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Journal ArticleDOI

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

01 Mar 2002-American Journal of Clinical Pathology (Am J Clin Pathol)-Vol. 117, Iss: 3, pp 401-408
TL;DR: Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses, showing that automated analyses were highly reproducible and the Carryover rate was 0.17% or less.
Abstract: The S-FCM uses flow cytometry technology to measure sperm concentrations. Semen samples from 104 men attending a male infertility clinic were used to evaluate the reproducibility of results and the carryover rate with the S-FCM by performing between- and within-run imprecision analyses. In addition, sperm concentrations measured with the S-FCM were compared with those obtained by manual analyses with the Makler chamber and the improved Neubauer hemacytometer. The results showed that automated analyses with the S-FCM were highly reproducible and the carryover rate was 0.17% or less. In within-run imprecision assays, the coefficients of variation for the S-FCM were less than 5% at all sperm concentrations, while those for the Makler chamber were between 17.7% and 28.7% at lower sperm concentrations. The overall correlation between values measured with the S-FCM and those measured with the Makler chamber and improved Neubauer hemacytometer was excellent, but at lower sperm concentrations the correlation was lower. The S-FCM performed sperm concentration analyses in 110 seconds compared with 5 minutes for the Makler chamber and 10 minutes for the improved Neubauer hemacytometer. The S-FCM is suitable for quantitative measurement of lower sperm concentrations. Sperm counts are an essential step in the evaluation of male fertility and usually are performed by microscopic examination of semen by trained personnel. The World Health Organization (WHO) recommends the use of a hemacytometer for determining sperm concentrations in semen, 1 and several methods of using these devices have been described. 1-3 However, the accuracy and reproducibility of analytic data obtained with hemacytometers reportedly are not good enough to compare results from different institutions. 4-6 A number of studies have shown that manual counting is associated with the greatest potential for gross analytic errors in measuring sperm concentrations. 4-8 Consequently, computer-assisted semen analyzers were developed to overcome the subjectivity of sperm counting, but most of these analyzers use some sort of counting chamber or hemacytometer that is itself a source of intrinsic errors. When using a counting chamber or hemacytometer, only spermatozoa located in it can be counted, and only very small quantities of specimens are used to estimate the sperm concentration in 1 mL of semen. Recently we developed a new device (S-FCM) for measuring sperm concentrations that uses flow cytometry technology and an argon laser as the light source. To improve accuracy, the S-FCM device uses 90 times greater quantities of semen than are used with a counting chamber or hemacytometer. In this study, we compared the accuracy and reproducibility of the S-FCM with that of the Makler sperm counting chamber. We also evaluated the feasibility of using the S-FCM in clinical settings by comparing the analytic data with those obtained by using the improved Neubauer hemacytometer or the Makler chamber.
Citations
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Journal ArticleDOI
TL;DR: 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.

94 citations


Cites background or methods from "Automated sperm concentration analy..."

  • ...The poor precision of the Makler chamber also appears to affect the accuracy and it’s use in a validation study could lead to an underestimate the accuracy of the new method [19]....

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  • ...In recent years, different types of haemocytometers have been used for validation of new protocols for determination of sperm concentration by flow cytometry [15–22]....

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  • ...Several authors [15–20] have recently validated various procedures for flow cytometric determination of sperm concentration....

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Journal ArticleDOI
TL;DR: A new flow cytometric method has been developed to rapidly determine sperm concentration and viability in semen from bulls and boars, and it was found that this system was two and four times more accurate than the spectrophotometer and the electronic cell counter, respectively.
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.

74 citations

Journal ArticleDOI
TL;DR: The automated SQA-V analyzer is more precise and shows the ability to accurately classify normal versus abnormal sperm morphology and can be used interchangeably with manual semen analysis methods for examining sperm concentration and motility.

46 citations

Journal ArticleDOI
TL;DR: The information covered in the article includes sample preparation and the use of manual counts, spectrophotometers, computer-assisted semen analysis, NucleoCounter, and flow cytometry.

34 citations


Cites background or methods from "Automated sperm concentration analy..."

  • ...[8,31,34,35,37]; FSC + other 3 stains [33]; FSC + SSC + positive DNA stain of permeabilized cells [39]; FSC + SSC + 4 viability with DNA marker + cytoplasma marker; FSC + SSC + viability with two DNA 5 markers....

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  • ...9 x 10 (6)/mL, were analysed [33], it is not clear whether 20 this rate would be higher when animal samples with higher sperm concentrations are analysed....

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  • ...Sample preparation: undiluted; diluted [32]; diluted and stained [8,31,33-38]; diluted, 20 permeabilized, and stained [30,39,40]....

