Laboratory Application of Chemiluminescent Assays

From the above descriptions, it will be evident that while chemiluminescent probes such as luminol and lucigenin cannot yield specific data on ROS generation by human spermatozoa, they are nevertheless sensitive, semiquantitative indicators of redox activity in these cells and many publications have shown that the chemilumi-nescent activity detected in their presence is inversely related to sperm function and significantly elevated in the cases of male infertility [23, 46-54]. A unique prospective study, conducted in the days before assisted conception therapy was readily available, went so far as to demonstrate an excellent relationship between spontaneous pregnancy rates over a 4-year follow-up period and measurements made at the beginning of the study of luminol-dependent chemiluminescent signals generated by the spermatozoa in couples characterized by a normal female factor [55]. The predictive power of the luminol-dependent assay used in this study was all the more impressive because within this data set the conventional criteria of semen quality (count, morphology, and motility) were of no diagnostic significance whatsoever.

As discussed above, the major caveat with the clinical interpretation of such studies is that if the level of leukocyte contamination has not been meticulously assessed, then the source and significance of the enhanced chemiluminescent activity are open to question. Much will depend on when, where, and how the leukocytes were activated [56]. Another clinically significant issue with chemiluminescence as a diagnostic tool is that the readout of individual luminometers depends heavily upon the response characteristics of the photomultipliers used to monitor the chemilumines-cent response. Since every individual luminometer is different in this respect, any attempt to describe a threshold level of chemiluminescence as a universal reference point for normal sperm function, though desirable, is simply not achievable [57],

14.2.3.1 Chemical Interference with Chemiluminescence Assays

While the sensitivity of chemiluminescent probes is extremely valuable, it renders such systems very susceptible to interference in a manner that may distort their diagnostic information content. Some of these confounding factors are described below:

1. Time to analysis. Chemiluminescent activity tends to decline with time following the isolation of spermatozoa from seminal plasma [46]. As a result, such assays are best conducted within 1 h of sperm isolation [58].

2. Bovine serum albumin. Supplementation of culture media with bovine serum albumin (BSA) has the potential to generate spurious chemiluminescent signals in the presence of human seminal plasma. These signals are generated because most commercial BSA preparations are heavily contaminated with polyamine oxidase(s), which will generate luminol-dependent chemiluminescence on contact with the polyamines (spermine and spermidine) in human seminal plasma and/or coated onto the surface of human spermatozoa [29, 59] .

3. MediumpH. Chemiluminescence systems are sensitive to changes in pH; for example, a change of 1 pH unit from pH 7.4 to 8.4 has a dramatic effect on the quantum yield of luminol [29] .

4. In the luminol-peroxidase system, the presence of electron donors such as NADPH or cysteine will generate massive chemiluminescent signals without any need for spermatozoa; it is entirely a chemical artifact. Alternatively, many small molecular mass-free radical scavengers, such as ascorbate or uric acid, will quench cellular chemiluminescence as will the common pH indicator, phenol red [29]. In light of this susceptibility to nonspecific interference, it is always advisable to run cell-free controls as an integral part of chemiluminescence assays.

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