Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay

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 observe d on different days by the same observers) variability; interdonor, intraob server (replications of the same sample on the same day) variability; and i nterobserver (multiple observers on the same day with the same sample) vari ability of the luminol-dependent chemiluminescence assay and to establish a n optimal semen age and sperm concentration. Semen samples were collected f rom 6 healthy donors for 108 measures of ROS. ROS levels were measured by t he 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 ev aluate the effect of sperm concentration on ROS production, ROS levels were measured in 10 donor sample aliquots with sperm concentrations ranging fro m 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 reliab ility of 97.5% (coefficient of variation [CV] = 0.9%). The intraobserver (b etween 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 9 3.8% (CV = 2.0%). There was no statistically significant inter-observer, in traobserver, or interassay variation (P > .80). ROS levels decreased signif icantly with time; a dramatic decline in ROS production was seen in the spe cimens 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-ol d specimens. A linear relationship was seen between the ROS levels and sper m concentration in 8 of the 10 samples analyzed (R-2 =.99). Our results dem onstrate that the luminol-dependent chemiluminescence assay for ROS measure ment is both accurate and reliable when the sperm concentration is greater than 1 x 10(6)/mL and the samples are analyzed within the first hour after specimen collection.