Liz Posted June 30, 2011 Share Posted June 30, 2011 Hello again,WARM, Peg and ELU:I have the kits and read the inserts and all the literature.WARM is giving me a headache.So about PeG: at what step do I use it if I have Warm autos and want to detect and ID the allos (if present). Please start imagining me holding the tube of whole blood. which direction do I go: autoadsorb or what??Thanks Link to comment Share on other sites More sharing options...
jcdayaz Posted June 30, 2011 Share Posted June 30, 2011 Sorry to be the bearer of bad news, but in my experience you will maintain your headache if you pursue the WARM procedure. The PEG procedure is much easier, quicker, etc etc. In my humble opinion, the step in which you use the PEG procedure is after a panel has been performed to check for "real" specificity and a positive auto-antibody has been confirmed. Then you can use the PEG process to show any underlying alloantibodies. Link to comment Share on other sites More sharing options...
rravkin@aol.com Posted June 30, 2011 Share Posted June 30, 2011 (edited) Hey Liz,I think that this is a problem for Malcolm, but as he would say, "I'll give it a go." So I will assume that you have confirmed a Warm Auto present in your specimen that you hold in your hand. Once the Warm Auto is adsorbed from the plasma content of your specimen you are left with Warm Auto Antibody Free Plasma. Now you can perform your usual antibody screening technique and if you get an inconclusive result do to week reactivity then there are several ways to enhance that reactivity, one of which can include the use of PeG. The same holds true for performing the DAT on the rbc portion of your now Warm Auto adsorbed specimen. Some other reaction enhancement techniques involve the use of Albumin or LISS with increased incubation time at 37C. The Elu would be used if your DAT is still positive and you can enhance reactivity with your eluate and screening cells using PeG as well as any other suitable enhancement techniques.I hope this helps a little. ): Edited June 30, 2011 by rravkin@aol.com Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 Sorry to be the bearer of bad news, but in my experience you will maintain your headache if you pursue the WARM procedure. The PEG procedure is much easier, quicker, etc etc. In my humble opinion, the step in which you use the PEG procedure is after a panel has been performed to check for "real" specificity and a positive auto-antibody has been confirmed. Then you can use the PEG process to show any underlying alloantibodies.Thank you for your reply and for confirming the persistence of a headache, so let me elaborate: I perform the panel without PeG, then if I have a positive AC I perform the panel again with Peg? Does this relieve me of autoadsorbing and the headache? or am I dreaming? Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 Hey Liz,I think that this is a problem for Malcolm, but as he would say, "I'll give it a go." So I will assume that you have confirmed a Warm Auto present in your specimen that you hold in your hand. Once the Warm Auto is adsorbed from the plasma content of your specimen you are left with Warm Auto Antibody Free Plasma. Now you can perform your usual antibody screening technique and if you get an inconclusive result do to week reactivity then there are several ways to enhance that reactivity, one of which can include the use of PeG. The same holds true for performing the DAT on the rbc portion of your now Warm Auto adsorbed specimen. Some other reaction enhancement techniques involve the use of Albumin or LISS with increased incubation time at 37C. The Elu would be used if your DAT is still positive and you can enhance reactivity with your eluate and screening cells as well as other any other enhancement techniques.I hope this helps a little and I will most certainly look for Malcolm's post. ):hahahaha You have a good imagination. The tube I am holding will give me a headache; thus, as you assumed I know it has a warm auto. So I summarize:1. I auto adsorb with WARM2. I perform the Panel3. I may repeat the Panel with PeG to enhance.Is this right?BUT:I read in this forum that Peg would by some miraculous way help me over-ride the auto adsorbtion step.... ??? Many said they stopped WARM and are now using Peg, yes it did not make sense to me, so here I am with my questions ... help....help....please Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 "over-ride" meaning in my sentence do without.... Link to comment Share on other sites More sharing options...
