Mary** Posted April 17, 2009 Share Posted April 17, 2009 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. Link to comment Share on other sites More sharing options...
David Saikin Posted April 17, 2009 Share Posted April 17, 2009 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. Link to comment Share on other sites More sharing options...
bxcall1 Posted April 21, 2009 Share Posted April 21, 2009 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. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted April 24, 2009 Share Posted April 24, 2009 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. Link to comment Share on other sites More sharing options...
salman Posted May 27, 2014 Share Posted May 27, 2014 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 Link to comment Share on other sites More sharing options...
Joanne P. Scannell Posted May 27, 2014 Share Posted May 27, 2014 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. Link to comment Share on other sites More sharing options...
goodchild Posted May 27, 2014 Share Posted May 27, 2014 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... Link to comment Share on other sites More sharing options...
mollyredone Posted May 27, 2014 Share Posted May 27, 2014 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. Link to comment Share on other sites More sharing options...
cthherbal ☆ Posted May 28, 2014 Share Posted May 28, 2014 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. Link to comment Share on other sites More sharing options...
Teristella Posted May 29, 2014 Share Posted May 29, 2014 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. Link to comment Share on other sites More sharing options...
goodchild Posted May 29, 2014 Share Posted May 29, 2014 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. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted May 29, 2014 Share Posted May 29, 2014 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. Link to comment Share on other sites More sharing options...
JEMarti Posted June 3, 2014 Share Posted June 3, 2014 (edited) 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 L106 and David Saikin 2 Link to comment Share on other sites More sharing options...
goodchild Posted June 3, 2014 Share Posted June 3, 2014 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? cthherbal 1 Link to comment Share on other sites More sharing options...
David Saikin Posted June 3, 2014 Share Posted June 3, 2014 I have rarely seen contaminated antisera in 40+ yrs in the field. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted June 3, 2014 Share Posted June 3, 2014 Neither have I David - but we do use disposable tips. I suspect if disposable tips are NOT used, then there is more chance. Link to comment Share on other sites More sharing options...
Dansket Posted June 3, 2014 Share Posted June 3, 2014 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. Link to comment Share on other sites More sharing options...
mollyredone Posted June 3, 2014 Share Posted June 3, 2014 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. Link to comment Share on other sites More sharing options...
Eagle Eye Posted June 3, 2014 Share Posted June 3, 2014 There was a abstract in 2008 Transfusion supplement regarding the cost comparision between Gel and tube method. Link to comment Share on other sites More sharing options...
Mabel Adams Posted June 5, 2014 Share Posted June 5, 2014 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. Link to comment Share on other sites More sharing options...
Likewine99 Posted June 5, 2014 Share Posted June 5, 2014 Same as David and mollyredone. Link to comment Share on other sites More sharing options...
David Saikin Posted June 6, 2014 Share Posted June 6, 2014 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 Link to comment Share on other sites More sharing options...
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