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Manufacture of Phenotyping Reagents


Blood_Banker

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Hi all

I'm hoping someone can help answer several questions that I have about the manufacture of phenotyping reagents.

Currently our procedures require us to test raw plasma donations containing alloantibodies for antibodies to low incidence antigens. These raw donations are then used to manufacture phenotyping reagents. So my question revolves around whether this procedure is worthwhile. I've been trying to find a reference that specifically states what low incidence antigens should be tested to see if this procedure has some sort of basis in order to eliminate it or modify it. The UK BTS lists the following:

"Where appropriate, the following requirements should also be included in performance evaluation:In the case of polyclonal antibodies, contaminating antibodies to antigens having a prevalence of greater than 99% in the general population of the UK should be excluded by negative results in tests using samples of red cells from four different individuals who lack the antigen corresponding to the antibody specificity under test. Tests for the presence of contaminating ABO antibodies should be performed with red cells from a minimum of two individuals of group A1 and two of group B who lack the antigen corresponding to the antibody specificity under test.If tests using all methods recommended for use by the manufacturer do not exclude the presence of antibodies to the following antigens, these antibody specificities should be stated in the package insert as not having been excluded in specificity testing:Xga; Doa; Yta; Cob; Wra; and Vw"

The strange thing is that our procedure specifically states to test against Bg(a+) cells specifically but where on earth did this come from. And it presents plenty of problems as we lack Bga antisera to even consider being able to easily perform this test.

Secondly as part of this testing we also perform testing against papainised cells but immediate spin testing. Is this really relevant? Are phenotyping reagents used against papainised cells? Are we likey to be able to detect relevant contaminating antibodies if we extend this to a 2 stage IAT with papainised cells? Or are we likely to find all relevant contaminating antibodies using a simple IAT with no enzymatically treated cells.

I guess that leads me to my next question - has anybody ever had their polyclonal antisera react to a low incidence antigen?

Plenty of questions but I see there are plenty of great minds who can hopefully provide me with some sort of answers to my post.

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I am not familiar with UK regulations, but in the USA blood grouping reagent specifications are listed under 21 CFR 660 Subpart C. The other document available is older, but it is currently the only guidance I'm aware of that indicates which low incidence antibodies are required to be ruled out. Bg antibodies are not required, but we try our best to rule them out to prevent problems for customers. There has been the rare occassion where a polyclonal reagent does contain a low incidence contaminant that is not required to be ruled out. The availability of rare cells can be a problem and where does it end with all the lows known to exist. No enzyme testing is required.

http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/UCM080926.pdf

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What are the references for your policy/procedure? You may find you answers with those.

I've already tried to go down this path but the current procedure appears to have been so truncated that the references that may have existed have been removed. Now it would be great if I could go to the very first edition of the procedure but our archiving procedures then were very poor so it will take a lot of work to find this edition. I was hoping people might have some answers for me here - also there are a lot of people here who use these reagents so I was hoping to get a general feel for what is expected from these reagents.

Also some of my questions are more broader such that people with long experience in blood banking would be able to answer (something that I sadly lack). In particular, whether 2 stage IAT papain testing would help detect relevant contaminating antibodies that simple IAT testing otherwise wouldn't. That is why I'm curious to know if any people use phenotyping reagents to test papainised cells or even if people test their QC reagents with papainised cells (even though IFU don't indicate that these reagents should be used this way!). If the answer is no, then really there is no point in using papainised cells for testing these reagents.

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I am not familiar with UK regulations, but in the USA blood grouping reagent specifications are listed under 21 CFR 660 Subpart C. The other document available is older, but it is currently the only guidance I'm aware of that indicates which low incidence antibodies are required to be ruled out. Bg antibodies are not required, but we try our best to rule them out to prevent problems for customers. There has been the rare occassion where a polyclonal reagent does contain a low incidence contaminant that is not required to be ruled out. The availability of rare cells can be a problem and where does it end with all the lows known to exist. No enzyme testing is required.

http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/UCM080926.pdf

Yes in my investigations, I have also found these guidelines but thank you for the link. And your point is absolutely right about the number of lows known to exist - how long is a piece of string. I mean in our particular procedure it even says to test against SARA, which is just crazy considering that there are only a handful of people that have this low incidence antigen. That's why the more I think about it, the more I'm inclined to think that someone just selected some random low incidence antigens to throw in the procedure. But with the crazy amount of regulation in our company now, to modify this procedure is extremely difficult as you have to confront a panel of 10 control members (most of which lack any sort of blood banking knowledge) and have one moron disagree with you to get your change thrown out and have to restart you justification.

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