Evaluation of a disposable plastic Neubauer counting chamber for semen analysis

Transcription

1 TECHNIQUES AND INSTRUMENTATION Evaluation of a disposable plastic Neubauer counting chamber for semen analysis Jackson Kirkman-Brown, Ph.D., a and Lars Bj orndahl, M.D., Ph.D. a,b a Centre for Human Reproductive Science, The Assisted Conception Unit, Birmingham Women s Health Care Trust, and Division of Reproductive and Child Health, The Medical School, The University of Birmingham, Birmingham, West Midlands, United Kingdom; and b Centre for Andrology and Sexual Medicine, Karolinska University Hospital, Huddinge, and Karolinska Institutet, Stockholm, Sweden Objective: To evaluate whether disposable plastic counting chambers effectively could replace nondisposable, time-consuming, and potentially dangerous glass hemocytometers. Design: Evaluation of equipment in modern laboratory andrology. Comparison of results obtained with plastic chambers with results obtained with gold-standard glass hemocytometer counts. Setting: Diagnostic laboratory for andrology. Patient(s): Twenty-one patients undergoing investigation for infertility problems. Intervention(s): No interventions with patients; sperm in diluted semen samples were used when patients had allowed the use for research and training. Main Outcome Measure(s): Sperm concentration, difference from results obtained with standard equipment. Result(s): In the first three experimental series, with use of standard routine phase-contrast microscopy, significantly lower count results were obtained consistently from the plastic chambers than from standard In the fourth series, with use of specialized equipment, equivalent results were obtained but with a considerably greater time commitment because of difficulties in distinguishing sperm adjacent to the gridlines in the plastic Conclusion(s): The plastic disposable chamber type was not suitable for routine semen analysis because results are variable depending on the microscope used, and increased time is necessary to do the assessment accurately. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Sperm concentration assessment, semen analysis, quality control, standardization, improved Neubauer hemocytometer Development of robust and standardized methods in diagnostic andrology was initiated several decades ago. These methods most recently have been revised and enhanced by recommendations from the World Health Organization (1), the Nordic Association for Andrology, and the European Society of Human Reproduction and Embryology (2). Continued efforts to improve the efficiency of these standard analyses must be combined with clear evaluation and validation of any procedural simplifications or use of less complicated materials to avoid any negative impact on the quality and consistency of the measurements. Two main issues have been identified as important for the correct assessment of sperm concentration: sampling of an Received September 13, 2007; revised November 22, 2007; accepted November 26, Presented as a poster at the Annual Meeting of the Nordic Association for Andrology in Stockholm, Sweden, August 31 September 1, Reprint requests: Lars Bj orndahl, M.D., Ph.D., Centre for Andrology and Sexual Medicine, Karolinska University Hospital, Huddinge M52, S Huddinge, Sweden (FAX: ; Lars. Bjorndahl@ki.se). exact semen volume for dilution and an exact volume to assess in the counting chamber. The former is best achieved with a positive displacement pipette for sampling semen (3). The hemocytometer is considered the standard counting chamber (1, 2, 4, 5) because of the high degree of accuracy with regard to chamber depth. Most other types of chamber are 10 to 20 mm deep, compared with the 100 mm of hemocytometers. Using 100 mm as chamber depth has several advantages. A slight inaccuracy in coverslip attachment will have a relatively small effect on the total depth. Furthermore, the high volume contained in the chamber enables assessment of five times as many sperm in the same area, compared with a 20-mm chamber, provided the same dilution is used. In addition, it is known that 20-mm chambers filled by capillary action consistently give lower results than hemocytometers (e.g., Tomlinson et al. [6]). Recently, it was suggested that this outcome depends on flow dynamics in the low-depth chambers, a phenomenon that does not occur in 100-mm chambers (7, 8). Even if hemocytometers give more accurate and consistent results, there are aspects that could be improved. Before /09/$36.00 Fertility and Sterility â Vol. 91, No. 2, February doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 assessing a sample, the chamber must be assembled, and attachment of the coverslip requires much care. A properly positioned coverslip should have several iridescence lines visible where the coverslip is attached to the counting chamber and should not be easily dislodged, for example, by a simple touch of the pipette tip used to fill the chamber. Sperm are very prone to adhering to glass surfaces and can stay attached even after extended exposure to detergents. Therefore the chamber has to be cleaned thoroughly after use. This procedure usually includes gentle physical cleaning (e.g., with a fingertip in a rubber glove) of the glass surface. To be sure that no sperm remain in the cleaned chamber, microscopic inspection is recommended. Both the mounting of the coverslip and the cleaning of chamber and coverslip involve a not negligible risk of injury by broken glass and thus a risk of infection. A disposable plastic chamber with a fixed cover would avoid the problems of cleaning and would thus decrease the risk of injury and infection, as well as eliminating the risks of inaccuracies associated with poor coverslip placement. The aim of this study was to evaluate a new disposable plastic 100-mm hemocytometer as an alternative to a conventional glass hemocytometer. MATERIALS AND METHODS This study used anonymous semen samples produced for diagnostic tests at the andrology laboratories of the Assisted Conception Unit, Birmingham Women s Health Care Trust Birmingham, West Midlands, United Kingdom, and the Centre for Andrology and Sexual Medicine, Karolinska University Hospital, Stockholm, Sweden. All samples included in the study were used with the consent of the patients concerned, and no ethical approval was required because the methodology fell within the bounds of routine laboratory validation procedures. All dilutions were analyzed on the day the sample was produced. Initial volumes of semen were taken with a positive displacement pipette and diluted with either a defined dilution medium containing formaldehyde (Stockholm [2]) or with distilled water (Birmingham). Either diluent will immobilize sperm, and the differing protocols were due to local hospital regulations for the use of chemicals. Directly after dilution each suspension was mixed on a vortex mixer (setting: maximum; duration: at least 5 seconds). Before loading the counting chambers, each dilution was mixed again by vortexing for at least 15 seconds. Neubauer hemocytometers (1, 2), which are currently used as standard in the both laboratories (examples of suppliers: VWR International, Lutterworth, Leicestershire, UK; VWR International AB, Stockholm, Sweden, were used as control. Coverslips were mounted and checked for presence of iridescence lines before loading. The test chambers, plastic 100-mm deep disposable chambers (incyto C-Chip DHC-N01, batch 12GD12; Digital Bio Technology Co. Ltd., Seoul, Korea, were supplied in individually packed slides (two chambers per slide) with the same improved Neubauer pattern as the glass slides. Glass chambers and plastic slides were loaded in immediate sequence for each sample. Where the same dilution was used for several chambers in parallel, glass and plastic slides were loaded alternately, and the loading pipette (air displacement pipette, 10 ml) was used to stir up the suspension between filling each chamber. After loading, the FIGURE 1 Individual results from 18 consecutive samples in glass and plastic chambers, respectively (series 1). Left: pairwise scatter diagram; right: Bland-Altman plot showing systematic error. 628 Kirkman-Brown and Bj orndahl Techniques and instrumentation Vol. 91, No. 2, February 2009

