H. Kobayashi et al., Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay, J ANDROLOGY, 22(4), 2001, pp. 568-574
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.