ANDROLOGY LAB CORNER*: Validation of Sperm Counting Methods Using Limits of Agreement

Abstract

A variety of methods exist for counting sperm. Since the introduction of semen analysis, one of these methods, the hemocytometer, has been regarded as the gold standard by andrology laboratories and the World Health Organization (WHO, 1999). The flexible features of this approach, involving fixation and immobilization of sperm, dilution of highly concentrated samples, and the counting of sperm in a single plane, contribute to the accuracy of the Improved Neubauer hemocytometer and its relative ease of use. This method is strongly accepted within andrology clinics and has been clinically validated by a number of studies (Dunphy et al, 1989; Tomlinson et al, 1996, 1999; Guzick et al, 2001). As technology and techniques improve, manufacturers are continually trying to develop newer, simpler, quicker and more accurate methods for determining sperm concentration. Busy assisted reproduction technology (ART) laboratories in particular would find a quicker yet comparatively accurate method highly desirable, since sperm counting is an essential part of the semen preparation process. Although modern methods may be faster, since unfixed, undiluted semen is used, some labs find these analyses more difficult to use and believe that the counting of motile sperm may produce erroneous results. Furthermore, the WHO states that the newly introduced methods “are convenient in that they can be used without dilution of the specimen, but that they may lack the accuracy of the hemocytometer technique especially for highly viscous and/or heterogeneous specimens. If such chambers are to be used, their adequate accuracy and precision must be established by comparison with hemocytometers” (WHO, 1999). In addition, it is now a requirement of laboratory accreditation systems that laboratories provide clinical validation for all methods used (ie, demonstrate that they are “fit for purpose”). Currently, the only sperm counting method with a considerable body of evidence to support and clincally justify its use is the hemocytometer (Mortimer, 1994; WHO, 1999). When using the hemocytometer, the sperm number is calculated using a fixed volume of semen under the coverslip and counting the sperm in a single plane. A significant association between pregnancy and the sperm concentration measured has consistently been shown for this method (Dunphy et al, 1989; Tomlinson et al, 1996, 1999; Guzick et al, 2001). Thus, 64% of laboratories involved in the analysis of semen use this method routinely (Keel et al, 2000). However, despite its validity as a method, the use of hemocytometry is thought by many to be inconvenient, in that the hemocytometer must be cleaned and assembled prior to each counting event and it involves the use of dilution techniques that can introduce errors, either due to poor technique or the viscous nature of the semen itself. Mathematical mistakes can occur when applying the correction factor to determine the eventual counts, and the recommended dilution method uses fixatives, such as formal saline, which are often a reason for rejection of the method by embryologists in IVF laboratories. Comparisons of other counting chambers with the hemocytometer, particularly those marketed as easy-to-use 1-step methods have generally not been favorable. Makler sperm counts have been shown to be generally higher than the corresponding counts obtained with the hemocytometer (Coetzee and Menkveld, 2001; Sukcharoen et al, 1994). Indeed, Ginsburg and Armant (1990) have found the Makler chamber counts to be 62% higher than those obtained with the hemocytometer when using latex beads. Other methods, such as the Leja slide (Gynotec Malden, Nieuw-Vennep, The Netherlands) or the Microcell (Conception Technologies, San Diego, Calif) have been shown to produce significantly lower average sperm counts as compared to the hemocytometer. In particular, marked differences were seen at high concentrations (Tomlinson et al, 2001). A number of potential sources of error have been suggested to cause these discrepancies when using 1-step methods. First, the recommendation that motile sperm are counted, which may mean a single spermatozoon is counted more than once or not counted at all. Second and perhaps more significantly is the phenomenum that affects capillary-loaded chambers, such as the Leja and Microcell slides, which is known as the Segre-Silberberg (SS) effect (Segre and Silberberg, 1961). The SS effect results in high-gradient fluid flow in thin capillary-loaded slides, which results in the sperm suspension being forced transversely towards the walls, causing uneven cell dispersion throughout the chamber. New measures have recently been introduced to compensate for this phenomenon. A correction factor is used, which makes allowances for changes in sample viscosity and appears to improve the performance of there slides in terms of agreement with the hemocytometer (Douglas-Hamilton et al, 2005). Third, many of the studies that have compared the performances of various methods with the gold standard of the hemocytometer are problematic in terms of the choice of statistics. Unfortunately, many of these studies have focused on analysing differences across a range of sperm counts, whereas a proper and more detailed analysis is one that compares each and every individual count and measures the agreement between these 2 parameters. Therefore, in the present study, we employ limits of agreement (LoA) to analyse comparative data. This approach, which is based on graphical techniques and simple calculations, allows comparisons between new measurement techniques (ie, the Leja and Makler chambers) and an established method (ie, the hemocytometer). Thus, we can evaluate whether the methods agree sufficiently for the new method to replace the old method and we can decide if the differences between the 2 methods are sufficiently small for the methods to be used interchangeably (Bland and Altman, 1986). The objectives of the present study were: 1) to determine whether the sperm counts obtained using the Leja slide and Makler chamber compare favorably with the counts obtained using the hemocytometer; 2) to determine whether the accuracy and reliability of sperm counts obtained using the Leja slide and the Makler chamber are improved by prior fixation and dilution of the specimen; 3) to determine whether the sperm counts obtained using the Leja slide compared more favorably with the counts obtained using the hemocytometer when correction is made for the SS effect; and 4) to determine by the use of LOA whether these methods can be considered to be interchangeable, thereby providing validation for their routine clinical application. In all, 163 semen samples were obtained from patients who attended the NHS Fertility Clinic and Nurture Assisted Conception Unit at the University Hospital, Nottingham, United Kingdom. Patients were referred to the laboratory either by their local GP after a period of suspected infertility or by the Fertility and Nurture Clinics themselves. Samples were produced by masturbation and ejaculation into a sterile, wide-mouthed, nontoxic 60-mL plastic container (Alpha Laboratories, Eastleigh, United Kingdom). On arrival at the laboratory, the samples were allowed to liquefy for up to 1 hour at room temperature. The samples were then well mixed in their original container prior to analysis. Specimen acceptance criteria were defined as follows: the sample had to be less than 1 hour old, complete, and produced in the recommended container after a period of abstinence of between 2 and 5 days. The double Improved Neubauer hemocytometer used for analysis was prepared for counting according to the WHO guidelines. The coverslip was applied by horizontal sliding. Correct positioning of the coverslip was confirmed by the presence of Newtons rings on both sides of the chamber (WHO, 1999). Before loading, the sample was diluted in 3% formal saline. Throughout the procedure, positive displacement pipettes were used for the transfer of semen (Gilson Microman; Anachem, Luton, United Kingdom). For most semen samples, a standard dilution of 1:20 was suitable. The dilution was adjusted appropriately according to the estimated concentration, to allow the enumeration of a mimium of 200 sperm. Each side of the counting chamber was then loaded until it was full (8–10 μL). Care was taken to ensure that the chamber was not underfilled and that the sample was not allowed to spill into the central trough. The hemocytometer was left for 5–10 minutes in a humid chamber, to allow the cells to sediment before counting and to prevent drying out. Duplicate counts using the same 163 specimen samples were made on the Leja slide according to the manufacturers instructions. Initially, the microscope was calibrated using an eyepiece reticule and stage micrometer, in order to obtain the objective correction factor. Positive displacement pipettes were used to fill the Leja chamber and filling time was recorded in seconds immediately after dispensing the semen. The number of sperm counted within the 10 × 10 grid was noted, with a minimum of 200 spermatozoa counted for each evaluation. The concentration was calculated according to the manufacturers' instructions and the formula (average number of sperm per small square × microscope factor × F), where F is the factor compensating for the SS effect. The counts were repeated using specimens fixed and diluted in 1:1 formal saline (3.5%). The Makler chamber was used according to the manufacturers instruction. Well-mixed semen (5 μL) was transferred to the Makler chamber. The cover was applied promptly, since a delay has been shown to be a potential source of error that results in a higher sperm concentration (Matson et al, 1999). When applying the cover, care was taken to avoid the formation of bubbles. Once again, the counts were repeated using specimens fixed and diluted in 1:1 formal saline. Each sample received for sperm concentration analysis was also assessed for sperm motility. The scepticism surrounding counts obtained from the Leja slide and Makler chamber is thought to be associated with the use of motile sperm, yet there are very few publications on this topic. Therefore, by determining the motility of each sample in an accurate manner, it is possible to elucidate whether increased motility leads to greater discrepancies or inaccuracies. The motility of each sample was assessed in the Improved Neubauer hemocytometer using the WHO classification system for a minimum of 200 spermatozoa (WHO, 1999). For consistency, all readings were carried out at 37°C using a heated microscope stage United Kingdom). is essential to that a single can obtain the same results when repeated are made using the same method under and 2005). Thus, in counts between chambers can be to the of the chambers than poor of the 163 samples obtained from the clinics were used in a Each sample was and were counted 5 in the hemocytometer, 5 on the Leja slide and then 5 in the Makler chamber. This was repeated after the sample was diluted 1:1 in with the counts being repeated on the Makler and Leja This a of counts for each counts obtained using the methods were compared using LOA according to the methods of and In this analysis, the differences between the methods being compared (ie, the Leja hemocytometer, and Makler are the mean of the 2 methods, since the is not known and this is the of this (ie, the sperm would be a to the between each as the differences be to which a et al, The limits within which of the differences are to are also limits are estimated from the mean and standard of the and are as horizontal 2 standard either side of the the an of the of between the methods. This method was used to compare the Leja Leja Makler and Makler 1:1 counts to the corresponding counts obtained using the Improved Neubauer hemocytometer. The SS counts were then compared to the hemocytometer, to whether