Questions About Adding Albumen To Neutralize Enzymatic Activity
We’ve received numerous questions about adding albumen to the liquified sample in the Qwik Check Liquefaction kit and have asked Dr. Rabinovitch, our Chief Technology Officer Emeritus to answer the question for us. Dr. Rabinovitch states:
Though adding albumin for termination the enzymatic process is well known, this guidance is not provided by WHO. Historically this was added to the product insert to provide guidance for some of our customers who inquired how to stop the enzymatic process when liquefaction is achieved. I do not believe majority of our customers implemented this step.
Concerning the questions, my answers are following:
Question: The package insert for the QwikCheck Liquefaction kit states, “Once the sample has liquefied (5-10 minutes), immediately perform automated testing or neutralize the enzymatic activity (optional) by adding of Human Serum Albumin (HSA) (not provided in this kit).” However the insert does not state how much to add, or to what volume of semen. Is there any additional guidance on this? Could Bovine Serum Albumin be used (we already have that)?
Answer: We will be removing this guidance from our product insert. Adding BSA has not been tested by our lab, so we cannot advise.
Question: What are the consequences of not adding albumin? Generally we can run the sample immediately on the instrument as the insert states, but I am currently working on our semi-annual validation study, and wondering about impact of the liquefaction enzyme on the manual method. I can only do the manual motility and sperm count so fast. Will using the liquefaction kit without albumin impact results if testing cannot be performed immediately?
Answer: In fact, using chymotrypsin for treatment of highly viscous or not completely liquefied semen samples results in limited proteolysis that does not impact assessment of semen parameters (please see attached technical release bulletin). The WHO 5th ed. manual does not provide any guidance of adding albumin for termination of the enzymatic process initiated by the protease enzyme for sample liquefaction. Adding albumin is a kind of precaution if the samples analysis takes a long time and that is why it was recommended as an optional procedure.
Question: I may be seeing things, but it seems to me that when I do the manual hemocytometer count, there are a larger number of sperm heads without tails than there were initially when I did the motility. I have seen this more than once. Is it possible that the liquefaction kit enzyme is digesting the sperm tails over time? I think I may have asked one of you this question before but I just saw it happen again…. If this is happening and I could prevent it by adding albumin I’d be interested in doing that, I’m just not sure how.
Answer: The phenomenon of tailless heads seen when counting sperm using hemocytometer is unlikely to happen due to chymotrypsin treatment. Most probably it caused by impact of fixative media (formalin) if this was added in advance long before examination (please check percentage of formalin in the fixative media). Intensive sample mixing by vortex can also contribute to disintegration of tails.