Emmendörffer A, Nakamura M, Rothe G, Spiekermann K, Lohmann-Matthes M L, Roesler J
Department of Immunobiology, Fraunhofer Institute, Hannover, Germany.
Cytometry. 1994 Sep 15;18(3):147-55. doi: 10.1002/cyto.990180306.
Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)-patients (4 CGD+), and from 18 X-linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry. Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed. Cells were activated to produce H2O2 by the phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria. To evaluate the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured. The results were compared to those from other methods (NBT-testing, cytochrome c-reduction, and especially chemiluminescence). Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 +/- 59 (S.D.), 97% < 890, for pediatric patients. Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, x = 830 +/- 52). For CGD+ (n = 4) the results were clearly far below the normal range. In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence. Cytochrome b558-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5. The normal range was 97% > 485, 97% < 680, peak channel fluorescence. We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods.
对50名吞噬细胞功能正常的儿科患者、150名健康成年人、10名慢性肉芽肿病（CGD）患者（4名CGD阳性）以及18名CGD的X连锁携带者的中性粒细胞，使用二氢罗丹明123染色及随后的流式细胞术检测其过氧化氢生成情况。此外，还评估了3名髓过氧化物酶缺乏症患者的中性粒细胞。通过佛波酯肉豆蔻酸佛波醇酯（PMA）以及吞噬大肠杆菌来激活细胞以产生过氧化氢。为评估该方法的敏感性，测定了不同浓度二苯基碘鎓（DPI）抑制的中性粒细胞过氧化氢生成情况。将结果与其他方法（硝基蓝四唑试验、细胞色素c还原试验，尤其是化学发光试验）的结果进行比较。根据峰值通道荧光评估正常数值和散射图谱范围：儿科患者中，97%＞700，x = 840±59（标准差），97%＜890。婴儿（＜1岁，n = 6，x = 830±52）的少量血样也得出了正常定量值。对于CGD阳性患者（n = 4），结果明显远低于正常范围。在表明活性氧中间体生成减少方面，该方法至少与光泽精增强化学发光法一样敏感。用单克隆抗体7D5染色后，通过流式细胞术确定患者和健康对照中性粒细胞的细胞色素b558表达。正常范围为97%＞485，97%＜680，峰值通道荧光。我们得出结论，与传统方法相比，CGD的流式细胞术常规诊断可轻松提高对该疾病识别的可靠性和信息获取量。
