Antigen Typing in Gel

[Mary**]

Would anyone be willing to share your procedures for special antigen typings (K, C, Lea, etc.) in gel? I am looking for both room temperature and coombs reactive procedures. I would like to use less of the now very expensive antisera. Thanks in advance.

[David Saikin]

I use 50uL of cells and 25uL of antisera. The anti-IgG gel cards are used for AHG reactive antisera. The buffered gel card for non-AHG reactive antisera. 15 minute incubation for the IgG cards. The buffered gel cards are incubated as per antisera instructions. All are spun for 10 minutes. You will need to validate all your antisera reactivity. I recommend running homozygous, heterozygous and negative cells with each antiserum for your validation, but how and what you do is up to you.

[bxcall1]

I have validated most all of my antisera for use on the Provue. As the previous post stated, use the IgG cards for those antisera that reguire AHG and use the buffered gel cards for those that don't.

If saving antisera (MONEY) is your goal, run crossmatches using your patient serum/plasma, then type only the units that are compatible.

[Malcolm Needs ☆]

I agree with both of these posts, but, as David says, validation is an absolute MUST.

We do almost all of our typing in gels now, with very few now performed in tubes.

[salman]

I use 50uL of cells and 25uL of antisera. The anti-IgG gel cards are used for AHG reactive antisera. The buffered gel card for non-AHG reactive antisera. 15 minute incubation for the IgG cards. The buffered gel cards are incubated as per antisera instructions. All are spun for 10 minutes. You will need to validate all your antisera reactivity. I recommend running homozygous, heterozygous and negative cells with each antiserum for your validation, but how and what you do is up to you.

 

David

 

Could you provide me your validation procedure .Thanks

[Joanne P. Scannell]

I use 50uL of cells and 25uL of antisera. The anti-IgG gel cards are used for AHG reactive antisera. The buffered gel card for non-AHG reactive antisera. 15 minute incubation for the IgG cards. The buffered gel cards are incubated as per antisera instructions. All are spun for 10 minutes. You will need to validate all your antisera reactivity. I recommend running homozygous, heterozygous and negative cells with each antiserum for your validation, but how and what you do is up to you.

Ditto, once again. 

[goodchild]

In my recollection, aren't buffered gel cards fairly expensive? Has anyone checked cost savings per test? Is this method change being driven for cost or for standardization/easier review?

 

I would love to do our antigen typing in gel...

[mollyredone]

I just recently validated Fya, Fyb and S in IgG gel cards.  I used 20 units and 20 patients.  My controls were panel cells, 1 negative and 1 heterozygous positive.  We will be switching to Jka and Jkb 5 minute incubation antisera soon, so I didn't test them. 

[cthherbal ☆]

We do the AHG reacting antisera testing in gel IGG cards. This process has been in use for probably 10 years and works very well. Pipetting is same as David's above.

[Teristella]

In my past life as a reference lab tech we would routinely phenotype new donors this way. We used the same procedure as David above. Saved us a lot of time finding the full phenotypes of multiple donors (we used plate testing to screen large batches for little c and little e negative units and would complete phenotype in gel). Write up a procedure and do a validation comparing results in tube to results in gel and you should be fine. The number of samples you choose to run is really up to you, but as someone said above, it would be a good idea to run some heterozygous samples. 

[goodchild]

Since I've never done phenotyping in gel cards, how easily is a mixed field reaction interpreted? e.g. phenotyping a patient who has been recently transfused.

[Malcolm Needs ☆]

It's very easy to see the mixed-field goodchild, very easy indeed.  The problem is knowing whether the positive cells are native, or the negative cells are native.  In other words, you can't, with any degree of certainty.  I'm afraid the only way to do it is by molecular techniques.

[JEMarti]

In my recollection, aren't buffered gel cards fairly expensive? Has anyone checked cost savings per test? Is this method change being driven for cost or for standardization/easier review?

 

I would love to do our antigen typing in gel...

Gel cards might seem expensive but they are cheap compared to the antisera.  Considering that a drop from a dropper is ~100ul you can cut your antisera costs by 75% per test by using 25ul in a gel card.  WIth some antisera running around $1000 per vial the antisera cost is potentially the biggest component of the test. 

 

Also, in a past life I did a cost study on antigen typing and in interviewing bench techs discovered (surprise) that techs often rely upon their training for how they do Ag typing and not the package insert(or their SOP).  So while the package insert says to use 1 drop and hold the dropper vertically, they will hold the dropper at an angle and use up to 2 drops (such was the way I was trained back in the 80's).  That can triple your cost per test really fast.  I found that average technique ran somewhere between the package insert and old school training (in truth almost right down the mddle). Gel testing allows you to control antisera usage in a way that tube testing cannot so not only will you use less but your usage will be more predictable.

 

If you want another eye opener, take a vial of antisera and see how many drops you get out of it and how that varies by your technique. Some manufacturers will give you very different results with a variance in technique others less so but there is always a difference.  Holding a dropper at an angle can decrease the number of drops/tests per vial by 30% or more.

 

Full disclosure: My current company is not one that sells antisera or blood typing reagents, although I have worked for one of those companies in the past.

Edited June 3, 2014 by JEMarti

[goodchild]

In my direct observations for competency assessment I've seen the same things JE.

 

Another follow up question regarding this topic that was brought up when I discussed this with our senior techs. For institutions that do their antigen typing in gel how often do you see contaminated antisera?

[David Saikin]

I have rarely seen contaminated antisera in 40+ yrs in the field.

[Malcolm Needs ☆]

Neither have I David - but we do use disposable tips.  I suspect if disposable tips are NOT used, then there is more chance.

[Dansket]

 

I just recently validated Fya, Fyb and S in IgG gel cards.  I used 20 units and 20 patients.  My controls were panel cells, 1 negative and 1 heterozygous positive.  We will be switching to Jka and Jkb 5 minute incubation antisera soon, so I didn't test them. 

 

ORTHO Monoclonal anti-Jka 5-minute spin antiserum didn't work in Gel for me (verified this with an ORTHO scientist). Antiglobulin antisera does work in Gel.

[mollyredone]

It seems the Jka/b 5 minute is cheaper, although maybe not as much volume per bottle, so we decided to not do IgG method on it.  We still have the other IgG antisera, but since we're switching I decided not to validate.

[Eagle Eye]

There was a abstract in 2008 Transfusion supplement regarding the cost comparision between Gel and tube method.

[Mabel Adams]

Part of my calculations would also include whether or not we currently (using tube testing) expire 1/2 the bottle of anti-S rather than use it up.  In that case using less per test doesn't save anything and the cost of the gel card has a bigger impact.  I guess we would have more expired antiserum in the vial to donate to the local MT school.  

[Likewine99]

Same as David and mollyredone.

[David Saikin]

   

ORTHO Monoclonal anti-Jka 5-minute spin antiserum didn't work in Gel for me (verified this with an ORTHO scientist). Antiglobulin antisera does work in Gel.

 

It worked for me but I let it sit for 15 minutes before centrifugation