Detection of reticulated platelets in whole blood of rats using flow cytometry

Abstract

As opposed to erythropoiesis, which is regularly assessed in the peripheral blood of animals by reticulocyte count, thrombopoiesis is rarely assessed in assays that detect immature platelets in the peripheral blood. An assessment of recent thrombopoiesis is feasible with the analysis of reticulated platelets in the peripheral blood via flow cytometry, but rarely performed. The aim of this study was to establish an assay for the detection of reticulated platelets in whole blood of rats via flow cytometry, using a two-color staining method with a platelet-specific antibody (CD61-PE) and thiazole orange to detect RNA-containing platelets. Platelets were detected in K3EDTA-anticoagulated, paraformaldehyde-fixed samples, using a CD61-PE antibody as well as a gate specific for the light scatter properties of platelets. The intra-assay coefficient of variation varied between 3.6% and 8.3% (n=6 animals). The stability of the assay was determined by storing blood prior to staining, storing stained samples for up to 2h at room temperature, and by diluting the blood prior to analysis with autologous plasma to create samples with artificial anemia and thrombocytopenia. Only samples stored at room temperature prior to analysis showed a significantly lower percentage of reticulated platelets. Percentage of reticulated platelets in the reference population (n=41 rats) was 10.0+/-1.3% reticulated platelets (mean+/-SD; min=6.2%; max=12.5%). These data show that the detection of reticulated platelets in whole blood of rats using a platelet-specific antibody is feasible. This test presents a minimal-invasive method to assess thrombopoiesis in rats that can be used for example in preclinical toxicological studies.