Semen analyses and Leja Comparison of different counting

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 • Amount = (better choice of) Quality 2

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 3

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

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

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

Haemocytometer I 7

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 8

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

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 10

Coulter Counter I 11

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 12

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

SP 100 (Nucleocounter) I 14

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 15

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 16

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

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 Nucleo. Counter SP-100 for determination of sperm concentration of boar semen a, *, T. Vermeidenb, J. Vermeiden , C. Simmet, B. C. Day, H. Feitsmab C. Hansen a The National Committee for Pig Production, Danish Bacon and Meat Council, Axeltorv 3, DK-1609 Copenhagen V, Denmark 18

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 19

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

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 21

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! 22

Leja slides III 23

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

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

Slide production II Curing cover slip by UV induction 26

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

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 28

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

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