SIMPLE AND RELIABLE ENUMERATION OF MICRONUCLEATED RETICULOCYTES WITH A SINGLE-LASER FLOW CYTOMETER

A flow cytometric procedure for scoring micronuclei in mouse periphera l blood erythrocytes, especially reticulocytes, is described. The meth ods reported herein were developed in an effort to simplify the techni ques and to reduce the equipment requirements associated with automate d micronucleus analyses. With this procedure, fluorescein-conjugated m onoclonal antibodies which bind to the CD71-defined antigen (the trans ferrin receptor) are used to label reticulocytes, The nucleic acid dye propidium iodide is used to identify cells with micronuclei, Given 48 8 nm excitation, four populations of erythrocytes are clearly resolved : normochromatic erythrocytes with and without micronuclei, and reticu locytes with and without micronuclei. Since the method is capable of s imultaneously providing the incidence of micronuclei in both mature an d immature erythrocyte populations, it is compatible with either chron ic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood mi cronuclei, an experiment with the model clastogen methyl methanesulfon ate is described. Additionally, a reconstruction experiment was perfor med whereby three mouse blood samples were spiked with successively gr eater volumes of blood from a clastogen-treated animal so each prepara tion differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cyt ometry so that the two methods could be directly compared. Collectivel y, the results from the methyl methanesulfonate experiment and the rec onstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughput scoring methodology are clearly indicated.