3 slides were kept 15 to 30 minutes in a humidified chamber to allow sedimentation of cells before assessment. Assessment was done under phase-contrast microscopy (Olympus BX40 with standard phase-contrast optics [20 objective 0.40 Ph1 N/0.17] or Olympus BX50 [20 objective: UPlan Fl, 0.50 Ph1; N/0.17]) (Olympus, Tokyo, Japan). A Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan) (objective: Plan Fluor 10/0.25, Ph1 DL N/0.17 WD 16, with an intermediate lens giving a total magnification of 200 in oculars and corresponding magnification and quality in the camera) was used for photomicrography (Sony SSC- DC58AP color video camera [Sony, Tokyo, Japan]; image capture with Picsara 8.81 rev. 2, Bildanalyssystem AB; Euromed Networks, Stockholm, Sweden). All counting was done according to recommended standards, meaning that for each final reading, two chambers were assessed (1, 2). FIGURE 2 Results from multiple assessments of different sperm dilutions. In series 2 (n ¼ 7) and series 3 (n ¼ 5) significantly lower concentrations (a: P<.05) were obtained with the plastic In series 4 (n ¼ 5) a microscope with high-quality optics was used (Olympus BX50), and efforts were made to trace sperm that could be hidden because of optical phenomena (b: P>.05). In this last series no difference was observed between the two types of The initial experiment (series 1) was a straightforward comparison of the two Eighteen samples were assessed with use of both glass and plastic slides by a single operator. When the results showed statistically significant differences between the chambers, further experiments were devised to investigate the cause(s) including the possibility of bias due to, for instance, systematic differences in loading of chambers and time for sedimentation (series 2 and 3). When such sources of errors were not confirmed, the influence of difference in visual perception was investigated (series 4). In series 2, a series of seven assessments of one single suspension was performed in parallel in glass and plastic slides by one investigator. In series 3, another investigator made a series of five assessments of another single suspension in parallel in glass and plastic slides. In series 4, the first investigator did a similar series of five assessments as in series 3, using a microscope with high-resolution positive phase-contrast optics (Olympus BX50). In this series considerable time and effort were invested in searching for sperm concealed in bright lines in the plastic TABLE 1 Comparison between results obtained with glass and plastic counting chambers, respectively. Series 1 Series 2 Series 3 Series 4 No. of samples No. of slides per sample Optics High-quality phase Group mean (10 6 /ml) Glass Plastic Group SD Glass Plastic P value.0003 a.0387 b.0079 c.4209 b a Glass compared with plastic, Wilcoxon matched-pairs test. b Glass compared with plastic, t-test. c Glass compared with plastic, Mann-Whitney U-test. Fertility and Sterility â 629

