Method for the enumeration of micronucleated erythrocyte populations with a single laser flow cytometer

A single laser flow cytometric method for the enumeration of micronuclei events in erythrocyte populations, the method comprising the steps of:

• a) obtaining a sample of erythrocyte populations from an individual, wherein the sample is from an origin selected from the group consisting of peripheral blood, and bone marrow;
• b) fixing the erythrocytes in said sample in a primary alcohol at a temperature of from about -70.degree. C. to about -90.degree. C. so as to cause the erythrocytes to be in suspension and free of aggregates, be permeable to a nucleic acid dye and RNase, maintain cell surface markers in a form recognizable by antibody, and exhibit low autofluorescence;
• (c) simultaneously degrading RNA of reticulocytes in the fixed erythrocytes with RNase, and contacting the reticulocytes with a fluorescent labeled antibody having binding specificity for a surface marker for erythroblasts/reticulocytes;
• (d) staining in the erythrocyte populations DNA representing micronuclei with a nucleic acid staining dye in a concentration range detectable by flow cytometry;
• (e) exciting the nucleic acid staining dye and the fluorescent label associated with the erythrocyte populations with a focused laser beam of appropriate excitation wavelength for both the nucleic acid staining dye and the fluorescent label to produce fluorescent emission;
• and
• (f) detecting the fluorescent emission and light scatter produced by the erythrocyte populations and calculating the number of specific erythrocyte populations in said sample, wherein the specific erythrocyte populations are selected from the group consisting of mature normochromatic erythrocytes, reticulocytes, micronucleated normochromatic erythrocytes, micronucleated reticulocytes, and a combination thereof.