Standardization of the use of opsonized zymosan as stimulus in the 1,2,3-dihydrorhodamine technique for the assessment of neutrophil respiratory burst

Uriel Pérez-Blanco, Jenniffer Yissel Girón, Guillermo Juárez-Vega, María Jiménez, Carlos Sánchez, Ricardo Rioja, Sara Espinosa-Padilla, Lizbeth Blancas-Galicia
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引用次数: 0

Abstract

Introduction: Chronic granulomatous disease is a defect in phagocytosis due to deficiency of gp91phox, p22phox, p47phox, p40phox, and p67phox (classic form of the disease). Recently, EROS and p40phox deficiency were described as responsible for the non-classical form of the disease. The 1,2,3-dihydrorhodamine oxidation technique, with phorbol-12-myristate-13-acetate as a stimulus, is performed to diagnose the classic chronic granulomatous disease. However, oxidation mediated by EROS and p40phox requires stimuli such as zymosan, Escherichia coli, or Staphylococcus aureus.

Objective: To optimize the 1,2,3-dihydrorhodamine technique using zymosan to assess neutrophil respiratory burst and detect the non-classical chronic granulomatous disease.

Materials and method: Blood was obtained from five healthy subjects after the signature of the informed consent. The 1,2,3-dihydrorhodamine technique was performed with phorbol-12-myristate-13-acetate as control and different quantities of opsonized zymosan (150, 100, 50, 20, and 10 μg). We obtained through flow cytometry the mean fluorescence intensity of rhodamine 1,2,3 oxidated in the neutrophil population and calculated the oxidation index. The Kolmogorov-Smirnov test, ANOVA, and Tukey’s post-hoc analysis were used. We considered a p value ≤ 0.05 as statistically significant.

Results: The phorbol-12-myristate-13-acetate increased the rhodamine 1,2,3 mean fluorescence intensity in healthy subjects. Among the different zymosan conditions tested, we selected 50 μg as the optimal and reproducible amount in all controls according to the statistical analysis and cytometric findings.

Conclusions: We present the optimization of the 1,2,3-dihydrorhodamine technique using zymosan. We propose its implementation in clinical diagnostic laboratories to expand the diagnosis of chronic granulomatous disease.

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[1,2,3-二氢罗达明技术中作为刺激物的异位zimosan的标准化,以评估中性粒细胞的呼吸爆发]。
介绍。慢性肉芽肿病是由gp91phox、p22phox、p47phox、p40phox和p67phox(典型形式)缺乏引起的吞噬细胞病的一种缺陷。最近,EROS和p40phox的缺陷被描述为非经典形式的原因。1,2,3-二氢罗达明的氧化技术——使用福波12-槲皮酸-13-乙酸作为兴奋剂——被用于诊断经典的慢性肉芽肿病。然而,为了检测EROS和p40phox介导的氧化,需要其他刺激物,如zimosan、大肠杆菌或金黄色葡萄球菌。目的:对1,2,3-二氢罗达明技术中的zimosan刺激进行标准化,以评估中性粒细胞的呼吸爆破。材料和方法。在知情同意的情况下,从5名健康受试者身上抽取血液。采用1,2,3-二氢罗达明法,以福波-12- miristate -13-乙酸酯为对照,并使用不同剂量的异位胞嘧啶(150、100、50、20和10微克)。通过流量计,我们得到了中性粒细胞群中罗达明1,2,3的平均荧光强度,并计算了氧化指数。采用Kolmogorov-Smirnov检验、方差分析和Tukey post-hoc分析。p≤0.05被认为具有统计学意义。结果:Forbol -12-miristato-13-乙酸酯提高了健康受试者中罗达明1,2,3的平均荧光强度。在测试的不同条件下,通过统计分析和细胞测定结果,50 μg是所有对照组的最佳和可重现量。结论:本文介绍了1,2,3-二氢罗达明技术的优化,使用zimosan作为刺激剂。
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