优化流式细胞术测定保存血小板CD62p表达  

Modified Flow Cytometry Assay for CD62p Expression of Preserved Platelets

作　　者：欧阳锡林[1] 刘景汉[1] 石群[1] 高大勇[2] 

机构地区：[1]解放军总医院输血科,解放军临床输血中心北京100853 [2]中国科技大学热科学及能源工程系低温生物医学研究所

出　　处：《中国实验血液学杂志》2002年第5期462-465,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目编号 39970 812

摘　　要：CD62p阳性率是保存血小板质量监测的重要指标之一。为在静脉全血内血小板CD62 p测定方法的基础上优化建立流式细胞术 (FCM)测定保存血小板表达CD62p的方法 ,在血小板检测标本内加入 0 .1mmol/L潘生丁稳定血小板 ;对TBS溶液进行了改良 ,添加了 1 .1mmol/L的EDTA和 1 0 0 μmol/L潘生丁 ,用以代替PBS ;采用新鲜富含血小板血浆 (FPRP)制备的阴、阳性对照 ;进行方法学评价。结果表明 :加入 0 .1mmol/L潘生丁和 1 .1mmol/L的EDTA可以有效防止实验过程中的血小板人为激活 ;采用新鲜富含血小板血浆 (FPRP)制备的阴、阳性对照既可优化FCM分析方法 ,还可用于检验所购荧光抗体的质量 ,保证实验结果的有效性。采用泛血小板特异性标志物CD61可灵敏特异地识别血小板 ,提高了实验抗干扰能力。制备保存血小板时采用的是专用大号采血针 ,抽血流畅 ,对血小板CD62p表达测定人为影响很小 ,明显优于用注射器采集患者静脉全血的做法。CD62 p测定灵敏度可达 1 % ,制备的标本可稳定 48小时。结论 :利用该流式细胞术可以灵敏而特异地测定保存血小板的CD62P表达率 ;该方法简便易行 ,提高了结果的准确性 。CD62p expression was an important monitoring parameter for preserved platelets quality. To set up an optimized flow cytometry assay for preserved platelets based on the CD62p expression on platelets, the platelet samples were collected, 0.1 mmol/L persantine and 1.1 mmol/L EDTA were added into the modified TBS used to replace PBS dilution; the methodological evaluation were carried out. Results showed that 0.1 mmol/L persantine and 1.1 mmol/L EDTA achieved to prevent platelets activation during the test procedure. The favorable negative or positive samples were prepared for check of fluorescence antibody′s quality to ensure the validity of results. CD61 was used to identify platelets for assay and improve veracity of assay. The special injector was also replaced by special big syringe needle for blood collection to reduce in vitro artifacts, and the prepared sample can be steady going for 48 hours at 4℃ after fixed by 1% paraformaldehyde. It is concluded that this flow cytometry assay for CD62p positive platelets is simple and efficient.

关 键 词：流式细胞术 血小板保存 CD62p阳性率 质量监测 测定方法 

分 类 号：R331.143[医药卫生—人体生理学] R45[医药卫生—基础医学]