Is flow cytometry really adapted to the determination of sperm concentration?

It has been reported that flow cytometry can be used as a reference procedure to determine sperm concentrations in quality control schemes in andrology laboratories, but there are no convincing quality control data. To understand comprehensively whether flow cytometry can be used to determine sperm concentration, sperm concentrations of 85 human semen samples were detected using three different methods, namely flow cytometry, computer-assisted semen analysis (CASA) and manual counting with a cell-VU chamber. The bead concentrations of both low [(18+/-2.5)x10(6)/mL] and high [(35+/-5)x10(6)/mL] pre-calibrated standard latex bead solutions were also determined with flow cytometry. The results showed that bead concentrations of both low and high pre-calibrated standard latex bead solutions counted five times with flow cytometry were (21.37+/-0.85)x10(6)/mL and (45.95+/-1.76)x10(6)/mL, respectively. Coefficient variances (CVs) and relative errors (REs) were 4%, 15.51% and 3.84%, 31.3% for low and high latex bead solutions, respectively. The overall correlation between values measured with flow cytometry and values measured with the cell-VU chamber and the CASA system was significant. However, flow cytometry overestimated the sperm concentration by 109% compared to the results with the cell-VU chamber. Moreover, for the azoospermic samples analysed, the sperm concentration was estimated at 0.12 (range from 0.04 to 0.24)x10(6)/mL. In conclusion, the data demonstrated that flow cytometry can result in an overestimation of both bead counting and sperm concentration, suggesting that flow cytometry is an inappropriate method for sperm counting, especially in the case of azoospermia.