Kobayashi H, Gil-Guzman E, Mahran A M, Nelson D R, Thomas A J, Agarwa A
Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Urological Institute, The Cleveland Clinic Foundation, OH 44195, USA.
J Androl. 2001 Jul-Aug;22(4):568-74.
A total of 28 donor semen samples were used to evaluate the characteristics of laboratory variability in measuring reactive oxygen species (ROS). The objectives of this study were to assess the interassay (same sample observed on different days by the same observers) variability; interdonor, intraobserver (replications of the same sample on the same day) variability; and interobserver (multiple observers on the same day with the same sample) variability of the luminol-dependent chemiluminescence assay and to establish an optimal semen age and sperm concentration. Semen samples were collected from 6 healthy donors for 108 measures of ROS. ROS levels were measured by the assay using luminol as the probe. An additional assessment measured the effect of time (age of the sample) on ROS production in 12 donor samples at 60, 120, 180, and 240 minutes after the specimen was produced. Last, to evaluate the effect of sperm concentration on ROS production, ROS levels were measured in 10 donor sample aliquots with sperm concentrations ranging from 1 to 120 x 10(6)/mL. In the controls, the mean ROS level was 0.218 x 10(6) counted photons per minute; the interassay variability standard deviation (SD) was 0.077. The interobserver SD was 0.002 for an interobserver reliability of 97.5% (coefficient of variation [CV] = 0.9%). The intraobserver (between replication) SD was 0.001 for an intraobserver reliability of 98.7% (CV = 0.5%). The interassay SD was 0.005 for an interassay reliability of 93.8% (CV = 2.0%). There was no statistically significant interobserver, intraobserver, or interassay variation (P > .80). ROS levels decreased significantly with time; a dramatic decline in ROS production was seen in the specimens that were more than 60 minutes old (P < .001). ROS values decreased by 31% at 120 minutes and 62% at 180 minutes compared with the 60-minute-old specimens. A linear relationship was seen between the ROS levels and sperm concentration in 8 of the 10 samples analyzed (R2 = .99). Our results demonstrate that the luminol-dependent chemiluminescence assay for ROS measurement is both accurate and reliable when the sperm concentration is greater than 1 x 10(5)/mL and the samples are analyzed within the first hour after specimen collection.
总共使用了28份供体精液样本评估测量活性氧（ROS）时的实验室变异性特征。本研究的目的是评估检测间（同一观察者在不同日期对同一样本进行观察）变异性；供体间、观察者内（同一天对同一样本进行重复检测）变异性；以及观察者间（同一天对同一样本由多个观察者检测）变异性，这些变异性是基于鲁米诺依赖性化学发光检测法的，并确定最佳精液保存时间和精子浓度。从6名健康供体收集精液样本进行108次ROS测量。使用鲁米诺作为探针通过该检测法测量ROS水平。另一项评估测量了时间（样本保存时间）对12份供体样本在样本产生后60、120、180和240分钟时ROS产生的影响。最后，为了评估精子浓度对ROS产生的影响，在10份精子浓度范围为1至120×10⁶/mL的供体样本等分试样中测量ROS水平。在对照组中，平均ROS水平为每分钟0.218×10⁶个计数光子；检测间变异性标准差（SD）为0.077。观察者间SD为0.002，观察者间可靠性为97.5%（变异系数[CV]=0.9%）。观察者内（重复检测之间）SD为0.001，观察者内可靠性为98.7%（CV=0.5%）。检测间SD为0.005，检测间可靠性为93.8%（CV=2.0%）。观察者间、观察者内或检测间均无统计学显著差异（P>.80）。ROS水平随时间显著下降；在保存时间超过60分钟的样本中观察到ROS产生急剧下降（P<.001）。与60分钟龄的样本相比，120分钟时ROS值下降了31%，180分钟时下降了62%。在分析的10个样本中的8个样本中，ROS水平与精子浓度之间呈线性关系（R²=.99）。我们的结果表明，当精子浓度大于1×10⁵/mL且样本在采集后第一小时内进行分析时，基于鲁米诺的化学发光检测法用于测量ROS既准确又可靠。
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