A method for the determination of the number of reticulated platelets from whole blood

Abstract

Platelets still containing RNA and DNA remnants are considered to be the immediate progeny of megakaryocytes which have matured beyond the proplatelet formation stage. Numerous investigators have shown that platelets which stain with thiazole orange, a nucleic acid dye, have increased RNA content, and therefore are the youngest platelets in the circulation. These immature, or reticulated platelets, are analogous to red blood cell reticulocytes in their level of differentiation, but not their function. Platelet analysis using thiazole orange dye, in combination with flow cytometry, has been recently shown by a number of investigators to be an optimal method for detection of this immature platelet population. However, the many variations in protocols cause results to greatly differ between laboratories. In our studies, we used a similar whole blood analysis approach developed by Matic and colleagues to quantitate the amount of dye needed for optimal staining. Therefore, we used a dose-escalation of thiazole orange (0.25 μg–50 μg) in combination with CD61 conjugated with phycoerythrin without any wash steps, which assures minimal platelet loss. CD61 was used in this setting in order to set an initial gate on the platelet population. There was a direct, dose-response relationship between the amount of thiazole orange added and the number of reticulated platelets observed. A plateau was reached with 5 μg thiazole orange per 5 μL of whole blood, while 12.5 μg was found to be optimal. Increasing amounts of thiazole orange produced a population shift, or mean channel shift, that needed to be accounted for when setting gates for thiazole orange positive events. We observed that nearly 7% of the platelets in normal resting BDF1 mice were reticulated. This compares to a reticulated population in humans of less than 10%. This protocol combines the ease of sample preparation with stability and accuracy for determining the number of reticulated platelets, and should be a useful method in efforts to standardize the enumeration of reticulated platelets from whole blood. Furthermore, the use of thiazole orange for determining reticulated platelet numbers provides a reproducible and validated method for the analysis of platelet precursors, and, thus, may be a useful tool for predicting platelet recovery post-chemotherapy.