Reticulated platelet determination: methodologies and applications for the evaluation of thrombocytopenic disorders]

Reticulated platelets retain some residual mRNA in their cytoplasm and are thought to be newly produced platelets. In recent years, it has been reported that the reticulated platelet count (RP) correlates well with platelet production. For that reason, the measurement of RP (%) is considered useful for analyses of platelet kinetics and differential diagnoses of thrombocytopenic disorders. However, certain technical difficulties exist because fluorochrome thiazole orange (TO), which is used for staining purposes, stains platelet granules nonspecifically, and so far, only a few reports have documented the study of precision staining techniques. We evaluated staining criteria precisely in an effort to solve the issue of nonspecific staining by TO, and concluded that the important points for effective staining were (1) fixation of platelets, (2) 1:8 dilution of TO (ReticCount), (3) incubation for 1 to 2 hours, and (4) the capture of platelets using anti-CD42b monoclonal antibody. We stained reticulated platelet samples by the above method and achieved intra-assay reproducibility of 3.4-5.1% RP (%) in normal subjects was 8.7 +/- 2.2%. It was significantly higher (23.6 +/- 13.3%) in patients with idiopathic thrombocytopenic purpura (ITP), and elevated in 87% of all evaluated ITP patients. Our method is sensitive, provides reproducible results, and can be effectively utilized for the analysis of platelet kinetics and differential diagnosis of thrombocytopenia.