Whole blood analysis of reticulated platelets: improvements of detection and assay stability.

Abstract

The flow cytometric analysis of reticulated platelets based on the fluorescent derivatization of their RNA content is increasingly used for the diagnostic classification of patients with thrombocytopenia as well as the monitoring of thrombopoiesis recovering under therapy. Many different modifications of the analytical protocol have been published following the first description in 1990 but consensus on the method has not yet been established. We have now reevaluated the assay's methodology in order to optimize sensitivity and specificity and reduce the time length of incubation and washing procedures. In the modified experimental approach native whole blood is incubated for 15 min with an increased amount of thiazole orange (1 microg/ml) in the presence of phycoerythrin labeled antibodies directed against the constitutively surface expressed antigen GPlb. Data acquisition on the flow cytometer can be started immediately after stopping and stabilization of the reaction by paraformaldehyde fixation. Thiazole orange fluorescence was not significantly changed in thrombin-activated, degranulated platelets compared to resting platelets indicating no significant non-specific staining of platelet granules under the selected test conditions. In addition, experiments employing RNAse digestion demonstrated specificity of thiazole orange staining for platelet RNA.