Recombinant human erythropoietin (rHuEPO) is an effective drug for curing people with anemia (low blood count) due to kidney dysfunction. To test its activity, we used a standardized in vivo bioassay protocol on mice line B6D2F1 with an automated fluorescence flow cytometry. The purpose of the master thesis was to optimize the analytical assay in reticulocyte staining and to determinate the best appropriate time for mouse blood sampling. We have confirmed that the optimal sampling is 4 days after drug application. We have quantitatively evaluated the increase of reticulocyte proportion in blood samples, which occurs as a response to the injected rHuEPO. Different influences for reticulocytes dying conditions were also studied. Blood samples were stained with commercial dye BD Retic-Count and laboratory prepared fluorescent dye thiazole orange in different concentrations and left incubated under different conditions (time, light, and vortex). The study showed that the staining process with commercial dye BD Retic-Count is comparable but different to laboratory prepared dye. The best incubation time of sample stained with laboratory prepared dye is between 30 minutes and 150 minutes regardless of the light presence. Before measuring on the flow cytometer, a onetime short vortexing is recommended to ensure homogeneity of the sample. With the better understanding of analytical methods and equipment, we can ensure better quality control which is the base for safe and effective therapeutic product.
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