酸洗脱血小板HLA-Ⅰ类抗原的研究  

Establishment and assessment of a method for acid elution of platelet HLA class I antigens

作　　者：刘相富[1] 邹勇[1] 刘文达[1] 陆英[1] 覃雪玲[1] 李芳[1] 廖思红[1] 袁青[1] 刘贵莲[1] 林哲生[1] 黎结芳[1] 林东军[1,2] 

机构地区：[1]中山大学附属第三医院输血科,广东广州510630 [2]中山大学附属第三医院血液科,广东广州510630

出　　处：《中国病理生理杂志》2011年第10期1995-1999,共5页Chinese Journal of Pathophysiology

基　　金：广东省科技计划资助项目(No.2008B030301055)

摘　　要：目的:建立一种有效酸洗脱血小板表面HLA-Ⅰ类抗原的方法,评估最适的酸洗脱条件以及这一技术临床应用的可行性。方法:血小板用不同pH值(pH=2、3、5、7)的柠檬酸缓冲液在0℃进行酸洗脱,多色流式细胞技术检测血小板表面HLA-Ⅰ类抗原及P-选择素(CD62P)的表达,Annexin V染色检测血小板的早期凋亡,电阻法检测血小板的最大聚集率。结果:随着柠檬酸缓冲液pH值的降低(从pH=7到pH=2),血小板表面HLA-Ⅰ类抗原的表达显著减少,但同时血小板的活化率(CD62P表达)和早期凋亡率(Annexin V表达)均显著增加。相对PBS处理,利用pH=3的柠檬酸缓冲液处理血小板可显著减少血小板表面HLA-Ⅰ类抗原的表达(P<0.05),尽管血小板活化率和早期凋亡率均显著增加(均P<0.05),但其聚集功能未受显著影响(P>0.05)。结论:利用pH=3的柠檬酸缓冲液0℃洗脱血小板HLA-I类抗原可作为预防血小板输注无效的方法进行尝试。这项酸处理技术在临床应用前尚需进一步改进和标准化。AIM：To establish an effective method for acid elution of platelet HLA class I antigens,and to evaluate the optimal condition and the feasibility of clinical application of the acid-elution technique.METHODS： Platelets were treated with citric acid buffer at different pH levels（pH=2,3,5,7）.Expression of HLA class I antigens and P-selectin（CD62P） on the platelet surface was analyzed by multicolor flow cytometry.The proportion of early apoptotic platelets was detected by Annexin V staining.The maximum platelet aggregation rate was determined by electrical impedance aggregometry.RESULTS： With the decrease in the pH levels of the citric acid buffer（from pH=7 to pH=2）,the expression of HLA class I antigens on the platelet surface was remarkably decreased.However,the rates of platelets activation（CD62P expression） and early apoptosis（Annexin V expression） were both significantly increased.Compared with PBS,treatment of the platelets with citric acid buffer at pH 3.0 remarkably reduced the expression of platelet HLA-class I antigens（P〈0.05）.Although the rates of the platelet activation and apoptosis were also significantly increased（P〈0.05）,the aggregation of platelet was not remarkably reduced（P〉0.05）.CONCLUSION： Acid elution of platelet HLA-class I antigens with citric acid buffer at pH 3.0 at 0 ℃ can be use as an attempt to produce HLA-eluted platelets.This technique of acid-elution needs further improvement and standardization before clinical use.

关 键 词：血小板 人类白细胞抗原 CD62P 细胞凋亡 酸洗脱 

分 类 号：R331.143[医药卫生—人体生理学]