滤白机采血小板消除白细胞干扰血小板RNA实验的研究  

Study on the elimination of leukocyte interference with apheresis platelet RNA assay

作　　者：林凤秋 李晓丰 李剑平 LIN Fengqiu;LI Xiaofeng;LI Jianping(Liaoning Blood Center,Key Laboratory of Blood Safety Research of Liaoning,Key Laboratory of Blood Safety Research of Shenyang,Shenyang 110044,China)

机构地区：[1]辽宁省血液中心、辽宁省血液安全研究重点实验室、沈阳市血液安全研究重点实验室,辽宁沈阳110044

出　　处：《中国输血杂志》2024年第10期1164-1168,共5页Chinese Journal of Blood Transfusion

基　　金：辽宁省自然科学基金项目(2021-MS-366);沈阳市科技创新平台建设计划项目(21-104-0-15)。

摘　　要：目的探讨滤白机采血小板中混入的少量白细胞对血小板RNA相关检测的影响及进一步去白细胞的方案。方法将滤白机采血小板进行分组处理。1)对照组:直接离心收集血小板细胞;2)离心纯化组:将血小板分别以不同离心力进行离心,收集沉淀细胞组分、回收悬浮细胞组分;3)磁珠纯化组:用磁珠法去除血小板中的CD45阳性白细胞,回收CD45阴性血小板细胞。分别提取以上细胞的总RNA,并逆转录合成cDNA。以cDNA为模板PCR扩增管家基因β-actin、血小板标志物ITGA2B、白细胞标志物PTPRC基因片段。根据扩增产物电泳结果判断血小板中的白细胞对血小板mRNA检测的影响,以及离心纯化法和磁珠纯化法去白细胞的效果。结果滤白机采血小板直接离心所得细胞、滤白机采血小板以不同条件离心后收集的沉淀细胞和悬浮细胞,其cDNA中白细胞标志物PTPRC cDNA检测均呈阳性;磁珠法纯化后血小板cDNA中PTPRC cDNA检测呈阴性。结论滤白机采血小板中混入的少量白细胞可干扰血小板RNA相关检测。简单离心法不能去除血小板中的白细胞污染,但磁珠法可有效进一步去除白细胞。Objective To investigate the effect of a small amount of leukocyte mixed in platelets on platelet RNA related study,and to further propose a method to remove leukocyte contamination.Methods The collected platelets were divided into groups and then processed.1)Control group:Cells were collected by direct centrifugation;2)Centrifuge purification group:Centrifuge with different centrifugal forces to collect precipitated cells and recover suspended cells;3)Magnetic bead purification group:Remove CD45 positive leukocyte from platelets using magnetic bead method,and recover CD45 negative platelet cells.Total RNA was extracted from the cells,and cDNA was reverse transcribed from the extracted RNA.The fragments of housekeeping geneβ-actin,platelet marker ITGA2B and leukocyte marker PTPRC were PCR amplified using cDNA as a template.The results of the PCR amplification were used to determine the influence of leukocyte contamination in platelet mRNA study and the effect of centrifugation purification and magnetic bead purification.Results PTPRC gene could be detected in cDNA from platelet cells obtained by direct centrifugation.PTPRC gene was also detected in cell segments collected from both precipitated and suspended cells after centrifugation under different conditions.But PTPRC was not detected in platelet after removal of CD45 positive cells by magnetic bead.Conclusion A small amount of leukocyte contamination in platelets can interfere with the mRNA study of platelets.The simple centrifugation could not remove the leukocyte contamination in platelets,but the magnetic bead had a better purification effect.

关 键 词：滤白机采血小板纯化 白细胞混入 血小板 RNA 

分 类 号：R457.1[医药卫生—治疗学] R331.143[医药卫生—临床医学]