Method for enumeration of mammalian micronucleated erythrocyte populations, while distinguishing platelets and/or platelet-associated aggregates

A method for the enumeration of micronucleated erythrocyte populations while excluding false-positive micronuclei scoring events caused by platelet and platelet-associated aggregates, the method comprising:

• providing a sample comprising erythrocyte populations including mature normochromatic erythrocytes, reticulocytes, micronucleated normochromatic erythrocytes, micronucleated reticulocytes, or combinations thereof, with the erythrocyte populations being in suspension and substantially free of aggregates, permeable to a nucleic acid dye and RNase, with cell surface markers in a form recognizable by an antibody, and able to exhibit substantially low autofluorescence;
• substantially degrading RNA of reticulocytes in the sample with RNase;
• contacting the sample with a first reagent comprising a fluorescent label and having binding specificity for a surface marker for reticulocytes and with a second reagent comprising a fluorescent label and having binding specificity for a surface marker for platelets, wherein the fluorescent emission spectrum of the fluorescent labels of the first and second reagents do not substantially overlap;
• staining cellular DNA with a nucleic acid staining dye having a fluorescent emission spectrum which does not substantially overlap with the fluorescent emission spectrum of the fluorescent labels of the first and second reagents;
• exciting the nucleic acid staining dye, the fluorescent label associated with the reticulocytes, and the fluorescent label associated with platelets using light of appropriate excitation wavelength for both the nucleic acid staining dye and the fluorescent labels to produce fluorescent emission;
• and
• detecting the fluorescent emission and light scatter produced by the erythrocyte populations and platelets, and counting the number of cells from one or more erythrocyte populations in said sample while excluding scoring events caused by platelets and platelet-associated aggregates from scoring events for micronucleated erythrocytes.