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Semen analyses and Leja®
Comparison of different counting methods
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Correct counting of semen
• There is a relationship between number of cells and human fertility.
• There is a relation between preferred method of treatment and number of cells:– Waiting– IUI, IVF, ICSI
• Amount = (better choice of) Quality
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Important aspects ofsemen analysis
• Semen is a suspension
• Semen need to be diluted
• Precision:– Reproducibility of obtained results
• Accuracy:– Correctness of results = correlation to golden
standard– Measurements taken by calibrated instrument
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Fertility of a semen sample
• Sperm count
• Motility– Hyper activation
• Morphology
• Stability of fertility when diluted and in uterine tube
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Counting
• What do we want to know?– Number of normal cells– Number of moving cells– Number of white blood cells
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Methods of counting
• Haemocytometer
• FACS
• Coulter Counter
• Corning
• SP 100
• CASA system with Leja® slides
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Haemocytometer I
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Haemocytometer II
• Golden standard
• Time consuming
• Error-sensitive: precision in making dilutions
• 2 x 200 cells to count
• Statistical error of counting alone ± 25%
• No differentiation between dead/alive; motile/non-motile cells
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Haemocytometer III
In conclusion, the haemocytometer is only usable to calibrate other counting systems.
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FACS
• Measures the fluorescence of individual cells• Uses an internal standard• Measures 2000 – 5000 cells per measurement• Cells remain alive (X – Y separation)• The fluorochrome needs to be specific and are
limited to 5 different• Staining of nucleus, dead/alive staining, DNA
lesions, proteins, receptors
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Coulter Counter I
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Coulter Counter II
• Counting large numbers is possible: smaller counting errors
• Differentiation between particle size is possible
• Differentiation between different cell types is not possible
• Suitable for pure suspensions
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Corning
• Measuring the optical density
• One is not sure about what exactly is being measured
• Very high error sensitivity
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SP 100 (Nucleocounter) I
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SP 100 II
• Measures haploid cells (= semen) using quantitative fluorescence microscopy
• Effect of laminar flow (SS-effect) not researched
• Measures many cells, relative low error.
• Special cassettes are needed
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Accuracy
• All systems have been calibrated to haemocytometer
• Each manufacturer is obliged to show calibration data
Conclusion 1:
• All systems count cells, but not motility
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CASA systems
• Measures concentration
• Measures motility
• Uses disposable counting chambers i.e. Leja® slides
• Fluorescence microscopy:– FACS possibility at a fraction of the costs
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Conclusions so far
Instrument FACS Improved Neubauer
Corning Sperm Vision
Ultimate SP 100
CV 2.72 % 7.14 % 10.4 % 8.13 % 5.40 % 3.11 %
95 % confidence
interval
2.45
–
3.05
6.42
–
8.03
9.40
–
11.7
7.32
–
9.14
4.87
–
6.06
2.81
–
3.49
Comparison of FACSCount AF System, Improved Neubauer hemacytometer, Corning 254 photometer, Sperm Vision CASA System, Hamilton Thorne Ultimate
CASA System and NucleoCounter SP-100 for determination of sperm concentration of boar semen
C. Hansen a, *, T. Vermeidenb, J. Vermeiden , C. Simmet, B.C. Day, H. Feitsmab
a The National Committee for Pig Production, Danish Bacon and Meat Council,
Axeltorv 3, DK-1609 Copenhagen V, Denmark
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Leja I
• Leja is the company that develops and produces in favour of assisted reproduction
• Leja’s ideas originate in an academic setting and are translated to products that work in everyday practice
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Leja II
• Leja is ISO 9001:2000 certified
• All Leja slides are CE-marked
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Leja slides I
• Two sheets of glass cured together at fixed distance– Using non-toxic resin– resin holds spacers of known defined
diameter– Only sharpened glass is completely flat
• Very small variations in chamber heights occur
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Leja slides II
• The glass surface is treated:– Cleaning– Coating
• No direct contact between sperm cell and glass• Regular microscopic glass is surface active:
– Sperm cells bind to clean glass– Residues of soap / oil are toxic to sperm– Quality is not consistent
glass directly from regular stock isnot suitable to determine motility!
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Leja slides III
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Leja quality control
• Semi-automated production leaves room for both human error and permanent quality control:– Printing errors– Chamber height– toxicity
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Slide production IProduction facilities in
clean room environment
Screen printing of ink and glue layers
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Slide production II
Curing cover slip by
UV induction
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Newton bands
Using monochromatic light, Newton bands are
an indication of chamber height
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R&D quality controlRandom testing of
chamber heights by special interference
meter (only 3 on earth)
Toxicity testing:<7% less motility after
6 minutes
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Quality certificate
Each order of Leja® slides
comes with a quality
certificate(available to download
on www.leja.nl)
