A tetrazolium-based colorimetric assay for metabolic activity of stored blood platelets

Abstract

Introduction: Much work has been carried out in an attempt to establish the optimum cryopreservation of platelets. To estimate the surviving numbers of stored platelets, the metabolic redox activity of resting platelets was measured and compared with the aggregation response. Materials and methods: The activity was determined using a simple colorimetric assay based on reduction of a tetrazolium salt, WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-[4-nitrophenyl]-2 H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), to water-soluble formazan and its quantification with a spectrophotometer. The aggregation response of the platelets was measured at 37 °C with an aggregometer. Results: Colored WST-8 formazan was produced in proportion to the number of intact fresh platelets, but only a little or none was produced by dead platelets lysed by sodium dodecyl sulfate (SDS), by platelets in which glycolysis had been blocked by the inhibitor monoiodoacetic acid (MIA), and by frozen platelets. The assay in conjunction with platelet aggregometry could distinguish between platelets which had lost only the aggregation response and those which had lost both aggregation response and redox activity. Freezing platelets in the presence of cryoprotectant Banbanker resulted in approximately 70% fresh platelets with redox activity and aggregation response. Conclusions: These results indicate that the WST-8 assay can be useful for identifying viable platelets independent of the aggregation response and may contribute to clarifying the mechanism of the loss of function of stored platelets.