Comparison of sperm concentration using the Makler and the Neubauer counting chambers
Cardona-Maya W, Berdugo J, Cadavid A
Actas Urol Esp. 2008;32(4):443-445
COMPARISON OF SPERM CONCENTRATION USING THE MAKLER AND NEUBAUER COUNTING CHAMBERS
Objective: To establish the concordance between the sperm count obtained with the Makler chamber and with the Neubauer chamber, in the ejaculates of fertile individuals.
Design: A prospective study conducted by the
Reproduction Group of the
Methods: A total of 112 ejaculates from fertile individuals were analyzed. The sperm count was carried out with the Makler chamber and with the Neubauer chamber – comparison between the two chambers being made with the Bland-Altman plot test.
Results: The mean concentrations were 107.8 and 106.2 x 106 sperm/ml, using the Makler and de Neubauer chambers, respectively. Concordance between the two chambers was established.
Conclusion: Our results indicate that the determination of sperm concentration with the Makler chamber is as accurate as the count obtained with the Neubauer chamber, and that both can be used for routine semen analysis.
Key words: Sperm concentration. Makler chamber. Neubauer chamber. Fertile males. Spermatozoa.
Sperm concentration is one of the most important parameters in the evaluation of male fertility. Traditionally, sperms counts have been made with the Neubauer® chamber, which has become the gold standard and is the method recommended by the World Health Organization (WHO)1,2. However, other chambers have also been developed, such as the Makler® (Sefi Medical Instruments, Haifa, Israel), Microcell® (Concepción Technologies, Natick, MA, USA) and Horwell® chamber (Horwell Ltd., London, UK), for determining sperm concentration. The advantage of the new chambers over the Neubauer® chamber is that they use undiluted samples – thereby increasing the precision of the procedure and the reproducibility of the results. However, the introduction of these alternative chambers for the determination of sperm concentration has generated controversy, due to the reporting of non-uniform results when using the Makler® chamber. It is important to compare the results obtained with different chambers, and to select the method best suited to each laboratory. The present study examines concordance between the sperm counts obtained with the Makler® and Neubauer® chambers in application to ejaculates of fertile males.
MATERIALS AND METHODS
Samples were collected from 112 males of
confirmed fertility programmed for vasectomy in the Clínica Profamilia (
The grid of the Makler® chamber measures 1 x 1 mm, with a depth of only 0.01 mm, and is subdivided into 100 squares, each measuring 0.1 x 0.1 mm. Thus, the total volume in the Makler® chamber is 0.01 µl, or equivalent to 1/10 that of the Neubauer® chamber. Sperm counting was made following the instructions of the manufacturer. A volume of 3-5 µl of semen sample previously immobilized by incubation at 60ºC for 10 minutes was transferred to the chamber, and counting was performed after 5 minutes in 10 squares of the chamber. The total sperm count is the end concentration expressed as 106 spermatozoa/ml.
The Neubauer® chamber
has a depth of 0.1 mm. It consists of 25 large squares and 16 small squares;
each large square measures 1 x 1 mm in size. Preliminary visual calculation was
made of the sperm concentration via microscopic observation of the undiluted
sample, before preparing the chamber. The samples were diluted in HAM F-12
medium (Sigma Chemical Company,
The results relating to sperm concentration are expressed as the mean ± SD (standard deviation). The limits of concordance were deduced calculating 2 x DS of the differences between the Makler® and Neubauer® chambers, and the dispersion diagrams of the difference between the chambers (Y-axis) and the mean of the two chambers (X-axis) were plotted to visualize the data, using the Bland-Altman method. On the other hand, we separated the samples under and over 40 x 106 spermatozoa/ml, and evaluated the statistical difference and correlation using the Student t-test and Pearson test, respectively. The results were analyzed using the Prism® 4.0 program.
The mean sperm counts were 107.8 ± 73.64 and 106.2 ± 74.24 x 106 spermatozoa/ml using the Makler® and Neubauer® chambers, respectively. The Bland-Altman plot indicated that the intervals obtained with either chamber were similar, with a bias of -1.55 and a 95% confidence interval of between -46.8 and 43.7 (Fig. 1).
