Maekawa Yuichi, Yagi Kyouko, Nonomura Ayako, Kuraoku Rie, Nishiura Emi, Uchibori Emi, Takeuchi Kikuko
Department of Clinical Laboratory Science, Osaka Prefecture College of Health Science, 3-7-30 Habikino, 583-8555, Osaka, Japan.
Thromb Res. 2003 Mar 15;109(5-6):307-14. doi: 10.1016/s0049-3848(03)00257-3.
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.
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]-2H-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 degrees C with an aggregometer.
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.
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.
为了确定血小板的最佳冷冻保存方法，已经开展了大量工作。为了估算储存血小板的存活数量，对静息血小板的代谢氧化还原活性进行了测量，并与聚集反应进行了比较。
该活性通过一种简单的比色测定法来确定，该方法基于将四唑盐WST-8（4-[3-(2-甲氧基-4-硝基苯基)-2-[4-硝基苯基]-2H-5-四唑]-1,3-苯二磺酸钠盐）还原为水溶性甲臜，并使用分光光度计对其进行定量。在37℃下用血小板聚集仪测量血小板的聚集反应。
有色的WST-8甲臜的产生量与完整新鲜血小板的数量成正比，但由十二烷基硫酸钠（SDS）裂解的死亡血小板、被抑制剂碘乙酸（MIA）阻断糖酵解的血小板以及冷冻血小板产生的甲臜很少或不产生。该测定法与血小板聚集测定法相结合，可以区分仅失去聚集反应的血小板和同时失去聚集反应和氧化还原活性的血小板。在冷冻保护剂Banbanker存在的情况下冷冻血小板，会产生约70%具有氧化还原活性和聚集反应的新鲜血小板。
这些结果表明，WST-8测定法可用于独立于聚集反应识别存活血小板，并可能有助于阐明储存血小板功能丧失的机制。
