Sperm Concentration Flashcards
What methods can be used to count sperm?
1) . Screening kit home test.
2) . Automated methods.
3) . Counting chambers.
What might cause errors variation in sperm counting results?
The method used, technique (technical error) and counting enough sperm (sampling error) may all effect the results.
List some of the most commonly used counting chambers.
1) . Microcell.
2) . Leja.
3) . Horwell.
4) . Makler.
(All above examine a motile fraction of sperm).
5). Neubauer haemocytometer.
What is the most commonly used counting chamber for counting sperm? What are the basics of using this chamber?
The Improved Neubauer Haemocytometer is most common. This method relies on fixing and diluting specimen so you immobilise the sperm and reduce the numbers down to numbers easier to count.
What can affect the reproducibility of sperm counting?
UKNEQAS has shown sperm counting to be very variable between labs.
Reproducibility is affected by counting chamber, technique/SOP, sampling, sample quality (viscosity), EQA/IQC and also care and training.
Haemocytometer counting is currently the gold standard method of counting sperm. Why?
Validity - Data has shown that the results obtained using the haemocytometer can relate to a clinical picture (fertility or infertility).
Reliability - The results seem to be reliable in achieving the right target answer.
Repeatability - Individual is able to achieve the answer over and over again.
Reproducibility - Different individuals in the same or different centres are able to achieve the same answer.
Give evidence to back up the claim that the haemocytometer method of sperm counting is valid.
The WHO decided to adopt the haemocytometer due to the range of data proving its validity. The WHO has also carried out extensive studies to further validate the haemocytometer for its 2010 manual.
Cooper et al examined samples from more than 4500 men in 14 countries over 4 continents .
Give evidence to back up the claim that the haemocytometer method of sperm counting is reliable.
It has been shown that the haemocytometer can be reliable. Studies have shown that the haemocytometer gives similar answers to flow cytometry (Eveson et al; 1983).
Coulter counters (Parks et al; 1985) and the haemocytometer have been should to be reliable in many other fields of laboratory medicine (biochemistry, microbiology, haematology).
We can also use quality control beads of a fixed concentration to determine whether the haemocytometer will achieve the target concentration assigned to those beads. Something that can be done regularly to check counting chambers.
What can be done to back up the claim that the haemocytometer method of sperm counting is repeatable.
We can count the same sample multiple times with the same operator and dilution and check whether the results are similar.
How can we minimise error in sperm counting with a haemocytometer and ensure the results are repeatable?
1) . Use the same chamber type and method.
2) . Use automation to help.
3) . Reduce technical errors by having robust procedures and quality control.
4) . Reduce sampling error by making sure we count enough sperm for any given analysis. WHO manual shows that counting more sperm reduces the sampling error. It is recommended that a minimum of 200 sperm are counted for any given analysis (preferably 400).
Describe the standard WHO method for the preparation of a sample and haemocytometer for sperm concentration measurement.
1) . Dispense fixative into 2 vials using an air displacement pipette.
2) . Mix the semen sample well by swirling and vortexing allowing no time for the spermatozoa to settle out of suspension.
3) . Use a positive displacement pipette to remove the volume of semen required (usually 50ul) to make your dilution.
4) . Wile the semen off the outside of the pipette tip, taking care not to touch the opening.
5) . Dispense the semen (appropriate to the dilution) and rinse the pipette tip by reaspirating and expressing,
6) . Repeat for the replicate.
7) . Prepare chamber by breathing on the haemocytometer and then sliding the coverslip on. Firm adherence can be checked by inverting the chamber and/or the presence of Newton’s rings.
8) . Mix first dilution thoroughly by vortexing for 10 seconds at maximum speed.
9) . Immediately remove approximately 10ul of fixed specimen then depress the plunger of the pipette slowly, allowing the chamber to fill by capillary action.
10) . Take the second dilution and repeat for the other side of the chamber, then leave in moist environment for 5 mins.
11) . Count the chamber under the microscope.
Describe the standard WHO method for the counting of sperm down the microscope using a haemocytometer (not including sample and haemocytometer perpetration).
Count whole sperm only.
Predominance of pinheads or free heads should be noted. Do not count pinheads.
Ignore debris.
Careful when sperm entangles within debris.
When counting sperm that touch the middle tramlines only count those on the same 2 sides of the square such as the lower and right sides for example.
The number of squares you count is governed by the dilution and the need to count a minimum of 200 sperm.
Concentration = number of sperm / correction factor
The correction factor depends on the number of squares counted and the dilution.
When using methods other than the haemocytometer for counting sperm how must they be validated?
Other methods must be validated against the haemocytometer.
May also use quality control beads to validate other methods.
Why is it beneficial to count sperm? What types of sperm should we be counting to give us a good indication of the quality of the sample?
Spermatocytes count is one of the most important indicators of sperm quality.
Studies have shown a direct relationship between sperm concentration and chances of conception. However, the relationship is not that simple.
Concentration of ‘good sperm’ is more important than total numbers of sperm. For example the concentration of progressively motile sperm, the concentration of sperm fee from antisperm antibodies, the concentration of morphologically normal sperm and the concentration of sperm with intact cell ultrastructure.
Total motile count = conc x volume
Recent evidence has pointed to the total number of normal/good sperm in the ejaculate being the single most predictive factor in terms of either normal or assisted conception.
