Automated analysis of reticulocytes using fluorescent staining with both acridine orange and an immunofluorescence technique.

We have used an automated system to measure serial reticulocyte samples from phlebotomized rats and rats treated with the chemotherapeutic agent actinomycin-D. In performing the automated samples, we utilized two different methods. The first method employed a fluorescent dye, acridine orange, which stains RNA in a manner similar to supravital stains presently used in performing reticulocyte counts. The second method is unique in that we utilized an immunofluorescent assay with antitransferrin receptor antibody which identifies the transferrin receptor found on the cell surface of reticulocytes but not on mature red cells. Both methods gave reproducible values in estimating the increase in reticulocytes seen after phlebotomy and the initial reticulocytopenia followed by recovery of the peripheral blood reticulocyte count seen after treatment with actinomycin-D. Also, since the antitransferrin receptor antibody appears to recognize the more immature reticulocytes, both methods taken together may provide an estimate of the maturity of a population of reticulocytes.