多重微球流式免疫荧光法检测外周血细胞因子影响因素分析与对策

Analysis and countermeasures of influencing factors in the detection of peripheral blood cytokines by multi-microsphere flow immunofluorescence assay

为提高临床利用多重微球流式免疫荧光法检测细胞因子结果的准确性,收集临床患者的外周血标本,比较血清、血浆、不同保存条件以及干扰物质对12种细胞因子(IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70、IL-17、IFN-γ、IFN-α和TNF-α)检测结果的影响。结果显示,血浆和血清中IFN-α、TNF-α、IL-1β、IL-2、IL-4、IL-5、IL-10、IL-12p70和IL-17水平差异无统计学意义(P>0.05),而IL-6和IL-8在血清中的水平显著高于血浆(P<0.05);EDTA-K2抗凝全血在4℃保存24 h对部分细胞因子浓度有一定影响,而离心获得的血浆在4℃保存48 h细胞因子水平显著降低(P<0.05);对检测过程中微球丢失或微球荧光信号异常增强的血浆标本经小鼠免疫球蛋白预处理后,检测信号与分析结果均恢复正常。该研究提示,临床检测细胞因子建议采用EDTA-K2抗凝的血浆标本,且细胞因子在4℃保存24 h较稳定;对于检测过程中出现微球丢失或荧光信号异常增强的标本,采用小鼠免疫球蛋白进行预处理是有效的解决方法。

This study aims to improve the accuracy of multi-microsphere flow immunofluorescence assay in the clinical detection of cytokines.To this end,peripheral blood samples from patients were collected,and the effects of serum,plasma,different storage conditions,and interfering substances on the results of 12 cytokines(IL-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-10,IL-12p70,IL-17,IFN-γ,IFN-α,and TNF-α)were analyzed.The results showed that the levels of IL-6 and IL-8 in the serum sample were significantly higher than those in the plasma sample(P<0.05),while the levels of the other cytokines including IFN-α,TNF-α,IL-1β,IL-2,IL-4,IL-5,IL-10,IL-12p70,and IL-17 showed no difference between serum and plasma(P>0.05).The levels of some cytokines changed slightly when EDTA-K2 was used as anticoagulation in whole blood samples stored at 4℃for 24 h.However,the cytokines levels in plasma decreased significantly after being stored at 4℃for 48 h(P<0.05).The detection signal and analysis results of abnormal samples(missing microspheres or abnormally enhanced fluorescence signal)were returned to normal after pretreatment with mouse immunoglobulin.The study indicates that the cytokine levels in EDTA-K2 anticoagulant plasma samples or samples stored at 4℃within 24 h are more stable than those in whole blood samples.In addition,pretreatment with mouse immunoglobulin is an effective method to solve the loss of microspheres or abnormal fluorescence signals of microspheres.