Method for discriminating between intact and damaged cells in a sample

Claim 1

A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of: a) taking a sample of peripheral blood from an individual; b) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye; c) adding a fixative to the sample; d) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR2## wherein n is 0; m is 1; R is a lower alkyl; R 1 and R 2 are lower alkyls; R 3 is hydrogen; Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and X represents an anion; e) analyzing the sample in an automated instrument capable of passing the cells in the sample through a sensing region substantially one at a time and capable of detecting and recording both fluorescence and light scattered in more than one direction by each cell passing through said sensing region; and f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.

Claim 2

A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of: a) taking a sample of peripheral blood from an individual; b) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye; c) adding a fixative to the sample; d) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR2## wherein n is 0; m is 1; R is a lower alkyl; R 1 and R 2 are lower alkyls; R 3 is hydrogen; Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and X represents an anion; e) analyzing the sample in an automated instrument capable of passing the cells in the sample through a sensing region substantially one at a time and capable of detecting and recording both fluorescence and light scattered in more than one direction by each cell passing through said sensing region; and f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.

Claim 10

A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of: a) taking a sample of peripheral blood from an individual; b) lysing erythrocytes in the sample; c) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye; d) adding paraformaldehyde to the sample; e) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR3## wherein n is 0; m is 1; R is a lower alkyl; R 1 and R 2 are lower alkyls; R 3 is hydrogen; Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and X represents an anion; e) analyzing said sample in a flow cytometer equipped with a single laser tuned to 488 nm; and f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.

Claim 11

A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of: a) taking a sample of peripheral blood from an individual; b) lysing erythrocytes in the sample; c) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye; d) adding paraformaldehyde to the sample; e) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR3## wherein n is 0; m is 1; R is a lower alkyl; R 1 and R 2 are lower alkyls; R 3 is hydrogen; Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and X represents an anion; e) analyzing said sample in a flow cytometer equipped with a single laser tuned to 488 nm; and f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.