不同预处理方法消除流式细胞术检测细胞因子分析干扰的效果

Effects of interference elimination using different pretreatment methods during cytokine determination using flow cytometry

目的 采用不同方法预处理流式细胞术多细胞因子检测结果异常升高的血浆样本,比较其消除分析干扰的效果。方法选取流式细胞术多细胞因子检测结果异常升高的血浆样本32例,分别采用样本稀释、20%聚乙二醇6000 (PEG-6000)沉淀法和嗜异性抗体阻断管(HBT)预处理上述血浆样本后,检测白细胞介素(IL)-2、IL-4、IL-6、IL-10、IL-17、γ干扰素(IFN-γ)和肿瘤坏死因子(TNF) 7种细胞因子的表达水平,并与电化学发光法的IL-6检测结果进行比较。结果 流式细胞术细胞因子检测结果异常升高的血浆样本经过倍比稀释后,7种细胞因子检测结果与稀释倍数未呈线性关系的比例>60%;与稀释倍数未呈线性关系的血浆样本经过PEG-6000沉淀法和HBT预处理后,与未经预处理的样本相比,7种细胞因子流式细胞术检测结果均明显下降(均P <0.05);与稀释倍数呈线性关系的血浆样本经过PEG-6000沉淀法和HBT预处理后,与未经预处理的样本相比,IL-6、IL-10、IFN-γ和TNF亦呈下降趋势(均P <0.05)。PEG-6000沉淀法和HBT预处理后血浆样本的流式细胞术IL-6检测结果与电化学发光法检测结果均呈较好的相关性,且PEG-6000沉淀法预处理后的相关性高于HBT。结论 应用PEG-6000沉淀法和HBT预处理样本,有助于消除流式细胞术多细胞因子检测中的分析干扰。

Objective To compare the effects of interference elimination by using different pretreatment methods on plasma samples with abnormally elevated flow cytometry-detected cytokine levels. Methods Thirty-two plasma samples with abnormally elevated cytokine levels were selected. The expression levels of seven cytokines were detected using flow cytometry:interleukin(IL)-2,IL-4,IL-6,IL-10,IL-17,interferon-γ(IFN-γ),and tumor necrosis factor(TNF). The samples were pretreated with either:dilution,20% polyethylene glycol 6000(PEG-6000) precipitation,or a heterophilic blocking tube(HBT). The IL-6 levels detected after pretreatments were compared with those detected by electrochemiluminescence. Results For plasma samples with abnormally elevated cytokines detected by flow cytometry,the percentages of the seven cytokines showing nonlinear relationships with the dilution ratio were all >60%. The levels of the seven cytokines in the plasma samples showing a nonlinear relationship with the dilution ratio after the pretreatments with PEG-6000precipitation method and HBT were significantly lower than those in the samples without pretreatments(all P < 0.05). In addition,IL-6,IL-10,IFN-γ,and TNF in the plasma samples showing a linear relationship with the dilution ratio also showed a downward trend after the pretreatments(all P < 0.05). Electrochemiluminescence detected that IL-6 after pretreatments with the PEG-6000 precipitation method and HBT showed a positive correlation,and the correlation was higher in the samples pretreated by PEG-6000 than those pretreated by HBT. Conclusion The PEG-6000 precipitation method and HBT can eliminate analytical interference when using flow cytometry to detect various plasma cytokines.