applying the SS correction factor increased the association between the Leja slide and hemocytometer. As the a they were using a The for the chambers were for using the Each method was compared in with the hemocytometer to evaluate significant The results were using for The Leja Leja Makler Makler and hemocytometer counts were for agreement using and the mixed This was as it is to in which are and measures are fixed (ie, the sample concentrations were and the measures in the chambers all be the The counts for each sample be that the the is to the more is the counting of were also to determine the of the counts relative to the mean for each The lower the the the between the counts, and the higher the The were also measured for each method, and in a to the and the of the repeated and that the hemocytometer the of of by the Leja slide and the Makler chamber This was confirmed by the of the with the hemocytometer the and the Makler the and the sample increased the for both the Makler and Leja Therefore, the hemocytometer had the with the Makler chamber the it being for the diluted method 1 and The 163 semen samples measured using the chambers are Since the hemocytometer is accepted as the gold the counts have been in order of concentration by 1 a between the methods but also that the Makler and Leja counts the concentration obtained using the Improved Neubauer hemocytometer. The Leja slide produced counts that were on average lower than those obtained using the hemocytometer, the Makler counts were on average The differences between the chambers to as the sperm concentration particularly for the Makler chamber, in the same was for sperm counts at the of the range and those in the sperm counts obtained on the Improved Neubauer hemocytometer, and Makler chambers using undiluted semen samples When the samples were diluted the hemocytometer counts were significantly for every chamber, with the of the Leja slide The Leja count was significantly lower than that of the hemocytometer the Makler was higher The introduction of to both methods improved their agreement with the gold to the that the Leja counts were significantly from those of the hemocytometer. This an significant between the methods a more method is to individual The mean between the Leja and hemocytometer was This that a sample to have a concentration of 5 × on the Improved Neubauer would be as an average of × on the A sample with × would be to an average of × using this method The standard was calculated as the limits of agreement between and This that in a specimens that 5 × on the hemocytometer have counts that between and × In the same the Leja slide would determine the concentration of a sample with × to between × and × LOA for the Leja slide using undiluted semen the Improved Neubauer hemocytometer The horizontal the mean the horizontal the 2 standard either side of the and the the at 5 × and × for and the it is that counts are of these Thus, as many of the Leja counts strongly with the hemocytometer counts than would be However, there is between the between the 2 methods and their Therefore, the sperm concentration of the semen not the accuracy of the Leja of the semen with 3% formal saline by analysis with the Leja method produced agreement between the 2 methods The Leja 1:1 method and hemocytometer had a mean of which that on their counts are in that the SS factor is applied their use. LOA for Leja 1:1 compared to the hemocytometer The horizontal the mean the horizontal the and the the at 5 × and × for and The limits of agreement were calculated to be between and are than for the analysis of undiluted samples, the Leja 1:1 method would determine a sample of 5 × as being between and × and a sample of × as being between × and × in a are 10 which the limits of the in it can be seen that the mean is The limits of agreement are between and are limits than for the Leja method, in a a sample of 5 × would be as being between × and × and a sample of × as being between × and × concentrations of 5 × and × were by the Makler to be 10 × and × were of these all of the which the of the Makler method to sperm LOA the Makler chamber and Improved Neubauer hemocytometer The horizontal the mean the horizontal the and the the at 5 × and × for and The is a that the in with However, it can be seen by the in 5 that there is a in as the mean (ie, semen concentration, This that the Makler to a greater as the semen concentration Therefore, LOA analyses were repeated for the samples once they were into one one and high one The hemocytometer and Makler the agreement the mean was at very sperm LOA for Makler 1:1 compared to the hemocytometer The horizontal the mean the horizontal the limits of and the the at 5 × and × for and The the in with The mean differences obtained with the hemocytometer for the and were and This that the Makler to a greater as the sperm concentration As the semen concentration the between the and lower limits of agreement increased as follows: lower concentration, to concentration, to concentration, to The Makler 1:1 method a mean of which is a than that obtained using undiluted semen. the that for diluted the Makler semen samples of 5 × as being × and × as being × on which is a small in terms of agreement as compared to using undiluted semen it can be seen that using counting methods has the potential to the In out of 163 in this was the counting method the Leja of the would be as and only would be as based on their hemocytometer In only was found to be using the Makler slide of its to sperm the specimens and using the Leja slide improved the the number as to using the Makler method this number increased to The of the present was to determine whether methods used for sperm counts in a laboratory be considered to be methods are in use in laboratories throughout the United and The and that all the counting methods have a particularly high of The hemocytometer was the most method, with the and This the hemocytometer as the gold standard method, its precision is