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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)

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Leja contact details

Leja Products B.V.

www.leja.nl

info@leja.nl