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Journal ArticleDOI
TL;DR: 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: 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.

18 citations


Cites background or methods from "Automated sperm concentration analy..."

  • ...Calculation of sperm concentration5(sperm count4bead count)6dilution factors [10]6fluorescent bead stock concentration (825610(3)/mL)....

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  • ...Flow cytometry has been used to investigate characteristics of sperm such as concentration [7,10], cell viability [11–13], plasma membrane integrity [14–16], mitochondrial function [17], capacitation and acrosomal status [18,19], Ca in sperm [20], chromatin structure Figure 2....

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  • ...Moreover, it is possible that the degree of overestimation of sperm concentration is related to particles and debris present in ejaculate [30], because some authors have reported that flow cytometry is suitable for quantitative measurement of lower sperm concentrations [10]....

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References
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Journal ArticleDOI
TL;DR: Data highlight the urgent need for improvement in overall quality of andrology testing and indicate that practical proficiency testing programmes can be made available on a large scale.
Abstract: Proficiency testing samples for antisperm antibodies (ASAB), sperm count, morphology and vitality were mailed to participating laboratories. The majority participating utilized Immunobead ASAB procedures (81 versus 14% mixed antiglobulin reaction and 5% 'other'), and there was 95.6 +/- 1.2% agreement on the presence or absence of ASAB. The majority of laboratories utilized manual (79%) versus computer assisted semen analysis (CASA; 15%) methods. Approximately 64% used the haemocytometer and 26% used the Makler counting chambers for manual counts. Coefficients of variation (CV) in sperm counts ranged from 24 to 138%, with CASA displaying lower overall CV (53 +/- 8%) than manual methods (80 +/- 9%). A wide variation in the reports of percent normal morphology was noted (CVs calculated from arc sin transformed means ranged from 15 to 93%). Participants using American Society of Clinical Pathologists (ASCP) criteria reported sperm morphology values that were clustered in the 'normal' range (11 out of 12 samples), while those using strict criteria were clustered in the 'abnormal' range (10 out of 12 samples). Good agreement was observed in sperm vitality (overall mean CV = 18%). These data highlight the urgent need for improvement in overall quality of andrology testing and indicate that practical proficiency testing programmes can be made available on a large scale.

97 citations

Journal ArticleDOI
TL;DR: A single-step computer system using visual evaluation of the microscopic field and manual tracking of sperm movement yields more reliable results, but technician expertise remains essential for correct performance.
Abstract: Objectivity in assessing critical sperm characteristics such as concentration, motility and morphology, has been the target of many systems and devices. Methods based on physical principles such as turbidimetry, spectrophotometry, laser Doppler technology, are too imprecise or technically too complicated to be applied routinely in the laboratory. Microscopic images providing photographical, cinematographical or video data acquisition followed by manual or computer-assisted image analysis (CASA) suffer from practical and/or device deficiencies, making them tedious and time-consuming, or unreliable because of confounding factors and uncontrolled artefacts. A single-step computer system using visual evaluation of the microscopic field and manual tracking of sperm movement yields more reliable results, but technician expertise remains essential for correct performance. The scientific basis of multiple errors in semen aspiration, chamber quality, data acquisition and analysis is given. Biased interpretations of results or incorrect handling of statistical data are criticised.

55 citations

Journal ArticleDOI
TL;DR: 20 MicroCell Chambers proved to be accurate and precise for determining concentration and motility of semen specimens whether analyzed manually or with CASA.

45 citations

Journal ArticleDOI
TL;DR: In this article, the authors developed methods of instrument calibration and standardization that enable the use of computer-aided sperm analysis (CASA) technology in multicenter studies of sperm motility.

44 citations

Journal ArticleDOI
TL;DR: The minimum number of spermatozoa needed for stable results, the variability of measurements and optimum methods of sampling the ejaculate were determined for one videomicrographic computer-automated semen analysis system.
Abstract: Videomicrographic computer-automated semen analysis systems allow quantitative description of sperm motility, velocity, progression, and head movement amplitude and frequency with unprecedented ease. The minimum number of spermatozoa needed for stable results, the variability of measurements and optimum methods of sampling the ejaculate were determined for one such system (Cell-Soft, CRYO Resources, New York, NY). Sampling a minimum of 225 spermatozoa yields stable measurements, and analyzing four microscope fields in triplicate provides data with the lowest coefficient of variation. The variability attributable to the instrument itself was acceptable for all measurements (6.2% to 15.4%) except mean amplitude of lateral head displacement. Limitations of these results and the potential utility of videomicrographic sperm movement analysis are discussed.

43 citations

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The S-FCM is suitable for quantitative measurement of lower sperm concentrations.