not The Leja was the most method, it was more without the which that this may introduce a of The analyses the Makler chamber to have significantly reliability than the hemocytometer or The of the Makler chamber was lower for both undiluted and diluted samples, and the and between the repeated The LOA analysis that the Leja slide be compared with the hemocytometer, particularly for the 1:1 However, despite that the Leja 1:1 and hemocytometer have a mean of the standard was calculated as Thus, the LOA between and which that in a specimens that 5 × in the hemocytometer have counts between and × the Leja method determine the concentration of a sample with × to be between × and × Although the of analysis may at to lack the same can be applied to counts using the same chamber. In other in most the between the chambers is than repeated measures using the same chamber. be and perhaps more when semen is the fluid in and this be due to one of a number of error or chamber When the agreement and of a method, it has to be in the of the of As there is in hemocytometer counts using either repeated measures or between we differences when this with other In this the Leja slide be as being with the hemocytometer, particularly if the sample is fixed and of the results very that applying the SS factor the of agreement between the Leja counts and hemocytometer counts, as This was more for the 1:1 diluted samples, for which the mean with the hemocytometer for the Leja This is since the Leja states that the correction were calculated by the Leja with the Improved Neubauer chamber. The Makler chamber poor agreement with all the other methods and had a to sperm In the a sample of × be found have between × and × which have for the or This studies that have found that the Makler high sperm counts and Sukcharoen et al, 1994; et al, and Menkveld, 2001; et al, with a latex with a fixed concentration of × the concentration on the Makler to be × on A study by et found that the Makler chamber the known concentrations by as as The counts using the Leja slide were significantly from the counts obtained using the hemocytometer they specimens were fixed and The Leja was significantly lower than that of the hemocytometer, and this was by the LOA analysis, which that the Leja on average the sperm count when undiluted semen was This was throughout the and was not to the sperm concentrations of the was that the Leja would the sperm count when compared to the hemocytometer, since a study by Tomlinson et has found Leja counts to be significantly lower than the corresponding hemocytometer these counts were using a of the Leja chamber and without of the SS factor. and the samples for analysis by the Makler method the mean and increased the of agreement with the hemocytometer the counts produced in the Makler chamber were significantly higher than those in the hemocytometer, of whether they were on undiluted or diluted specimens. In some laboratories are to on the accuracy of sperm in order to and The for methods of sperm counting and the of counting chambers may in many the of clinical and the of by a particular is known that many IVF labs the 1-step methods to their this in that based on sperm be from erroneous data. has been suggested that using to sperm concentrations is the cause of in sperm counts between laboratories et al, 2000). to between a be as by one and as by et al, This has been known for some time and is by the of the present concentration discrepancies between the Leja and hemocytometer the of patients in the present erroneous have for some of may be as to the WHO which in have and mean that some would not the in from studies that many of the methods in use a of which is high in comparison with many other of many of which are such as the viscosity and of in and are all is that the lack of and lack of evidence sperm concentration with either or assisted are due to either poor or the of many of the methods source of and the most is its very 1 small of semen may have very when compared to Therefore, only by the volume of and the number of sperm counted per analysis can we to improve the of accuracy and However, this has to whether there is a for such a of accuracy and also whether it is a use of The high of error the analysis, be it due to the method used, or a of the as to whether a be based the of a single semen in other such as whether the sample is complete, of and all of which are known to sperm that the between and sperm count is than perhaps it The of this is that semen analysis to demonstrate poor in terms of either or assisted and The hemocytometer method has been the accepted standard for the of sperm concentration. Indeed, the WHO in its to semen analysis that a hemocytometer, such as the Improved be used for sperm concentration an estimated 64% of laboratories used the hemocytometer, used the Makler chamber, and the used methods, such as the Leja slide (Keel et al, 2000). methods, such as the Leja and chambers, such as the have been as convenient The present study that there is agreement between all of these methods, with the between hemocytometry and the Makler If the standard Leja method was by and the the agreement with was very Therefore, if it can be shown that the 2 methods are to the same counts, the 2 methods be to be providing a of validation for the Leja The hemocytometer is in terms of all of the methods, has in terms of reliability and which may be to the fluid in In there is to between the hemocytometer and diluted Leja method in terms of average sperm which that the Leja slide may be for use in clinical In the Makler method very poor agreement throughout the range and be The agreement seen between all methods and their lack of precision is a cause for If the WHO to the hemocytometer as the gold standard method, then there at be a of the are often on poor technique or yet it is that all methods for sperm counting have must be in order to provide of their with proper the its and the of accuracy and in order to allow of their