Transcript Semen analyses and Leja&#174

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 of
semen 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
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•
•
•
•
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 is
not 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 I
Production 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 control
Random 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)
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Leja contact details
Leja Products B.V.
www.leja.nl
[email protected]
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