Automated sperm concentration analysis with a new flow cytometry-based device, S-FCM.

Abstract: Determination of sperm concentration by use of a haemocytometer or counting chamber is an important step in semen evaluation and is also used for calibration or validation of instruments. Three experiments were carried out to determine the precision and accuracy of the Makler chamber, the Thoma haemocytometers (50 and 100 microm deep, TH-50, TH-100) and the Burker-Turk (BT) haemocytometer. The first experiment confirmed that precision in sperm count by use of the haemocytometers (TH-50 and BT) can be increased if a higher number of sperm are counted. In contrast, the precision of the Makler chamber was relatively unaffected by the number of sperm counted and the coefficient of variation for this chamber was significantly (P < 0.05) higher than for the two haemocytometers. Experiment 2 confirmed the low precision of the Makler chamber and also showed that the TH-50 haemocytometer underestimated sperm concentration by approximately 25% in comparison to the Makler chamber and the BT haemocytometer. Experiment 3 demonstrated a slight underestimation of sperm count by the TH-100 haemocytometer in comparison to the BT haemocytometer, but both haemocytometers yielded acceptable precision (coefficients of variation were 10.4% and 9.4%, respectively). In comparison, the precision of the Makler chamber was significantly poorer (coefficient of variation 18.6%). When used for validation of a flow cytometric method which determines sperm concentration, the Makler chamber caused a higher degree of scattering of the points around the regression line than when the flow cytometric method was validated against the BT haemocytometer. It thus appears that the poor precision of the Makler chamber also affects the accuracy. It is concluded that duplicate counts by at least two technicians is recommended to achieve high precision but, that particular caution should be exerted with regard to the precision and accuracy of the used counting device.

Abstract: A new flow cytometric method has been developed to rapidly determine sperm concentration and viability in semen from bulls and boars. Sperm viability was determined on the basis of staining with SYBR-14 and propidium iodide (PI), and this allowed detection of live (membrane-intact) sperm, dying (moribund) sperm, as well as dead cells. Fluorescent microspheres (beads) were used to determine sperm concentration. The use of SYBR-14 at 50 nM and PI at 12 μM in combination with the FACSCount diluent in the counting tubes resulted in a uniform staining after 2.5–5 minutes at room temperature. Reagent staining was reproducible enough to allow subsequent semiautomated analysis of data using Attractors software. In experiment 1, this method was used to analyze semen from boars, rams, rats, rabbits, humans, and turkeys. In experiment 2, Attractors analysis was performed by the FACSCount AF flow cytometer, and sperm concentration determination with this system was compared with results obtained by a spectrophotometer and an electronic cell counter, which is routinely used by bull artificial insemination centers. When compared to microscopic counting in a hemocytometer, the FACSCount AF flow cytometer was two and four times more accurate than the spectrophotometer and the electronic cell counter, respectively. In addition, the FACSCount AF flow cytometer determined both sperm concentration (coefficient of variation 3.3%) and sperm viability (coefficient of variation 0.7%) with high precision.

Abstract: Objective To evaluate the performance of the automated semen quality analyzer system for assessing sperm quality. Design Double-blind prospective study. Setting Tertiary care hospital. Patient(s) Fifty healthy men donated semen samples. Intervention(s) None. Main Outcome Measure(s) Precision, accuracy and agreement between automated and manual semen analysis methods was assessed for sperm concentration, motility, morphology, and known concentrations of latex bead quality control media. Result(s) A good agreement was seen between the results of sperm concentration reported by the SQA-V automated analyzer (Spermalite/SQA-V; Medical Electronic Systems Ltd, Caesarea Industrial Park, Israel) and those obtained manually. A similar linearity was seen when the SQA-V results were compared with the manual data and also when the manual results of individual operators were compared with each other. The automated assessment of morphology showed high sensitivity (89.9%) for identifying percent normal morphology, and the precision of the SQA-V was considerably higher when compared with the manual method. The interoperator variability for manual assessment was significant. The automated analysis was quick compared with the manual method. Conclusion(s) The SQA-V can be used interchangeably with manual semen analysis methods for examining sperm concentration and motility. The automated SQA-V analyzer is more precise and shows the ability to accurately classify normal versus abnormal sperm morphology.

Abstract: This article is the result of the work of the andrology task-force of the Association of Applied Animal Andrology, American College of Theriogenologists, European College of Animal Reproduction, Society for Theriogenology, and National Association of Animal Breeders. It is intended to serve as a comprehensive reference on methods to evaluate sperm concentration and to contribute to the adoption of best practices in veterinary andrology laboratories. The information covered in the article includes sample preparation and the use of manual counts, spectrophotometers, computer-assisted semen analysis, NucleoCounter, and flow cytometry. Emphasis is given to the principles of the methods and equipment, performing the evaluation, and common mistakes and/or pitfalls. In addition, the precision and accuracy of the different methods are also discussed.

Abstract: 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.

Abstract: An external quality control study for semen analysis was performed involving 10 andrology laboratories in geographically separate locations. For the evaluation of sperm concentration, eight samples with different concentrations, and for the assessment of sperm morphology three slides prepared from different semen samples were distributed. Sperm motility was evaluated in five samples delivered cryopreserved to the participants. The coefficients of variation (CVs) for sperm counts varied with the sperm concentrations showing highest variability for low and lowest for high concentrations (range 23% to 73%). The CV for sperm morphology ranged from 25% for normal heads to 87% for abnormal midpieces. The CV for motility of sperm was 21%. For comparison the mean CVs for internal quality control were as follows: 10% for concentration, 8% for morphology (normal heads), and 8% for motility.

Abstract: To test the influence of parameter settings on results of automated semen analysis, microscopic images of 43 semen samples were videotaped. Tapes were analyzed by the CellSoft system (CRYO Resources Ltd., New York, NY) at 12 different parameter combinations for gray scale, number of frames analyzed, and minimum number of frames analyzed for velocity measurements. The data indicate that values obtained for concentration, percentage of motile sperm, velocity, and linearity depend, to varying degrees, on the settings of the computer. It is concluded that results of automated semen analysis may not be comparable among different laboratories unless identical parameter settings are used. The need for general agreement of these details among different users is stressed.

Abstract: Semen samples from 77 men were used to estimate the accuracy and precision of measurements of sperm density, percent motility, and motion characteristics using a new, fully automated, computer-assisted semen analyzer (CASA). Results are compared with traditional semen analysis methodology. Acceptable precision for count and percent motility was obtained using three to nine random fields of observation when there were more than 10 cells per high-power microscopic field. A highly significant correlation (P

Abstract: A new chamber for sperm count and motility estimation is described. This chamber, which is only 10 micron deep, enables free horizontal movement of spermatozoa in one focal plane and provides conditions for the examination of undiluted samples. Therefore, with the aid of this instrument it is possible to compare sperm motility in various samples from the same person or in different samples at different times. This can be done either by simple estimation or with any other method of motility evaluation chosen by the examiner. The sperm count can be made rapidly and directly from an undiluted, preheated sample by counting spermatozoa in the area of a grid located within the eyepiece; the count is expressed in millions per milliliter. Thirty-seven specimens were analyzed with this chamber. Statistical evaluation of the results revealed high precision, accuracy, and reliability of sperm counts when compared with the hemocytometric method. Better results were obtained when motility estimation was compared with the ordinary slide technique. Easy performance, rapid sperm counts, and improvement of motility estimation make this chamber a useful tool where sperm analysis is carried out.