高浓度胆红素对流式细胞术外周血淋巴细胞亚群检测的干扰和消除方法

Interference of high levels of bilirubin on lymphocyte subset determination in peripheral blood by flow cytometry and its elimination methods

目的探讨高浓度胆红素对流式细胞术(FCM)外周血淋巴细胞亚群检测的干扰和消除方法。方法选取51例血清总胆红素(TB)>200μmol/L且进行淋巴细胞亚群检测的患儿,将直接胆红素(DBil)升高的6例患儿纳入高DBil组,将间接胆红素(IBil)升高的45例患儿纳入高IBil组。以6名健康体检儿童(对照组)无黄疸血液样本为对照样本,将高DBil血浆加入对照样本中,制备成干扰样本。采用配对差异实验分析胆红素对FCM检测淋巴细胞亚群的干扰,并评估胆红素干扰的剂量。评估洗涤-染色-裂解法和染色-裂解-洗涤法对胆红素干扰的消除作用。结果采用FCM检测淋巴细胞亚群,高IBil组样本无影响,高DBil组样本均出现异硫氰酸荧光素(FITC)标记的CD3-与CD3^(+)细胞群荧光表达分界不清。采用染色-裂解法处理干扰样本后,出现FITC标记的CD3-与CD3^(+)细胞群荧光表达分界不清。DBil≤150μmol/L对FCM检测无影响,DBil≥200μmol/L对FCM检测的干扰较大。采用洗涤-染色-裂解法和染色-裂解-洗涤法处理均可消除DBil的干扰。经染色-裂解-洗涤法处理的干扰样本与对照样本比较,淋巴细胞亚群检测结果差异均无统计学意义(P>0.05),2种样本之间CD3^(+)、CD3^(+)CD8^(+)、CD3^(+)CD4^(+)、CD3-CD16/56^(+)、CD3-CD19^(+)细胞百分比和CD4/CD8比值呈显著正相关(r值分别为0.977、0.994、0.95、0.941、0.996、0.965,P<0.05)。结论高浓度DBil会干扰FITC的荧光信号,导致FCM检测FITC标记的CD3-与CD3^(+)细胞群荧光表达分界不清。采用染色-裂解-洗涤法处理可最大程度地消除DBil的干扰。

Objective To investigate the interference of high levels of bilirubin on lymphocyte subset determination in peripheral blood by flow cytometry(FCM)and its elimination methods.Methods Totally,51 patients with serum total bilirubin(TB)>200μmol/L and undergoing lymphocyte subset determination were enrolled,which included 6 cases of elevated direct bilirubin(DBil)(DBil group)and 45 cases of elevated indirect bilirubin(IBil)(IBil group).The non-jaundiced samples from 6 healthy children(control samples)were collected,and plasmas with high DBil were added to the control samples to prepare for interference samples.Paired difference experiments were used to analyze the interference of bilirubin on lymphocyte subsets determined by FCM and to assess the dose of bilirubin interference.The effects of washing-staining-lysis method and staining-lysis-washing method on bilirubin interference elimination were assessed.Results In elevated IBil group,lymphocyte subset determination was not interfered.But in elevated DBil group,the fluorescence expressions of fluorescein isothiocyanate(FITC)-labeled CD3-cells and CD3^(+)cells were not clearly demarcated.For interference samples treated by staining-lysis method,the fluorescence expressions of FITC-labeled CD3-cells and CD3^(+)cells were also not clearly demarcated.When DBil≤150μmol/L,no effect on FCM was found.When DBil≥200μmol/L,the interference was greater.Such interference could be eliminated to the greatest extent by both washing-staining-lysis method and staining-lysis-washing method.Staining-lysis-washing method was more operable,and no significant difference was found in lymphocyte subsets between interference samples and control samples(P>0.05),and the percentages of CD3^(+),CD3^(+)CD8^(+),CD3^(+)CD4^(+),CD3-CD16/56^(+),CD3-CD19^(+)cells and CD4/CD8 ratios between the 2 samples were positively correlated(r=0.977,0.994,0.950,0.941,0.996 and 0.965,P<0.05).Conclusions High level of DBil can interfere with the fluorescence expressions of FITC-labeled CD3-cells and CD3^(+)cells in lymphocyte subset determination in peripheral blood by FCM.Staining-lysis-washing method can eliminate such interference to the greatest extent.