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, signifi-
cantly lower count results were obtained consistently from the plastic chambers than from standard chambers.
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
chambers.
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 2009;91:627–31. 2009 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 diagnos-
tic 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 So-
ciety of Human Reproduction and Embryology (2). Contin-
ued efforts to improve the efficiency of these standard
analyses must be combined with clear evaluation and valida-
tion of any procedural simplifications or use of less compli-
cated 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, 2007.
Presented as a poster at the Annual Meeting of the Nordic Association for
Andrology in Stockholm, Sweden, August 31–September 1, 2006.
Reprint requests: Lars Bjo€ rndahl, M.D., Ph.D., Centre for Andrology and
Sexual Medicine, Karolinska University Hospital, Huddinge M52,
S-141 86 Huddinge, Sweden (FAX: 46-8-31-36-91; E-mail: Lars.
Bjorndahl@ki.se).
exact semen volume for dilution and an exact volume to as-
sess 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 hemocy-
tometers. Using 100 mm as chamber depth has several advan-
tages. 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 capil-
lary action consistently give lower results than hemocytome-
ters (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

0015-0282/09/$36.00 Fertility and Sterility Vol. 91, No. 2, February 2009 627
doi:10.1016/j.fertnstert.2007.11.076 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
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 cham-
ber and should not be easily dislodged, for example, by a sim-
ple 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 proce-
dure 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, micro-
scopic 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 di-
agnostic tests at the andrology laboratories of the Assisted
Conception Unit, Birmingham Women’s Health Care Trust
Birmingham, West Midlands, United Kingdom, and the Cen-
tre for Andrology and Sexual Medicine, Karolinska Univer-
sity Hospital, Stockholm, Sweden. All samples included in
the study were used with the consent of the patients con-
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.
Kirkman-Brown. Techniques and instrumentation. Fertil Steril 2009.
628 Kirkman-Brown and Bj€orndahl Techniques and instrumentation
cerned, and no ethical approval was required because the
methodology fell within the bounds of routine laboratory val-
idation procedures.
All dilutions were analyzed on the day the sample was pro-
duced. Initial volumes of semen were taken with a positive
displacement pipette and diluted with either a defined dilu-
tion medium containing formaldehyde (Stockholm [2]) or
with distilled water (Birmingham). Either diluent will immo-
bilize 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 (set-
ting: 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, www.vwr.com)
were used as control. Coverslips were mounted and checked
for presence of iridescence lines before loading.
The test chambers, plastic 100-mm deep disposable cham-
bers (iNCYTO C-Chip DHC-N01, batch 12GD12; Digital
Bio Technology Co. Ltd., Seoul, Korea, www.incyto.com)
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 di-
lution 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 sus-
pension between filling each chamber. After loading, the
Vol. 91, No. 2, February 2009
slides were kept 15 to 30 minutes in a humidified chamber to
FIGURE 2
allow sedimentation of cells before assessment.
Assessment was done under phase-contrast microscopy Results from multiple assessments of different
(Olympus BX40 with standard phase-contrast optics [20 sperm dilutions. In series 2 (n ¼ 7) and series 3 (n ¼ 5)
objective 0.40 Ph1 N/0.17] or Olympus BX50 [20 objec- significantly lower concentrations (a: P< .05) were
tive: UPlan Fl, 0.50 Ph1; N/0.17]) (Olympus, Tokyo, Japan). obtained with the plastic chambers. In series 4 (n ¼ 5)
A Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan) a microscope with high-quality optics was used
(objective: Plan Fluor 10/0.25, Ph1 DL N/0.17 WD 16, (Olympus BX50), and efforts were made to trace
with an intermediate lens giving a total magnification of sperm that could be hidden because of optical
200 in oculars and corresponding magnification and quality phenomena (b: P>.05). In this last series no
in the camera) was used for photomicrography (Sony SSC- difference was observed between the two types of
DC58AP color video camera [Sony, Tokyo, Japan]; image chambers.
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).
The initial experiment (series 1) was a straightforward
comparison of the two chambers. Eighteen samples were as-
sessed 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 possi-
bility 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 investi-
gated (series 4). In series 2, a series of seven assessments of Kirkman-Brown. Techniques and instrumentation. Fertil Steril 2009.
one single suspension was performed in parallel in glass and
plastic slides by one investigator. In series 3, another inves-
tigator made a series of five assessments of another single tion positive phase-contrast optics (Olympus BX50). In
suspension in parallel in glass and plastic slides. In series this series considerable time and effort were invested in
4, the first investigator did a similar series of five assess- searching for sperm concealed in bright lines in the plastic
ments as in series 3, using a microscope with high-resolu- chambers.

TABLE 1
Comparison between results obtained with glass and plastic counting chambers, respectively.
Series 1 Series 2 Series 3 Series 4
No. of samples 18 1 1 1
No. of slides per sample 1 7 5 5
Optics Standard phase Standard phase Standard phase High-quality phase
contrast 200 contrast 200 contrast 200 contrast 200
Group mean (106/mL)
Glass 64 75 19 67
Plastic 55 67 15 69
Group SD
Glass 43.19 6.30 1.55 1.91
Plastic 38.83 6.80 1.84 5.03
P value .0003a .0387b .0079c .4209b
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.
Kirkman-Brown. Techniques and instrumentation. Fertil Steril 2009.

Fertility and Sterility 629

Statistical Analysis
The Wilcoxon matched-pairs test was used for series 1 (pair-
wise 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 statisti-
cal calculations were performed with use of GraphPad Prism
version 4.03 for Windows (GraphPad Software, San Diego,
CA, www.graphpad.com).
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.

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 sam-
ple was assessed but in seven parallel slides to evaluate
whether there was a difference in both average and variabil-
ity. This was not the case, because the values for SD were
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).

Kirkman-Brown. Techniques and instrumentation. Fertil Steril 2009.

630 Kirkman-Brown and Bj€orndahl Techniques and instrumentation Vol. 91, No. 2, February 2009
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 obliga-
tion to test new methods and equipment. In some cases there
may be new and better methods, and sometimes disadvan-
tages 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 rou-
tine 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 out-
side 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 examina-
tion 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
Fertility and Sterility
‘‘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 King-
dom (www.labtech.co.uk), kindly provided the iNCYTO chambers (www.in-
cyto.com) for this study. The authors acknowledge the ongoing support of
their employers for their ongoing research work.
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