Evaluation of flow cytometric methods to measure human sperm concentration

Human sperm concentration is usually assessed using hemocytometry (HM). How ever, external and internal quality control schemes have shown that the acc uracy of this method is low overall. Flow cytometry (FC) is a rapid, accura te, and reproducible technology for the quantification of various cell popu lations. We used 3 FC methods for human sperm counting from a 1:1 mixture o f a diluted semen sample with a suspension of fluorospheres of known concen trations. The events that represented sperm cells were detected according t o 1) gating on size and granularity (FCM1), 2) gating on DNA staining by pr opidium iodide (FCM2), and 3) a combination of FCM1 and FCM2 (FCM3). Sperm concentration was calculated from the ratio of detected events to fluorosph ere counts and fluorosphere concentration. A pilot study undertaken by 12 t echnicians from different laboratories to compare FCM1 with HM showed a gen eral agreement between both methods, despite wide variations in sperm conce ntration exhibited by HM due to the use of unoptimized procedures. A second experiment indicated that the overall variability in sperm concentration a ssessment by FCM1 was lower than that produced by HM when performed by 2 te chnicians using optimal procedures for 3 preparations of the same semen sam ples. The overall mean coefficients of variation were 3.9% for FCM1 vs 8.0% for technician 1, 12.3% for technician 2 (P < .05), and 15.7% for both tec hnicians (P < ,05). FCM1, FCM2, and FCMS were compared with HM performed by a single trained technician for 39 semen samples (triplicates) of various quality. Compared with HM, FCM1 and FCM2 overestimated the sperm concentrat ion by 14% and 8%, respectively, against only 4% per million sperm for FCM3 , which was effective for the full spectrum of sperm concentrations (except azoospermia). In conclusion, this study demonstrates that human sperm conc entration can be accurately assessed by the FC method combining gating on c ell size, granularity, and DNA staining.