一种新的血小板活化释放试验监测阿司匹林的治疗反应

A new platelet activated-release test for monitoring aspirin response

目的建立一种新的血小板活化释放试验用于监测阿司匹林的治疗反应。方法采用ARA活化抗凝全血中血小板，应用血细胞分析仪检测MPC的变化。抽取5名健康志愿者外周血标本，取血后迅速混匀，用于MPC、LTAARA和血小板CD62P表达的检测，以优化检测条件，包括ARA活化浓度（分别为0、0．5、1．0、1．5、5．0、10．0mmol／L）的选择和ARA活化血小板时间的选择（37℃水浴中5、10、20、30、40、50、60、70、80、90min）；评价该方法的稳定性（取血后0、1、2、3h分别检测）、重复性（MPC、LTAARA、血小板CD62P表达的检测分别重复测定10次，计算批内及批间CV）；并通过体外乙酰水杨酸试验验证该方法监测阿司匹林治疗反应的可行性。ARA活化血小板后用CD旷PerCP和CD62P-PE标记，流式细胞仪测定CD62P，表达阳性的血小板百分率，对MPC与CD62P的相关性进行分析。选择口服阿司匹林至少7d的患者为服药组，共93例；选择未口服或停止口服阿司匹林7d以上的患者为对照组，共100例。用该方法检测服药组与对照组患者ARA（终浓度0．5mmol／L）活化前后MPC的变化，并与LTAARA方法进行比较，评价该方法的准确性。结果ARA终浓度为0．5mmol／L时，血小板活化充分，MPC与血小板膜表面CD62P呈负相关（r=-0．755，P〈0．01）。37℃水浴活化30min后MPC达到稳定，取血后室温3h内MPC保持稳定，新鲜全血MPC的批内CV为1．35％，固定质控全血批间CV为0．71％和0．74％。随着全血中乙酰水杨酸浓度升高（0～6．95μmol／L），ARA活化血小板后MPC逐渐升高，CD62P逐渐减低，二者呈负相关（r=-0．765，P〈0．01）。服药组MPC差值为（8．2±8．6）g／L，MPC变化率为（3．4±3．6）％，对照组分别为（37．4±10．3）g／L、（15．7±4．0）％，差异均有统计学意义（t=21．522、22．409，P均〈0．01）。MPC变化率ROC曲线下面积为0．992，当临界值为8．7％时，其监测阿司匹林治疗反应的敏感度为96．8％，特异度为99．0％。MPC变化率与LTAARA具有很好的相关性（r=0．720，P〈0．01）。结论本研究建立了一种新的监测阿司匹林治疗反应的血小板活化释放试验，可以替代LTAARA用于临床常规检测。

Objective To establish a new method for monitoring aspirin response by platelet activated-release experiment. Methods The platelets in whole blood were activated by ARA, and the MPC was measured by hematology analyzer. Blood samples were drawn from five healthy volunteers for measuring MPC, LTAARA and platelet membrane CD62P expression. Blood samples were mixed thoroughly right after venipuncture. The concentration of ARA（0,0. 5,1.0,1.5,5.0 and 10. 0 mmol/L） and the time for platelet activation（5,10,20,30,40,50,60,70,80 and 90 rain in 37 ℃ water bathe） were optimized to evaluate the stability（0,1,2 and 3 h after venipuncture） and reproducibility （MPC, LTAARA and platelet membrane CD62P were measured ten times and the CV was calculated）. Platelets were mixed with acetylsalicylic acid at different concentrations in vitro in order to verify the validity for monitoring aspirin response. The percentage of CD62P positive platelets after activated by ARA was measured using flow cytometer with CD61-PerCP and CD62P-PE antibodies. The correlation between MPC and CD62P was determined. 100 patients without taking or stop taking aspirin more than 7 days and 93 patients who took aspirin at least 7 days were enrolled. Duplicate measurements of platelet function were performed using the change of MPC （ ARA 0. 5 mmol/L） and LTA （ARA 0. 5 mmol/L） among two patient groups to evaluate the accuracy of the new method. Results Platelets were completely activated by ARA at final concentration of 0. 5 mmol/L. MPC negatively correlated with platelet membrane CD62P （ r = - 0. 755 ,P 〈 0. 01 ）. MPC was stable for 30 minutes in 37 ~C water bathe after ARA activation. The result of MPC was consistent at room temperature within 3 hours after blood collection. This method had good reproducibility. CV in batch using fresh whole blood was 1.35% and CV between batches using commercial control whole blood were 0. 71% and 0. 74%. With the concentration of acetylsalicylic acid increased （0-6. 95 μmol/L） , MPC increased as CD62P decreased, which showed negative correlation （ r = - 0. 765,P 〈 0.01 ）. The difference of MPC before and after ARA activation （AMPC） and MPC variance ratio of the group taking aspirin were （ 8. 2 ± 8. 6） g/L and （ 3.4 ± 3.6） % , and they were （37.4 ± 10. 3） g/L and （ 15.7 ± 4. 0）% in control group, respectively. AMPC and MPC variance ratio showed significant difference between the two groups （ t = 21. 522, 22. 409, all P 〈 0. 01 ）. Area under the ROC curve for MPC variance ratio was 0. 992 with sensitivity of 96. 8% and specificity of 99.0% for monitoring the aspirin response using the cut-off of 8.7%. MPC variance ratio had good correlation with LTAARA （ r = 0. 720, P 〈 0. 01 ）. Conclusions A new method for monitoring aspirin response by platelet activated-release experiment is established. It may replace LTAARA for routine clinical examination.