FIGURE 1. Correlation between the sperm counts
obtained in 112 fertile males using the Makler® and Neubauer®
The difference between the sperm counts using the Makler® and Neubauer® chambers (Y-axis) is plotted against the mean of the two counts (X-axis). The diagram shows the magnitude of the difference between the two chambers and the distribution close to zero.
In addition, the comparison of the samples under and over 40 x 106 spermatozoa/ml using the Makler® and Neubauer® chambers was not statistically significant (p>0.05), and the Pearson correlation coefficient was high (sample under 40 x 106 = 0.88, and samples over 40 x 106 = 0.93).
The Makler® chamber for determining sperm concentration requires a single aliquot of the original sample, and is easier to use than the Neubauer® chamber – affording a calculation that comes close to the true sperm concentration present. Variability in the determination of sperm concentration between the different chambers is inevitable, and the differences have been attributed to dilution of the semen samples, the chambers used to count the sperm, sperm sample properties such as viscosity, and differences among the technicians performing the procedure3. Thus, research in this field is justified with the purpose of validating one of the existing methods, or for designing new chambers capable of offering less variable results4.
No significant differences were found between the Makler® and Neubauer® chambers in performing the sperm counts. This does not coincide with the observations of other authors, however. In effect, Ginsburg and Armant5, in a study of 10 individuals, observed a statistically significant 62% increase on comparing the counts obtained with the Makler® chamber versus the Neubauer® chamber. Christensen et al.6 obtained similar results in application to bull sperm. In turn, Imade et al.7 reported significant differences in a study of 50 samples. On the other hand, Matson et al.8 showed that these differences could be reduced by carefully following the instructions of the manufacturer.
In contrast, our results coincide with those published by Sukcharoen et al.9 and Mahmoud et al.3, who found no statistically significant differences (p>0.05) between the two chambers. In addition, Sukcharoen et al.9 found that in samples with concentrations under 40 x 106 spermatozoa/ml, the count using the Makler® chamber was greater (p<0.0001) than in samples with a larger concentration of sperm. This may suggest that the Makler® chamber is inadequate for samples of this kind. Therefore, we divided the samples under and over this critical value, and found the count obtained with each chamber to be similar in both sample groups (p>0.05). This difference could be explained by the number of samples used by Sukcharoen et al.9 (n=55), compared with the 112 ejaculates in our series – 92 of which corresponded to the group with concentrations of under 40 x 106 spermatozoa/ml.
There is no agreement on the protocol to be used in the test recommended by the WHO as the gold standard for sperm counting2. Each laboratory must experiment with its own population in order to determine which chamber is most adequate. However, it must be pointed out that the Makler® chamber is rapid and simple to use. In sum, our results show that both chambers are precise, even when used in application to samples with low counts.
The authors thank the Clínica Profamilia (
This study was supported by the
1. OMS. Manual de Laboratorio de la OMS para el examen del semen humano y de la interacción entre el semen y el moco cervical. Bogotá,: Editorial panamericana; 1992.
WHO. WHO Laboratory Manual for the Examination of Human Semen and
Sperm–Cervical Mucus Interaction.
5. Ginsburg KA,
9. Sukcharoen N, Ngeamjirawat J, Chanprasit Y, Aribarg A. A comparison of Makler counting chamber and improved Neubauer hemocytometer in sperm concentration measurement. J Med Assoc Thai. 1994;77(9):471-6. [PubMed]
Correspondence author: Dr. Walter Cardona-Maya
Grupo Reproducción. Universidad de Antioquia -SIU-, Medellín.
Carrera 53 # 61-30, Colombia. Tel.: 57 (4) 2196685
Author e-mail: email@example.comfirstname.lastname@example.org
Papel information: Original – Andrology - infertility
Manuscript received: june 2007
Manuscript accepted: july 2007