4 Statistical Analysis The Wilcoxon matched-pairs test was used for series 1 (pairwise analysis) and the Mann-Whitney U-test (group-wise analysis) or t-test (when the Kolmogorov-Smirnov test verified Gaussian distribution) in the other series. All statistical calculations were performed with use of GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA, RESULTS In series 1, the results obtained in the plastic slides were on average 15% lower than those obtained in the glass slides. In total 17 of 18 samples revealed lower concentrations in the plastic slides (Fig. 1). In the second series, only one sample was assessed but in seven parallel slides to evaluate whether there was a difference in both average and variability. This was not the case, because the values for SD were equivalent whereas the averages differed significantly. These findings were verified in series 3 where assessments were done by another investigator. In the last series, a microscope with higher optical quality was used, and the investigator actively searched for sperm hidden in the bright lines of the plastic slides. In this series there was no statistically significant difference between the two types of counting slides. All data are summarized in Figure 2 and Table 1. In Figure 3 photomicrographs from a glass chamber (Fig. 3A) and a plastic chamber (Fig. 3B and 3C: different focal depths of the same field) illustrate the difficulty of locating and identifying sperm located on or adjacent to the bright lines in the plastic slides. DISCUSSION Careful evaluation and validation of methods and equipment are cornerstones of modern laboratory science. For the FIGURE 3 (A): Field from standard glass counting chamber (improved Neubauer pattern). Photomicrograph from Nikon Eclipse E600 microscope (objective: Plan Fluor 10/0.25, Ph1 DL N/0.17 WD 16; intermediate magnification (tube factor): 2) was used for photomicrography (Sony SSC-DC58AP color video camera; image capture with Picsara 8.81 rev. 2). (B, C): Field from plastic disposable counting chamber (improved Neubauer pattern) at different levels of focal depth. Arrow points at a position where a sperm head is hidden on a line in the chamber and therefore difficult to locate and identify. Same equipment for photography as for (A). 630 Kirkman-Brown and Bj orndahl Techniques and instrumentation Vol. 91, No. 2, February 2009

5 clinical user of laboratory results it may appear oversensitive to reject time-saving equipment that has a 15% error in sperm counting. However, clinical laboratory science has an obligation to test new methods and equipment. In some cases there may be new and better methods, and sometimes disadvantages of new ideas are demonstrated before another source of error has been introduced into a field already plagued by the fact that basic standardization and proper working methods still are not used in many laboratories (9). In the present investigation we found that, with use of routine phase-contrast optics, sperm on or close to the very bright lines of the disposable plastic counting chambers could not be distinguished, and this placed the accuracy of the counts outside of acceptable limits compared with standard methods. Although these new chambers save time by eliminating the need for mounting the coverslips, as well as for cleaning the slides, the time for assessment increased considerably because of the need to change focus repeatedly for all fields to be able to detect sperm hidden in bright lines. Our estimation is that this doubled the time needed for assessment. Any change in analytic methods should be motivated by easier performance, increased quality, or a combination of both. Our conclusion is that even with the best available optics the very bright lines of the disposable make it difficult to identify all sperm in the chamber. This uncertainty, in addition to the extra time needed for assessment, makes these plastic disposable counting chambers unsuitable for sperm concentration assessment under current guidelines. However, with careful assessment and benchmarking of disposable chambers versus improved Neubauer hemocytometers on individual microscopes, laboratories undertaking examination of known biohazardous samples could consider their implementation. If further revision of the 100-mm disposable chamber design by the manufacturer occurred, to reduce the bright-line effect, then implementation of these chambers might become a reality, but the current semen test 20-mm chip should not be used. Acknowledgments: Mr. Tom Connolly, B.Sc., M.Sc. (Assisted Conception Unit, Birmingham Women s Hospital, and Division of Reproductive and Child Health, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom) is acknowledged for patient assistance in counting sperm. Labtech International, Ringmer, East Sussex, United Kingdom ( kindly provided the incyto chambers ( for this study. The authors acknowledge the ongoing support of their employers for their ongoing research work. REFERENCES 1. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interactions. 4th ed. New York: Cambridge University Press, Kvist U, Bj orndahl L, eds. Manual on basic semen analysis. Oxford: Oxford University Press, Mortimer D, Shu MA, Tan R, Mortimer ST. A technical note on diluting semen for the haemocytometric determination of sperm concentration. Hum Reprod 1989;4: Mortimer D, Shu MA, Tan R. Standardization and quality control of sperm concentration and sperm motility counts in semen analysis. Hum Reprod 1986;1: Mahmoud AM, Depoorter B, Piens N, Comhaire FH. The performance of 10 different methods for the estimation of sperm concentration. Fertil Steril 1997;68: Tomlinson M, Turner J, Powell G, Sakkas D. One-step disposable chambers for sperm concentration and motility assessment: how do they compare with the World Health Organization s recommended methods? Hum Reprod 2001;16: Douglas-Hamilton DH, Smith NG, Kuster CE, Vermeiden JP, Althouse GC. Capillary-loaded particle fluid dynamics: effect on estimation of sperm concentration. J Androl 2005;26: Douglas-Hamilton DH, Smith NG, Kuster CE, Vermeiden JP, Althouse GC. Particle distribution in low-volume capillary-loaded J Androl 2005;26: Riddell D, Pacey A, Whittington K. Lack of compliance by UK andrology laboratories with World Health Organization recommendations for sperm morphology assessment. Hum Reprod 2005;20: Fertility and Sterility â 631