外周血B细胞上糖肌醇磷脂连接蛋白的表达在阵发性睡眠性血红蛋白尿症诊断中的应用

Estimation of Expression of Glycosylphosphatidylinositol-anchored Protein in BLymphocytes for Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

目的评价外周血B细胞上糖肌醇磷脂(glycosylphosphatidylinositol,GPI)连接蛋白之一CD59的表达能否作为诊断阵发性睡眠性血红蛋白尿症(paroxysmalnocturnalhemoglobinuria,PNH)的指标。方法采用流式细胞术双色免疫荧光分析法,结合B细胞特异性抗体CD20,分析92例外周血标本B细胞和中性粒细胞上CD59的表达,并通过临床指标评价方法,分析该指标临床应用的可行性。结果健康人B细胞上CD59均为完全阳性表达,表达率的参考范围为(96.3±1.2)%;PNH患者B细胞上CD59的表达率降低,均出现阴性或部分阳性病态细胞,病态细胞表达率为10.5%～92.3%;92.9%(26/28)非PNH贫血患者B细胞上CD59的表达均在正常参考范围内。该方法精密度的变异系数(coefficientvariation,CV)值小于4.4%,标本染色后无固定剂情况下可稳定保存24h,与常规检测CD59的方法(即中性粒细胞上CD59检测法)比较,PNH阳性和阴性检出符合率均高达100%。结论采用流式细胞术检测B细胞上CD59的表达具有简单、准确、重复性好的特点,可以成为诊断PNH的新指标。

To estimate whether the expression of glycosylphosphatidylinositol (GPI)-anchored protein in B lymphocytes could be an indicator for detecting paroxysmal nocturnal hemoglobinuria (PNH).Methods Flow cytometry and two -color McAbs were used to detect CD59expression in B lymphocytes and granulocytes of92clinical samples.The feasibility of the indicators for diagnosis of PNH was assessed.Results B lymphocytes from healthy individ uals mainly showed a single population of cells strongly positive for CD59,as the percentage of CD59expression was(96.3±1.2)%.In PNH patients,B lymphocytes showed decreased expression of CD59,as well as negative cells and partly-positive cells were observed,as the percentage of CD59expression was10.5%～92.3%.In26of28non-PNH anemia patients,CD59expression in B lymphocyte was normal,and in the other2patients,CD59expression were decreased(<95%),but no negative cell peak was observed.The precision CV of CD59in B lymphocyte assay is less than4.4%,stained samples could be kept stable for24h,the CD59in B lymphocyte assay was100%in accordance with routine assay(CD59in granulocyte assay).The sensitivity of the indicators(CD59in B lymphocytes and granulocytes)was100%,the specificity was97.4%,misdiagnosis rate was2.6%,the rate of failing to diagnosis was0%,the rate of positive forecast was87.5%,the rate of negative forecast was100%,the accuracy was97.8%.Conclusions The test of CD59expression in B lymphocytes by flow cytometry was simple,accurate and reproduceable.It could be a good marker for diagnosis of PNH because of high sensitivity and specificity.