应用全血法检测中性粒细胞表面膜蛋白酶3表达及其意义

Detection of Membrane Proteinase 3 Expression on Neutrophils Using a Whole Blood Method and Significance of mPR3 in ANCA-associated Vasculitis

目的建立应用洗涤的全血标本,通过流式细胞术检测中性粒细胞表面膜蛋白酶3(mPR3)的方法,并分析mPR3在抗中性粒细胞胞浆抗体(ANCA)相关性血管炎中的意义。方法采集55例全身型韦格纳肉芽肿病(genWG)、15例局限型韦格纳肉芽肿病(locWG)、20例Churg-Strauss综合征(CSS)患者和30例健康对照者(HC)的全血标本,用流式细胞术检测中性粒细胞表面的mPR3和CD63,记录阳性细胞的百分数和中位荧光强度(MFI)比。结果分离提取的中性粒细胞和洗涤全血中表达mPR3的中性粒细胞百分数(r=0.818,P<0.001)和MFI比(r=0.836,P<0.001)均显著相关。mPR3在中性粒细胞表面呈现单峰低表达、双峰表达和单峰高表达3种模式。mPR3+中性粒细胞百分数在genWG高于HC(P<0.001)、CSS(P=0.003)和locWG(P=0.004);反映mPR3表达水平的MFI比在genWG亦高于HC(P<0.001)、CSS(P=0.028)和locWG(P=0.028)。中性粒细胞表面mPR3+百分数和MFI比,在HC、CSS和locWG组差异均无统计学意义。结论全血法可检测"体内"循环状态中性粒细胞表面mPR3,方法简便快速、切实可行。中性粒细胞表面高表达mPR3仅见于genWG,在CSS和locWG未见增高。

Objective To determine whether the flow cytometric analysis of mPR3 expression on isolated neutrophils is applicable to whole blood（WB） samples by assessing the reproducibility and correlation of these two methods in healthy controls and to analyze mPR3 expression on neutrophils in ANCA-associated vasculitis.Methods Neutrophil form patients with generalized Wegener granulomatosis（genWG,n = 55）,localized WG（locWG,n = 15）,Churg Strauss Syndrome（CSS,n = 20） and healthy controls（n = 30） were analyzed.mPR3 and CD63 expression on surface of neutrophils were assessed by flow cytometric analysis on isolated neutrophils and whole blood.Results The percentage and median fluorescence intensity（MFI） ratio of mPR3 expression on isolated neutrophils and whole blood method correlated significantly（r = 0.818,P 0.001,and r = 0.836,P 0.001,respectively）.Three patterns of neutrophil mPR3 expression：monomodal low,bimodal,and monomodal high were identified with both methods.In genWG,both the percentages and MFI ratios of mPR3＋ neutrophils were significantly increased compared with healthy controls（P 0.001,and P 0.001,respectively）,CSS（P = 0.003,and P = 0028,respectively）,and locWG（P = 0.004,and P = 0.028,respectively）.There were no significant differences in percentages or MFI ratios of mPR3＋ neutrophils between healthy controls and CSS,locWG.Conclusion The WB method described in this report to quantify mPR3 on neutrophils is fast,reproducible and correlates excellent with isolated neutrophils method.High mPR3 expression on neutrophils is noted only in genWG,not CSS or locWG.