应用流式细胞术研究剪切力诱导的血小板聚集

Study of platelet aggregation induced by shear stress using flow cytometry

目的 建立流式细胞术方法检测剪切力诱导的血小板聚集 (SIPA) ,研究不同剪切力作用后血小板聚集率的变化规律 ,并初步探讨SIPA测定的临床意义。方法 用锥板黏度计对全血标本施加剪切力作用 ,使血小板产生聚集反应 ,再以荧光抗体CD6 1PerCP标记血小板 ,然后进行流式细胞术分析 ,测定血小板聚集率的大小 ;同时在显微镜下直接观察血小板聚集体形态。结果 不同的剪切力作用后 ,血小板产生聚集反应程度不同。在 10 0S-1剪切作用下 ,只有少量的血小板聚集 ;5 0 0S-1时 ,血小板聚集增加 ;在高于 2 0 0 0S-1的剪切作用下 ,30s即可发生明显的血小板聚集 ,30 0 0S-1剪切作用 3min时血小板聚集率为 (5 8 4± 5 3) % ,已接近最大值 ,明显高于 10 0S-1的(9 4± 1 3) %和 5 0 0S-1的 (2 6 4±3 2 ) %。随着剪切力的增大和作用时间的延长 ,血小板聚集体逐渐增大 ,结构由松散到致密。AMI、脑梗死、TIA、糖尿病等动脉血栓相关性疾病患者的高剪切力诱导的血小板聚集明显高于正常。结论 直接的剪切力作用能够诱导血小板发生聚集反应 ,聚集程度与剪切力的大小和作用时间成正相关 ;

Objective To establish a flow cytometric method for assay of platelet aggregation induced by shear stress(SIPA) and investigate its clinical value. Methods After the whole blood was subjected to a certain shear stress by a rotational viscometer, the platelets were activated and aggregated. Then the fluorescence antibody CD61 PerCP was added to stain the platelets. The free platelets and aggregates were counted respectively by flow cytometry, thereby the ratio of platelet aggregation was calculated. Results Shear stress-induced whole blood platelet aggregation varied with the increase of shear stress and time of stress. Only a small amount of platelet aggregation was detected at relatively low shear stress of 100S -1, and it was increased at 500S -1. However, a significant increase occurred with 2 000S -1. It approached to the maximum value (58.4%±5.3%) at 3 000S -1 with exposure of 3min, which was higher than the values at 100S -1(9.4%±1.3%)and 500S -1(26.4%±3.2%). At the same time, the aggregates increased in size and became more compact with increase in the shear stress and prolongation of time. High shear stress induced platelet aggregation(H-SIPA) was significantly enhanced in patients with AMI, cerebral infarction, TIA or diabetes, all of them were susceptible to coagulopathy, compared with normal control. Conclusion The results indicated that shear stress could induce platelet aggregation directly, and the degree of SIPA had positive correlation with the stress stength and exposure time.The measurement of H-SIPA was of some clinical value in the diagnosis of arterial thrombosis.