摘要
目的对应用干化学发光免疫分析仪与流式细胞仪比较检测人血小板膜分子表达的特异性、准确性和重复性等参数进行验证,开发基于共振能量转移技术的均相化学发光法检测血小板膜受体分子和活化功能的新方法。方法采用干化学发光免疫分析仪检测45例健康对照组、45例阳性刺激剂组外周血中血小板膜表面分子标志物CD41、血小板-白细胞聚集体分子标志物CD45、经凝血酶受体活化因子肽6(thrombin receptor activator peptide 6,TRAP6)活化前后的血小板活化标志物CD62P表达,与流式细胞术检测结果进行对比。结果干化学免疫发光仪检测得CD41、CD45和CD62P表达的校准曲线拟合度较好,CV%较理想。干化学免疫发光仪检测得CD45、TRAP6活化后的CD62P发光强度与流式细胞仪测得的平均荧光强度具有正相关性(r=0.3165,P=0.0341;r=0.3159,P=0.0345);百分比与流式细胞仪测得的表达百分比呈正相关性(r=0.3630,P=0.0142;r=0.4165,P=0.0044)。干化学免疫发光仪检测CD41、CD45、CD62P表达百分比的AUC分别为0.843、0.977、0.810。结论共振能量转移技术可有效分析血小板表面分子表达水平和活化功能,在临床具有一定的应用价值。
Objective To explore and evaluate the specificity,accuracy and reproducibility of the parameters for detecting platelet molecular expression using a dry chemiluminescence immunoassay and flow cytometry methods.We developed a novel homogeneous chemiluminescence method for detecting platelet membrane receptor molecules and activated functions based on chemiluminescence resonance energy transfer(CRET).Methods The peripheral blood samples of 45 healthy control subjects and 45 subjects treated with a positive stimulant were detected using a dry chemiluminescence immunoassay.The expressions of the corresponding platelet membrane surface molecular marker CD41,platelet-leukocyte aggregates molecular marker CD45 and platelet activation marker CD62P before and after TRAP6 activation were measured and compared with the results of flow cytometry.Results The calibration curves of CD41,CD45 and CD62P were well fitted and the CV%was ideal.The detection of CD45 and TRAP6-activated CD62P luminescence intensity by the dry chemiluminescence immunoassay was positively correlated with the average fluorescence intensity measured by the flow cytometer(r=0.3165,P=0.0341;r=0.3159,P=0.0345).The percentage of CD45 and TRAP6-activated CD62P was positively correlated with the percentage measured by the flow cytometer(r=0.3630,P=0.0142;r=0.4165,P=0.0044).AUCs of CD41,CD45,and CD62P expression percentages detected by the dry chemiluminescence immunoassay were 0.843,0.977,and 0.810,respectively.Conclusion The chemiluminescence resonance energy transfer technology can be used to effectively analyze the expression level of surface molecules on platelets,and show certain application value in clinical practices.
作者
何林燕
曲乐
左元玲
阮长耿
赵益明
HE Linyan;QU Le;ZUO Yuanling;RUAN Changgeng;ZHAO Yiming(The First Affiliated Hospital of Soochow University,Jiangsu Institute of Hematology,National Clinical Research Center for Hematologic Diseases,NHC Key Laboratory of Thrombosis and Hemostasis,Collaborative Innovation Center of Hematology,Suzhou 215006,China;Laboratory Medicine Center,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou Municipal Hospital,Suzhou Key Laboratory of Intelligent Critical Illness Biomarkers Translational Research,Suzhou 215006,China;The First Affiliated Hospital of Soochow University,Suzhou 215000,China)
出处
《标记免疫分析与临床》
2025年第1期164-168,共5页
Labeled Immunoassays and Clinical Medicine
基金
江苏省科教强卫工程-临床医学中心资助(编号:YXZXA2016002)。
