Antigen typing by gel methodolgy

[donellda]

Our system will most likely be switching to gel in the near future. I am a former gel user when I worked for another health system and I loved it. Our system Blood bank manager asked me if we did antigen typing by gel methods when I used gel. We did not but I don't see any reason why it couldn't be done. Does anyone use gel for antigen typing?

[Eagle Eye]

Before you use the gel for antigen typing you will need to validate the process. I know someone who did a little project on donor antigen typing. As long as you use AHG reacting antisera you will be able to reroduce the same result as tube technique.

It is going to be cheaper...as the cost of antisera goes up gel method is cheaper then tube technique because you are using 1/2 or 1/4 of the antisera comapre to tube method.

Also you have less operator error because you have a prinout(if you are using SA reader).

Only probelm you will encounter is when you have positive DAT on the donor. You might see 1+ typing for donor unit by gel and negative by tube. But in that case you can compare your antigen typing to positive control or run DAT on donor unit and call it positive or false pos or neg.

You will encounter the same problem with the patient if you are going to decide to use gel for patient phenotyping. I believe that you need to run patient's DAT as control everytime you use gel for phenotyping.

[KathleenSL]

We are a manual gel user. We do use both IAT and monoclonal reagents to do antigen typings in gel. We validated each antisera in gel. You will need to decide whether to use Diluent 2 or saline for you 0.8% when you validate. We chose saline (same as tube testing & reagent directions). We use Buffered Cards for the monoclonals and follow the reagent incubation temperature. We chose to use 15 minutes as the incubation time during reagent validation (fell with in the manufacturers' recommend times). We do not dilute any antisera. The IAT reagents work great in the IgG Cards. Occassionally we get some questionable results with the monoclonals in the Buffered Cards and need to repeat testing using tubes.

[donellda]

Thanks Kathleen! I think that since most antisera (AHG) are done without LISS, the saline would be the way to go.

[Sandy Jo]

Would anyone be willing to share their Gel Antigen typing validation and/or procedure?

I am planning to validate gel antigen typing and I would like to read over some helpful resources before starting the project. Thanks so much for your assistance...

fax (813)634-0127

email sandra.rothenberger@hcahealthcare.com

[bbbirder]

I would like to reopen this thread to see if anyone else has validated gel recently.

I would like to use gel for antigen typing, units especially.

Has anyone been using gel for antigen typing and encountered problems?

Thanks,

Linda Frederick

[galvania]

Hello everyone who wants to use gel for antigen typing. I just thought you might like a few tips. Depending on the antigens you are typing for, there are 3 different methods for use with the gel. Rh phenotype and K, as well as M and N are monoclonal reagents that use a modified LISS solution and excepting the fact that any monoclonal antisera might miss a few mutaions, there should not be any problems with these. Even someone with a 4+ DAT will not give you false positives. The only problems you will get is with someone with strong cold agglutinins. But then the control well will also be positive. For k, Kpa, Kpb, Lua, Lub, Jka, Jkb, P1, Lea, Leb you need to use an enzyme to get the reaction. Here you will have 'false' positives if the patient/donor has a strong positive DAT with IgG coating or if they have enzyme auto-antibodies. Again, the control will be positive, so this will indicate that the test is invalid. For Fya, Fyb, S and s, this is a Coombs technique, so clearly a DAT needs to be put up as a control. Obviously, the above only goes for our gel technique - other companies might have different limitations. Are all of these reagents available in the States? Hope these comments are of some use

Anna

[David Saikin]

I wonder what company's gel you are using? In the USA there is only one vendor. I know that in Europe there are at least 3 different suppliers. I have validated typings in gel using either the buffered gel cards or the anti-IgG cards available here. The only difficulty I encountered was with Lea typing (probably had to do with the antisera being outdated for a few years more than the gel). I did not need to use enzymes. Are these recommendations based on your personal validations or from your gel manufacturer(s)? Thanks for your valuable input.

[galvania]

Hello David. Well, I didn't mention the company's name because I work for the company, and so I think it constitutes advertising, which as you know is not allowed. Perhaps the people who run the site could actually clarify that point for me. Am I allowed to mention the company's name in response to a direct question like this one? Well, for now David, if you drop me a line at a.galvani@freesurf.ch I can tell you privately! All of the methods are the company's own methods and should not really need to be validated as they undergo extensive QC here in Switzerland, as well as being externally validated to obtain the CE mark. Maybe they're not available in the states directly? I'm not sure how that side of things works.

[Rizebal]

Maybe I am confused...If you are performing Fya, Fyb, S, s (or any Coombs phase typing) in buffered gel cards, how does that work? It is my understanding that there is no anti-IgG in buffered gel cards. I know that the Buffered Gel Card's package insert says that it can be used for warm antibody ID, but without the anti-IgG how is that possible...for the Ab ID or Ag typing? Thanks!

[yiams]

Donna,

Technically, no. Our pathologist has not yet signed off. We've played with it and it seems to work fine. The hints we've received from various sources are: 1. IgG gel cards with IgG antisera are handled as recommended by Ortho. 2. Buffer gel cards with non-IgG antisera (monoclonal or blend using 37C or RT) should be handled like a backtype; 50uL of RBCs and 50uL of antisera.

What our path has approved is the use of Ortho's Rh antigen cards. We can use their C cards, c cards and E cards. Does Ortho make any others? I don't know. My supervisor who does the orders has not mentioned other antigen cards to me.

Hope this helps some.

Randy

[Gerald]

When you use the buffer cards, it should be 50ul RBC's and 25ul antisera.

Take a look at the following link:

http://www.cbbsweb.org/enf/2005/gel_phenotype.html

[galvania]

In the DiaMed (ID MTS) gel cards (which are the ones I was talking about before), the Fya, Fyb, S and s are used with Coombs cards (either poly or IgG). There are specific antisera designed to be used with the cards. There is also a specific anti-M and anti-N liquid reagent for use with buffered cards (50ul + 50ul). All of the other antisera are already present in the gel so no other liquid antisera are neccessary. Otherwise the buffered cards take either 25ul or 50ul of antiserum/patient plasma depending on the test being carried out

Anna

[David Saikin]

I validated my buffered cards with 50uL cells/25 uL antisera.