CD109表达量及保存方法的相关性研究

The correlation study of the expression and preserve method of HPA-15 antigen

目的分析中国南方人群HPA-15基因型分布频率,研究血小板上HPA-15抗原表达量。寻找较好的血小板保存方法以维持HPA-15的高表达量,为单克隆抗体俘获血小板抗原技术(MAIPA)检测HPA-15同种抗体提供合适的供者血小板。方法 1)随机选取广州血液中心的健康血小板单采献血者109名,留取EDTA抗凝管全血。SSPPCR方法检测HPA-15基因型。流式细胞术检测血小板上HPA-15抗原表达量。2)另随机选取健康献血者的单采血小板67例,将每份血小板分为2份,1份重悬于EDTA-PBS中放于4℃保存作为检测组A,另1份留于血小板辫子中放于血小板振荡仪中作为检测组B,在d 0、1、2、3、7、14检测2组血小板上HPA-15的检测含量,比较2组HPA-15含量的变化。结果 1)109名随机健康献血者HPA-15a和HPA-15b的基因频率是0.495和0.505。流式细胞术检测HPA-15抗原的MFI值的范围2.44-20.4,阳性率3.2%-86.7%。不同HPA-15基因型之间,HPA-15抗原表达量有统计学差异(P<0.05)。2)分析比较AB这2个组,随着测量时间点的推移,2组HPA-15的表达量均有降低,但检测组B中HPA-15的表达含量高于检测组A,且2组的MFI值有统计学差异(P<0.05)。结论 HPA-15a和HPA-15b的基因分布频率相近,HPA-15抗原表达含量整体水平偏低且个体差异性大。体外保存方式下,HPA-15的表达含量随着时间推移逐渐降低,4℃保存方式下HPA-15含量降解尤为明显。血小板振荡仪中保存血小板的方法为MAIPA检测HPA-15抗体提供更合适的血小板抗原。

Objective To analyze the frequencies of HPA-15 genotypes and the HPA-15 antigen expression on platelet in southern China,and to find a better platelets preserve method which can sustain the HPA-15 expression,and to provide the suitable donor platelet for the detection of HPA-15 antibodies using MAIPA assay. Methods 1） A total of 109 healthy blood donors were collected in Guangzhou Blood Center,China. EDTA-anticoagulated blood was kept. SSP-PCR method was used to test HPA-15 genetype. The HPA-15 expression was determined on the platelets by flow cytometry. 2） Another 76 healthy blood donors were divided into two groups. Platelets in Group A were suspended in PBS-BSA and stored in 4℃. Platelets in Group B were stored in an platelet orbital shaker. HPA-15 expression of the two groups was determined by flow cytometry on days 0,1,2,3,7,14. Results 1） In the 109 donors,the allele frequencies of HPA-15 a and HPA-15 b are 0. 495 and 0. 505.The MFI of HPA-15 expression ranged from 2. 44 to 20. 4. The positive rates ranged from 3. 2% and 86. 7%. The expression of HPA-15 antigen have statistical difference among different HPA-15 genetypes（ P〈 0. 05）. 2） In the two compared groups,according to different determination time point,the MFI of the two groups has statistical difference during the storage（ P〈 0. 05）. Although the HPA-15 MFI of the two groups both declined during this period,the HPA-15 expression of group B was better than group A. Conclusions The HPA-15 a and HPA-15 b have similar frequency. The expression of HPA-15 on platelet is low and varies among different individuals. The HPA-15 expression decreases during storage,especially in 4℃. Platelets stored in platelet orbital shaker are better to provide donor platelet for detecting HPA-15 antibodies by MAIPA.