循环血小板活化与血小板-白细胞黏附的流式细胞分析法及其应用  

Flow cytometry for assessing circulating platelet activation and plateletleukocyte adhesion

作　　者：崔巍[1] 刘飒[1] 董磊[1] 袁慧[2] 杨平[2] 吴依娜[1] 李玉琳[1] 

机构地区：[1]首都医科大学附属北京安贞医院北京市心肺血管疾病研究所心血管重塑相关疾病教育部重点实验室,北京100029 [2]首都医科大学附属北京安贞医院检验科,北京100029

出　　处：《首都医科大学学报》2015年第3期414-419,共6页Journal of Capital Medical University

基　　金：国家自然科学基金项目(81230006,81000069)~~

摘　　要：目的建立流式细胞术(flow cytometry,FCM)分析循环血小板活化(circulating platelet activation,CPA)与血小板-白细胞黏附(platelet-leukocyte adhesion,PLA)方法,为临床开展血小板相关疾病研究奠定基础。方法选择50例健康志愿者,收集枸橼酸钠抗凝血,采用CD61/PAC-1/CD62P三色法检测CPA,分析血小板早期活化率(PAC-1^+CD62P^-)和晚期活化率(PAC-1^+CD62P^+);并设置0、5、10、15、20、30、60 min 7个时间点染色,分析样本放置时间对血小板活化的影响,建立参考值。采用CD61/CD3/CD19/CD11b/CD14/CD45六色法检测PLA,分析血小板一白细胞黏附CD45^+CD61^+(PLA)、血小板-T淋巴细胞黏附CD3^+CD61^+(PTLA)、血小板-B淋巴细胞黏附CD19^+CD61^+(PBLA)、血小板-单核细胞黏附CD14^+CD61^+(PMA)、血小板-中性粒细胞黏附CD45^(low+)CD11b^+CD61^+(PNA)表达率。结果宜选用15 min点建立健康人血小板活化参考范围,早期活化百分比为:3.75(1.00~9.80)%,晚期活化百分比为:0.10(0.00~0.10)%;FCM检测PIA、PTLA、PBLA、PMA、PNA表达率,其中PMA比例最高为:(10.75±3.23)%。结论 CPA检测血样放置时间不应超过15 min,PLA检测关键在于PMA分析。建立快速、稳定、准确的CPA和PLA检测方法是正确评价血小板功能的技术保障。Objective To assess the circulating platelet activation and platelet leukocyte adhesion by flow cytometry and lay the foundation for the clinical development of platelet associated diseases.Methods The sodium citrate anti-coagulation blood was collected from 50 cases of healthy volunteers,using CD61/PAC- 1/CD62 P to detect platelet activation;analysis the percentage of early activation（PAC- 1＋ CD62P-） and late activation（PAC- 1＋ CD62P＋）.Moreover,the different time points,such as 0 min,5 min,10 min,15 min,20 min,30 min,and 60 min were detected the platelet activation.Via CD61/CD3/CD19/CD11b/CD14/CD45 detected platelet activation,and analyzed the percentages of platelet leukocyte adhesion（CD45＋ CD61＋）,platelet-T lymphocyte adhesion（CD3＋CD61＋）,platelet-B lymphocyte adhesion（CD19＋ CD61＋）,platelet monocyte adhesion（CD14＋ CD61＋）,platelet-neutrophil adhesion（CD45low＋CDllb＋ CD61＋）.Results The time point of 15 minutes was detected to establish the reference range for healthy human platelet activation：the percentage of early activation was 3.75（1.00-9.80）%,late activation was 0.10（0.00-0.10）%.Detected the percentages of platelet leukocyte adhesion,platelet-T lymphocyte adhesion,platelet-B lymphocyte adhesion,platelet-monocyte adhesion and platelet-neutrophil adhesion showed platelet-monocyte adhesion was the highest proportion（10.75 ± 3.23）%.Conclusion Storage time of blood samples should not exceed 15 min to detect platelet activation.Platelet-monocyte adhesion was the key role in platelet activation analysis.Establishment the fast,stable,accurate evaluation method of platelet activation is important.

关 键 词：循环血小板活化 血小板-白细胞黏附 流式细胞术 

分 类 号：R558[医药卫生—血液循环系统疾病]