. The ratio of certain cell populations can be readily measured. Furthermore, it is also possible to express cell population values in terms of number per unit volume. This invention can be used to evaluate the hematotoxicity of drugs, chemicals, radiation, and other exogenous agents, or the effects that a suspected protective agent may have on induced hematotoxicity. Furthermore, the matrix of measurements provided by this invention is useful in estimating radiation dose, i.e., retrospectively.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The present invention relates a method for the enumeration of in vivo gene mutation. The method utilizes differential staining of GPI-anchor deficient erythrocyte populations to distinguish between wild-type and pig-a gene mutants. Quantitative analyses can be conducted on erythrocytes and/or reticulocytes, and is based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of mutant erythrocytes or reticulcoytes relative to the number of total erythrocytes or reticulocytes can be used to assess the DNA-damaging potential of an exogenous chemical agent, the DNA-damaging potential of an exogenous physical agent, the effects of an exogenous agent which can modify endogenously-induced DNA damage, and the effects of an exogenous agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

Abstract: A single laser flow cytometric method for the enumeration of micronuclei in erythrocyte populations, wherein a sample of peripheral blood or bone marrow is obtained and the cell populations in the sample are fixed. Reticulocytes in the fixed samples are treated simultaneously with RNAse and with a fluorescent labeled antibody having binding specificity for a surface marker for erythroblasts/reticulocytes. The erythrocyte populations are then stained with a nucleic acid stain which stains DNA representing micronuclei, if present. The stained and/or labeled erythrocyte populations are then exposed to a laser beam of appropriate excitation wavelength for both the nucleic acid staining dye and the fluorescent label to produce fluorescent emission. The fluorescent emission and light scatter produced by the erythrocyte populations are detected by the flow cytometer from which is calculated the number of specific erythrocyte populations in said sample.

Abstract: A single laser flow cytometric method for the enumeration of micronuclei in erythrocyte populations, wherein a sample of peripheral blood or bone marrow is obtained and the cell populations in the sample are fixed. Reticulocytes in the fixed samples are treated simultaneously with RNAse and with a fluorescent labeled antibody having binding specificity for a surface marker for erythroblasts/reticulocytes. The erythrocyte populations are then stained with a nucleic acid stain which stains DNA representing micronuclei, if present. The stained and/or labelled erythrocyte populations are then exposed to a laser beam of appropriate excitation wavelength for both the nucleic acid staining dye and the fluorescent label to produce fluorescent emission. The fluorescent emission and light scatter produced by the erythrocyte populations are detected by the flow cytometer from which is calculated the number of specific erythrocyte populations in said sample.

Abstract: Disclosed are a composition for the lyophilization of mammalian red blood cells comprising a hydrophilic polymer, a carbohydrate, and an organic solvent; and a method of using the composition to lyophilize red blood cells comprising mixing red blood cells with the composition, freezing the mixture, and drying the mixture by removing water by sublimation. Also disclosed are red blood cells lyophilized according to this method for lyophilization, and a method for reconstituting the lyophilized red blood cells. In particular, the composition used to lyophilize the red blood cells comprises a mixture of a hydrophilic polymer ranging from 1,450-20,000 Daltons at 5-50% w/v, a mono- or disaccharide or a mixture thereof from 0.01-0.2M and an organic solvent such as a primary alcohol, a secondary alcohol, dimethyl sulfoxide or combinations thereof at 0.5-20% v/v.

Abstract: Genotoxic chemicals are an existing wide-spread health hazard to the human population. Advances in genetic toxicology testing have made it possible to assay potential mutagens, carcinogens, teratogens and clastogens in the environment. The mouse micronucleus assay provides an example of an excellent test for genetic damage to cells. When chromosome breaks occur in the blood stem cell population, the damaged piece of chromosome remains behind as a micronucleus in the normally DNA deficient red blood cells. However, currently available manual micronucleus assays are costly, time consuming, and labor intensive. In addition, the statistics are often marginal since the number of micronucleii (MNs) in 1000 polychromatic cells are scored manually, yielding limited amounts of data. This invention discloses the means for assaying the change in micronucleated cells by high speed flow cytometry.