WHOLE-BLOOD ANALYSIS OF RETICULATED PLATELETS - IMPROVEMENTS OF DETECTION AND ASSAY STABILITY

The flow cytometric analysis of reticulated platelets based on the flu orescent derivatization of their RNA content is increasingly used for the diagnostic classification of patients with thrombocytopenia as wel l as the monitoring of thrombopoiesis recovering under therapy. Many d ifferent modifications of the analytical protocol have been published following the first description in 1990 but consensus on the method ha s not yet been established. We have now reevaluated the assay's method ology in order to optimize sensitivity and specificity and reduce the time length of incubation and washing procedures. In the modified expe rimental approach native whole blood is incubated for 15 min with an i ncreased amount of thiazole orange (1 mu g/ml) in the presence of phyc oerythrin labeled antibodies directed against the constitutively surfa ce expressed antigen GPIb. Data acquisition on the flow cytometer can be started immediately after stopping and stabilization of the reactio n by paraformaldehyde fixation. Thiazole orange fluorescence was not s ignificantly changed in thrombin-activated, degranulated platelets com pared to resting platelets indicating no significant non-specific stai ning of platelet granules under the selected test conditions. In addit ion, experiments employing RNAse digestion demonstrated specificity of thiazole orange staining for platelet RNA. (C) 1998 Wiley-Liss, Inc.