Slide preparation and staining procedures for reliable results using computerized morphology

The purpose of this study was to standardize slide preparation and staining procedures to improve the efficiency and effectivity of the IVOS system on normal sperm morphology readings with regard to the strict criteria. Semen samples from patients attending the Reproductive Biology Unit, Tygerberg Hospital, were used. In experiment 1, five different Diff-Quik staining procedures, including the standard procedure, were evaluated on each of 22 patients and the effect of slide preparation within 1 h or more than 5 h after collection and the effect of immediate fixation versus fixation after 24 h were observed. In experiment 2, the manual evaluation time per slide (n = 20) by two technicians was compared with the time taken by computer. In experiment 1 the median % normal for the 5 different staining procedures was 6, 6.5, 9.5, 8.5, and 5.5%. No significant difference was found between the different staining procedures (p =.60, nonparametric Friedman test). In experiment 2 the mean time for manual assessment by two technicians was 3 min:6 s and 3 min:53 s per slide as compared to 4 min:39 s by computer. For experiment 1, slides can be prepared immediately or after 5 h. Fixation time also does not interfere with the computer's ability to identify normal forms. For experiment 2, the IVOS system is competitive regarding assessment time. Standardization of optimum staining procedures is important to ensure repeatability and comparability. Therefore, slides should be prepared immediately after liquefaction and fixed immediately after air drying.