BACKGROUND Neutrophils ex vivo in whole blood specimens are widely understood to decay rapidly when compared to other leukocytes, requiring assessment of neutrophil activity to be performed shortly after blood collection. There is a disparity in evidence for decay rates in measurements and recommended time-frames for assaying neutrophil parameters in particular assays following blood collection. We, therefore, evaluated the decline in the neutrophil respiratory burst, typically screened for assessing congenital NADPH oxidase defects, over a shorter time-course than previously published experiments. METHODS The neutrophil respiratory burst was assessed by flow cytometric detection of DHR-123 oxidation to rhodamine-123 (Rho123), following stimulation of neutrophils by phorbol myristate acetate (PMA), in heparinized healthy donor blood specimens immediately following venipuncture, and then at 3 and 5 h later with ambient temperature or refrigerated specimen storage. RESULTS A consistent time-dependent decline in the Rho123 fluorescence of PMA-stimulated neutrophils was detected in the healthy donor specimens, indicating a decay in respiratory burst activity. Neutrophil oxidative indexes calculated for half of the specimens at 3 and 5 h of age, fell below our normal laboratory lower limit. We also found that Rho123 histograms of PMA-stimulated neutrophils from stored healthy donor specimens have a risk of misinterpretation due to mimicking the appearance of histograms from carriers of CGD and other NADPH oxidase defects. Refrigeration of specimens did not significantly minimize decay. CONCLUSIONS DHR assay of the neutrophil respiratory burst from blood specimens at 3 h post-venipuncture and beyond can generate unreliable clinical measurements due to decay. © 2019 International Clinical Cytometry Society.

中文翻译：

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延迟的血液处理导致通过Dihydrorhodamine-123还原测定法对中性粒细胞呼吸爆发的测量迅速恶化。

背景技术与其他白细胞相比，全血标本中离体的嗜中性粒细胞被广泛理解为迅速衰减，需要评估血液收集后不久要进行的嗜中性粒细胞活性。血液采集后特定测量中测定嗜中性粒细胞参数的测量值衰减率和建议时间框架方面的证据存在差异。因此，我们评估了嗜中性粒细胞呼吸爆发的下降，该过程通常比以前发表的实验在较短的时间过程中进行筛选，以评估先天性NADPH氧化酶缺陷。方法采用佛波肉豆蔻酸酯乙酸酯（PMA）刺激中性粒细胞后，通过流式细胞术检测DHR-123氧化为若丹明-123（Rho123）来评估中性粒细胞呼吸爆发。静脉穿刺后立即在肝素化的健康供体血液样本中进行采样，然后在环境温度或冷藏样本存储后的3和5小时内进行采样。结果在健康供体标本中检测到PMA刺激的中性粒细胞Rho123荧光的时间依赖性持续下降，表明呼吸爆发活性下降。在3和5小时龄时，一半标本的中性粒细胞氧化指数低于我们正常实验室的下限。我们还发现，由于模仿了CGD载体和其他NADPH氧化酶缺陷携带者的直方图的外观，来自存储的健康供体标本的PMA刺激的中性粒细胞的Rho123直方图具有误解的风险。标本的冷藏并没有显着减少衰变。结论静脉穿刺后3 h及以后，血液样本中嗜中性粒细胞呼吸爆发的DHR分析可能会由于衰变而产生不可靠的临床测量结果。©2019国际临床细胞计量学会。