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

A method for the enumeration of micronucleated erythrocyte populations, the method comprising:

• providing a blood or bone marrow sample that is enriched for erythrocyte populations comprising one or more of mature normochromatic erythrocytes, reticulocytes, micronucleated normochromatic erythrocytes, and micronucleated reticulocytes, wherein the erythrocyte populations in the enriched sample are rendered permeable;
• contacting the sample with (i) RNase to degrade RNA in the permeable erythrocyte populations, (ii) a first reagent comprising a fluorescent label and having binding specificity for a surface marker for reticulocytes, (iii) a second reagent comprising a fluorescent label and having binding specificity for a surface marker for platelets, and (iv) a nucleic acid staining dye, wherein said contacting with RNase is carried out prior to said contacting with the nucleic acid staining dye, and wherein the fluorescent emission spectra of the fluorescent labels of the first and second reagents and the nucleic acid staining dye are distinguishable from one another;
• 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 total number of reticulocytes labeled by the first reagent and the number of micronucleated reticulocytes labeled by the first reagent and the nucleic acid staining dye, while excluding normochromatic erythrocytes lacking a label by the first reagent from counted cells and excluding platelets and platelet-associated aggregates labeled by the second reagent from scoring events for micronucleated reticulocytes.