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Frequently asked questions for: LeucoScreen™

FAQ1: In some samples formation of bubbles makes it almost impossible to read the results under the microscope, what can I do to avoid this ?
Formation of bubbles is part of the chemical reaction taking place between the peroxidase and the hydrogen peroxide. Formation of bubbles is therefore correlated to the amount of peroxidase available in the sample. To avoid bubbles from interfering with the interpretation of the results, cover the semen/LeucoScreen as soon as possible after mixing and read the results immediately.

FAQ2: Can I test if the reagent of the LeucoScreen kit are still active ?
Yes you can !

Materials required for the test:
LeucoScreen test
Peroxidase from horseradish (e.g. Sigma code P8125)

Method (the test is performed at room temperature):
1. "Activate" 1mL of LeucoScreen dye (Reagent 1) by adding 30uL of hydrogen peroxide (Reagent 2) - to save reagent you may also use 0.33mL of reagent 1 and 10uL of reagent 2 or any other volume as long as the relative amounts stay the same
2. Dissolve 1mg of peroxidase into 1mL of demineralized or highly purified water, depending on the concentration indicated on the label of the bottle of peroxidase this will yield a concentration of between 50-150 U/mL of peroxidase
3. Add 10uL of the above peroxidase solution to the activated LeucoScreen reagents and mix well
4. Depending on the concentration of peroxidase in the initial dilution, the color of the LeucoScreen reagent will change from orange-pink to dark brownish-red within 2-4 minutes. The formation of a precipitate after a few minutes is normal

==> The color change indicates the reagent is still active

The image below gives you some idea of the color change you should expect from the QC test. The left vial shows the activated LeucoScreen reagent, next you see the color change after 1, 2 and 3 minutes exposure to the peroxidase.

FAQ3: What is the threshold?
According to the WHO, the threshold is 1x106 peroxidase positive cells/ml. However, this threshold is under debate, as some have found this value too low and other too high. Threshold levels from 0.2x106 - 2x106 peroxidase positive cells/ml have been reported. If the threshold is exceeded, additional tests can be done to investigate for accessory gland infection.

FAQ4: How is the concentration of round cells calculated?
Count the number of round cells whilst determining the sperm concentration in a routine semen analysis. If the concentration of round cells exceeds 1x106 cells/ml, additional testing with LeucoScreen is recommended.

FAQ5: Will LeucoScreen be taken out of production?
Yes, the product will be discontinued as from January 1st, 2020. The kit is replaced by the LeucoScreen Plus kit, which is equally suitable to distinguish between peroxidase-positive and -negative round cells. Moreover, it has the advantage of not containing a carcinogenic substrate.

FAQ6: Is differentiation between different peroxidase-negative cells possible?
No, this is not possible. Spermatids and spermatocytes can be differentiated with a papanicolaou staining or with Spermac Stain.

FAQ7: How is a pink cell with yellow/brown spots classified?
As positive. These cells contain peroxidase that stays in granulocytes. If the cells are fully yellow/brown, the granulocytes are no longer intact.

Resources

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