流式细胞术检测HLA-C分子表达水平的影响因素探讨  

Exploration on factors influencing HLA-C molecular expression level by flow cytometry

作　　者：李宇楠 江任辉 蔡思齐 刘洁 邓志辉[1] LI Yunan;JIANG Renhui;CAI Siqi;LIU Jie;DENG Zhihui(Shenzhen Blood Center,Shenzhen 518040,China;Department of Transfusion,The Eighth Affiliated Hospital,Sun Yat-Sen University,Shenzhen 518033,China)

机构地区：[1]深圳市血液中心,广东深圳518040 [2]中山大学附属第八医院输血科,广东深圳518033

出　　处：《中国输血杂志》2025年第1期79-84,共6页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金青年项目(32301234);广东省基础与应用基础研究基金委员会项目(2022A1515011045);深圳市科技创新委员会基础研究项目(JCYJ20190806152001762);深圳市医学重点学科(SZXK070);深圳市血液中心中心课题(SZBC202409)。

摘　　要：目的探讨流式细胞术检测HLA-C分子表达水平的影响因素。方法收集2024年3—5月外周造血干细胞志愿捐献者CD34细胞计数检测后剩余的单采造血干细胞悬液标本共12例,分别探讨读取有核细胞不同数量(50万、5万、0.5万个)、加入红细胞裂解液及抗体的先后顺序不同、距离失效期时长不同的HLA-C抗体,对HLA-C表达水平检测结果的影响,并采用Student t检验分析组间差异显著性。结果分别读取50万、5万、0.5万个有核细胞,3组数据组间HLA-C阳性细胞占比、平均荧光强度差异均无显著性(P>0.05);加入红细胞裂解液及抗体的先后顺序不同,对于HLA-C阳性细胞占比差异无显著性(P>0.05);但“先裂解红细胞再加抗体”的HLA-C MFI值显著低于“先加抗体再裂解红细胞”的MFI值(P<0.05);使用距失效期仍有24个月的HLA-C抗体所检出的阳性细胞占比及MFI值,显著高于距失效期仅剩有5个月的HLA-C抗体(P<0.05)。结论本文探讨了流式细胞术检测HLA-C分子表达水平的影响因素,研究结果对规范HLA-C表达水平的实验操作、提高检测结果的准确性和可比性具有良好的参考和应用价值。Objective To investigate the factors influencing the detection of HLA-C expression by flow cytometry.Methods A total of 12 hematopoietic stem cell suspension samples from peripheral hematopoietic stem cell volunteer donors were randomly collected after CD34+cell counting detection.The influence of detecting different number of nucleated cell(500000,50000 and 5000),sequential order of red blood cell lysis and antibody incubation,and the HLA-C antibody with varied remaining time from the expiration date on the detection results of HLA-C expression by flow cytometry were investigated,respectively.The significance of differences between different groups was analyzed through Student t test.Results There was no significant difference in the proportion of HLA-C positive cells and mean fluorescence intensity(MFI)among the three groups with different nucleated cell numbers detected(500000,50000 and 5000)(P>0.05).The sequential order of red blood cell lysis and antibody incubation had no influence on the proportion of HLA-C positive cells(P>0.05),but HLA-C MFI value was significantly lower when antibody incubation was performed after red blood cell lysis than that when antibody incubation was performed before red blood cell lysis(P<0.05).The proportion of HLA-C positive cells and MFI value detected by HLA-C antibody remaining 24 months from the expiration date were significantly higher than those detected by HLA-C antibody remaining only 5 months from the expiration date(P<0.05).Conclusion The present study has investigated the factors of influencing HLA-C expression level by flow cytometry,the results have important reference and application value for standardizing the experimental operation of HLA-C expression and improving the accuracy and comparability of detection results.

关 键 词：人类白细胞抗原(HLA) HLA-C基因 表达水平 流式细胞术 平均荧光强度(MFI) 

分 类 号：Q344.13[生物学—遗传学] R446.113[医药卫生—诊断学] R331.142[医药卫生—临床医学]