Suzuki Y, Kaneko K, Saionji K
Department of Pediatrics and Clinical Pathology, Juntendo University School of Medicine, Tokyo, Japan.
Nihon Jinzo Gakkai Shi. 1995 May;37(5):71-6.
It has been reported that anti-neutrophil cytoplasmic antibody (ANCA) is a specific serological marker for systemic necrotizing vasculitis, such as Wegener's granulomatosis, microscopic polyarteritis and idiopathic crescentic glomerulonephritis. Although the standard method for ANCA detection enplays an indirect immunofluorescence technique (IIF), the method is not objective. We attempted to detect ANCA by a more objective method using flow cytometry.
Thirty ANCA positive sera (cytoplasmic in 4, perinuclear in 26) and 20 ANCA negative sera including 2 sera with anti-nuclear antibody (ANA) detected by standard IIF were used for this study. For flow cytometric analysis, heparinized blood was taken from normal healthy volunteers. Neutrophils in the heparinized blood was incubated with human recombinant tumor necrosis factor alpha (rTNF alpha) in order to facilitate reaction between the antigens of ANCA and ANCA. Primed neutrophils were incubated with sera. After washing with phosphate buffered saline, immunofluorescence-labeled antibody against human IgG was added and incubated. The mean intensity of fluorescence (MIF) of the neutrophils was measured by flow cytometry (FACS, Becton-Dickinson CO., U.S.A.) for each sample. ANCA was considered positive by flow cytometric analysis if MIF of the sample was over the mean +2SD for normal human sera.
By flow cytometric analysis, ANCA was detected in all ANCA positive samples by IIF and in 3 ANCA negative samples by IIF, of which 2 had ANA. However, it was possible to discriminate serum with positive ANCA from serum with positive ANA by flow cytograms because ANA reacted with the nuclei of both lymphocytes and neutrophils showing two peaks on the histogram.(ABSTRACT TRUNCATED AT 250 WORDS)
据报道，抗中性粒细胞胞浆抗体（ANCA）是系统性坏死性血管炎的特异性血清学标志物，如韦格纳肉芽肿、显微镜下多动脉炎和特发性新月体性肾小球肾炎。虽然检测ANCA的标准方法采用间接免疫荧光技术（IIF），但该方法不客观。我们尝试用更客观的流式细胞术方法检测ANCA。
本研究使用30份ANCA阳性血清（4份胞浆型，26份核周型）和20份ANCA阴性血清，其中2份通过标准IIF检测出抗核抗体（ANA）。为进行流式细胞术分析，从正常健康志愿者采集肝素化血液。将肝素化血液中的中性粒细胞与人重组肿瘤坏死因子α（rTNFα）孵育，以促进ANCA抗原与ANCA之间的反应。致敏中性粒细胞与血清孵育。用磷酸盐缓冲盐水洗涤后，加入抗人IgG的免疫荧光标记抗体并孵育。通过流式细胞术（FACS，美国BD公司）测量每个样本中性粒细胞的平均荧光强度（MIF）。如果样本的MIF超过正常人血清平均值+2SD，则通过流式细胞术分析认为ANCA为阳性。
通过流式细胞术分析，在所有通过IIF检测为ANCA阳性的样本中均检测到ANCA，在通过IIF检测为ANCA阴性的3个样本中也检测到ANCA，其中2个样本有ANA。然而，通过流式细胞图可以区分ANCA阳性血清和ANA阳性血清，因为ANA与淋巴细胞和中性粒细胞的细胞核反应，在直方图上显示出两个峰。（摘要截断于250字）
