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Semen analyses and Leja ®

Semen analyses and Leja ®. Comparison of different counting methods. 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

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Semen analyses and Leja ®

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  1. Semen analyses and Leja® Comparison of different counting methods

  2. 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

  3. 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

  4. Fertility of a semen sample • Sperm count • Motility • Hyper activation • Morphology • Stability of fertility when diluted and in uterine tube

  5. Counting • What do we want to know? • Number of normal cells • Number of moving cells • Number of white blood cells

  6. Methods of counting • Haemocytometer • FACS • Coulter Counter • Corning • SP 100 • CASA system with Leja® slides

  7. Haemocytometer I

  8. 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

  9. Haemocytometer III In conclusion, the haemocytometer is only usable to calibrate other counting systems.

  10. 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

  11. Coulter Counter I

  12. 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

  13. Corning • Measuring the optical density • One is not sure about what exactly is being measured • Very high error sensitivity

  14. SP 100 (Nucleocounter) I

  15. 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

  16. 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

  17. CASA systems • Measures concentration • Measures motility • Uses disposable counting chambers i.e. Leja® slides • Fluorescence microscopy: • FACS possibility at a fraction of the costs

  18. Conclusions so far 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

  19. 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

  20. Leja II • Leja is ISO 9001:2000 certified • All Leja slides are CE-marked

  21. 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

  22. 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!

  23. Leja slides III

  24. Leja quality control • Semi-automated production leaves room for both human error and permanent quality control: • Printing errors • Chamber height • toxicity

  25. Slide production I Production facilities in clean room environment Screen printing of ink and glue layers

  26. Slide production II Curing cover slip byUV induction

  27. Newton bands Using monochromatic light, Newton bands are an indication of chamber height

  28. 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

  29. Quality certificate Each order of Leja® slides comes with a quality certificate (available to download on www.leja.nl)

  30. Leja contact details Leja Products B.V. www.leja.nl info@leja.nl

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