Prince H E, Lapé-Nixon M
MRL Reference Laboratory, Cypress, CA 90630, USA.
J Immunol Methods. 1995 Dec 15;188(1):129-38. doi: 10.1016/0022-1759(95)00210-3.
The oxidative burst generation capacity of granulocytes can be reliably detected by a flow cytometric procedure using lysed whole blood, dihydrorhodamine 123 (DHR), and phorbol myristate acetate (PMA). This assay is used to detect chronic granulomatous disease (CGD) and the CGD carrier state. To assess the feasibility of performing this assay in a reference laboratory setting, we investigated the influence of anticoagulant and specimen age on flow cytometric detection of granulocyte oxidative burst generation. Peripheral blood from 20 healthy controls was collected in acid citrate dextrose (ACD), ethylenediaminetetraacetate (EDTA), or sodium heparin (HEP) and held at room temperature. At 4, 24, 48, 72, and 96 h after collection, red cells were lysed, the white cells loaded with DHR, and then activated by PMA. Granulocyte-associated fluorescence, indicative of oxidative burst generation, was assessed by flow cytometry. Blood in any of the three anticoagulants tested gave reliable results (> 90% of granulocytes positive for fluorescence) at 4 h after collection; at 24 h after collection, HEP and ACD specimens performed slightly better than EDTA specimens. At later time points, HEP proved superior to ACD and EDTA for maintaining granulocyte oxidative burst capacity. As a demonstration of the practical utility of the assay, both CGD and the CGD carrier state were accurately detected using heparinized blood specimens analyzed 72 h after collection. These results show that heparinized blood specimens up to 72 h old can be used to reliably assess granulocyte oxidative burst generation.
使用溶血全血、二氢罗丹明123（DHR）和佛波醇肉豆蔻酸酯乙酸酯（PMA）的流式细胞术程序能够可靠地检测粒细胞的氧化爆发生成能力。该检测方法用于检测慢性肉芽肿病（CGD）和CGD携带者状态。为了评估在参考实验室环境中进行该检测的可行性，我们研究了抗凝剂和标本保存时间对粒细胞氧化爆发生成流式细胞术检测的影响。采集20名健康对照者的外周血，分别置于酸性枸橼酸盐葡萄糖（ACD）、乙二胺四乙酸（EDTA）或肝素钠（HEP）中，并在室温下保存。采集后4、24、48、72和96小时，裂解红细胞，用DHR加载白细胞，然后用PMA激活。通过流式细胞术评估指示氧化爆发生成的粒细胞相关荧光。在采集后4小时，三种测试抗凝剂中的任何一种血液都能给出可靠的结果（>90%的粒细胞荧光呈阳性）；在采集后24小时，HEP和ACD标本的表现略优于EDTA标本。在后续时间点，HEP在维持粒细胞氧化爆发能力方面被证明优于ACD和EDTA。作为该检测实用性的一个例证，使用采集后72小时分析的肝素化血液标本准确检测出了CGD和CGD携带者状态。这些结果表明，保存时间长达72小时的肝素化血液标本可用于可靠地评估粒细胞氧化爆发生成。
