Factors Affecting Chemiluminescent Reactions

It is important to have a reliable and valid method of detection of ROS [29]. The luminol assay is robust; however, there are various factors that can affect ROS detection by the chemiluminescent reactions [7, 30, 34-36]. Both the probes luminol and lucigenin have great sensitivities, although the specificity is limited because of the interference of other components such as infiltrating leukocytes. Some of these confounding factors are:

1. The luminometer instrumentation, its calibration, determination of sensitivity, dynamic range, and units used.

2. The concentration and the type of probe used.

3. The concentration and volume of the semen sample, use of reagent, and temperature of the luminometer.

4. Semen age, i.e., time to analysis after sample, is collected to the time of ROS measurement.

5. Viscous samples and poor liquefaction interferes with chemiluminescent signals.

6. Repeated centrifugation: Artificial increase in chemiluminescent signal because of the shearing forces generated by centrifugation [35] .

7. Serum albumin. Use of media that contain bovine serum albumin can generate spurious signals in the presence of human seminal plasma.

8. Medium pH. Luminol is sensitive to pH changes.

9. Nonspecific interference: Many compounds can artificially increase (cysteine, thiol-containing compounds) or decrease (ascorbate, uric acid) the chemilumi-nescent signal generated by the spermatozoa. Hence, it is necessary to run spermfree controls as integral part of the chemiluminescent assay.

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Pregnancy Guide

Pregnancy Guide

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