储存红细胞体内外质量指标相关性分析研究  

In Vivo-in Vitro Correlation in Quality Indexes of Leukocyte-reduced Suspended Red Blood Cells

作　　者：强书毓 董晏榕 张玉龙 詹林盛 王小慧 QIANG Shu-yu;DONG Yan-rong;ZHANG Yu-long(Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850)

机构地区：[1]军事科学院军事医学研究院卫生勤务与血液研究所,北京100850

出　　处：《临床输血与检验》2022年第2期182-188,共7页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：国家自然科学基金青年科学基金项目(No.81800183)资助。

摘　　要：目的建立生物素体外红细胞标记技术平台,探究不同储存时间4℃去白悬浮红细胞输血后24 h回收率(PTR_(24h))与红细胞凋亡、膜表面分子及能量代谢等体外质量指标之间相关性。方法设置不同生物素浓度:0,1,2.5,5,7.5,10,12.5μg/mL对小鼠红细胞(约1×10^(9)/mL)进行标记,并通过流式细胞术分析确定最佳标记条件。依据最佳条件对小鼠4℃不同储存时间(0,2,4,6,8,10,12,14 d)去白悬浮红细胞进行生物素化后输入小鼠体内,24 h后采血并采用流式细胞术分析计算其PTR_(24h)。同时测定和分析不同储存时间红细胞膜表面CD47分子、磷脂酰丝氨酸(PS)、细胞内ATP含量变化。最终利用SPSS软件分析不同储存时间红细胞体外相关质量指标与其输注后体内PTR_(24h)之间的相关性。结果综合分析后确定小鼠1×10^(9)/mL红细胞的生物素标记最佳浓度为:7.5μg/mL,标记率>99%。以溶血率及红细胞凋亡率为质量控制指标,生物素标记未造成大量红细胞破裂(溶血率<1.5%)及明显细胞凋亡。CD47分子平均荧光强度从第10天开始降低(P<0.05);PS占比则从第8天开始随储存时间延长而增加(P<0.05);ATP含量从第2天起降低,与新鲜红细胞相比差异具有统计学意义(P<0.05)。不同储存时间去白悬浮红细胞输入小鼠体内后,其体内PTR_(24h)呈下降趋势,储存10 d(65.8±2.9)%、12 d(70.3±5.3)%、14 d(52.9±7.9)%,红细胞PTR_(24h)与新鲜红细胞(94.0±0.6)%输注相比降低,差异有统计学意义(P<0.05),且低于75%。对体内外指标的Pearson相关性分析结果显示,CD47、ATP均与PTR_(24h)具有强的正相关性,相关系数r值分别为0.954(P<0.01)和0.938(P<0.01),而PS则与PTR24h呈负相关,r=–0.777(P<0.05)。结论4℃不同储存时间去白悬浮红细胞的膜表面分子CD47表达量、PS外翻率及ATP含量与其PTR24h具有强相关性,而上述红细胞体外质量指标的测定可为临床评估红细胞能否有效输注提供参考。Objective To establish biotin labeling for in vitro erythrocyte technology platform to investigate the correlation between the 24-hour post-transfusion recovery(PTR_(24h))of leukocyte-reduced suspended red blood cells(RBCs)stored at 4℃for different times and the in vitro quality indexes such as apoptosis,membrane surface molecules and energy metabolism.Methods We set different NHS-biotin concentrations 0,1,2.5,5,7.5,10,12.5μg/mL to label mouse red blood cells(about 1×10^(9)/mL),and determined the optimal labeling conditions by flow cytometry.Based on the optimal conditions,the mice RBCs stored at 4℃for different storage times(0,2,4,6,8,10,12,14 d)were biotinylated and then infused into mice.After 24 hours,blood was collected and PTR_(24h) was calculated by flow cytometry.The changes of CD47 molecule,phosphatidylserine(PS)on the surface of erythrocyte membrane and intracellular ATP content at different storage time were also determined and analyzed.The correlation between PTR_(24h) in vivo and quality indexes of RBCs in vitro was analyzed by SPSS.Results The optimal concentration of biotin labeling was 7.5μg/mL in 1×10^(9) RBCs/mL with positive labeling rate as high as 99%.Taking hemolysis rate and erythrocyte apoptosis rate as quality control,biotin labeling did not cause many RBCs rupture(hemolysis rate<1.5%)and obvious apoptosis.Compared to the mean fluorescence intensity(MFI)on fresh RBCs group,CD47 expression significantly declined from day 10(80.4%)(P<0.05).External exposure of membrane PS increased to(0.7±0.0)%from day 8(P<0.05).ATP decreased significantly from day 2,and the difference was statistically significant(P<0.05).The PTR_(24h) in vivo showed a decreasing trend with extended storage time.PTR_(24h) values on day 10(65.8±2.9)%were significantly lower than that of fresh RBCs(94.0±0.6)%(P<0.05),and less than 75%.The Pearson product-moment correlation coefficient showed that there was a strong positive correlation between CD47,ATP and PTR_(24h),with r=0.954(P<0.01)and r=0.938(P<0.01),respecti

关 键 词：生物素标记 红细胞 回收率 

分 类 号：R331.141[医药卫生—人体生理学]