jcdayaz Posted June 30, 2011 Share Posted June 30, 2011 The PEG I referred to in my previous post is not a regular PEG panel using neat serum. It is a PEG adsorption procedure. You can get the same (if not better) adsorbed plasma/serum to then use for your regular antibody id. I don't see a way around the adsorption step, but I do know the PEG adsorption can save hours! Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 The PEG I referred to in my previous post is not a regular PEG panel using neat serum. It is a PEG adsorption procedure. You can get the same (if not better) adsorbed plasma/serum to then use for your regular antibody id. I don't see a way around the adsorption step, but I do know the PEG adsorption can save hours!Yes, this must be what I read and am trying to understand. What I have is the PeG kit Immucor. How do I use it for autoadsorption.. (I think I am completely off) sorry. Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 jcdayaz, is this the procedure?AABB:"Procedure 1. Wash aliquots of red cells in large volumes of saline three times and centrifuge for 5 to 10 minutes at 1000×g. Remove all residual saline. 2. To 1 volume (eg, 1 mL) of red cells, add 1 volume of serum and 1 volume of PEG. Mix well and incubate at 37 C for 15 minutes. 3. Centrifuge the serum/PEG/cell mixture for 5 minutes and harvest the adsorbed serum/ PEG mixture. "after checking the serum for complete auto-adsoption we then test the adsorbed serum with a panel of cells. ? right?Liz Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted June 30, 2011 Share Posted June 30, 2011 The above all seems good to me.I would just add though, that everyone seems to be talking about auto-adsorption. It must be remembered that, if the patient has been transfused within the last three months, or, in the unlikely event that they are, or have been recently pregnant, then an alloadsorption must be performed, rather than an auto-adsorption. jayinsat 1 Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 yes malcolm. But how about the use of PeG:First: autoad with PegThen run AbId.Is that the magic of not performing the ZZAP autoadsorp? what about the need to initially remove the autoabs on the autologous red cells ? Is that done away with??? Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 (your audience was expecting more) Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted June 30, 2011 Share Posted June 30, 2011 Well, it's a bit difficult, because I NEVER use PEG!!!!! I always use enzyme-treated red cells for either an auto- or an alloadsorption, and have never had any problems, so have never switched. Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 Oooh ok. Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 I am referring myself (yes myself) back to the thread below, when Dave gave some very helpful info.Thanks to all!http://www.pathlabtalk.com/forum/showthread.php?4463-PEG-Adsorption&highlight=autoadsorption Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 I still have this question, please:"what about the need to initially remove the autoantibidies from the autologous red cells before autoadsorption ?" Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted June 30, 2011 Share Posted June 30, 2011 Ah, I can answer that one! You don't need to (unless you want to group them by IAT, in which case you can use dithiothreitol - or, of course, if you have the technology available, by molecular techniques), as there will be, in all but extreme cases, sufficient antigens available that are not blocked to take out the auto-antibody. Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 Thank you Malcolm.One for David: how do I calculate the number of autoadsoptions that I need to do with PeG?Thank you Link to comment Share on other sites More sharing options...
Liz Posted June 30, 2011 Author Share Posted June 30, 2011 Not just Dave, of course all responders' responses are very much welcome. Link to comment Share on other sites More sharing options...
David Saikin Posted June 30, 2011 Share Posted June 30, 2011 LizI love the PeG autoabsorption but use the procedure as first defined in Transfusion in 1999 (vol 39, Jan 1999 pp11-16). (Why you would wash the cells - to get rid of plasma - when you are going to add the same plasma back is beyond me). I have never had to perform more than 1 absorption, but you may repeat using the same cells as many times as you think. The only thing I use WARM for is to destroy Kell system ags. The enzyme pretreated absorption is very good and sometimes I have to use it, but it is more time-consuming. Malcolm has the "luxury" of being in a reference facility and so may utilize such (try the PeG Malcolm). Esp with gel, I cannot absorb out all the autoantibody, so once I have a +DAT with a panaggl in the serum, I switch to tubes and use PeG and Peg autoabsorptions. And Malcolm has a great point in that it is soooo important to know the transfusion/pregnancy history. I wish I had the opportunity to play with the alloabsorptions using R1R1, R2R2 and rr cells. You need to know the ag typings of all the cells you use - - - my mind boggles when trying to elucidate the results generated, but I think it is because I have never had any experience performing these. Link to comment Share on other sites More sharing options...
DFields Posted June 30, 2011 Share Posted June 30, 2011 We used to use ZZAP and then WARM, but have switched to Peg adsorptions and they are so much easier and faster.. Link to comment Share on other sites More sharing options...
Liz Posted July 1, 2011 Author Share Posted July 1, 2011 Dave, thank you for the useful info that I am going to use with my new kit. I have also pulled out the 1999 article. I hope this is the solution to my headache. And that I shall soon be the one answering questions about Peg I shall soon be the expert (false modesty.. ) Link to comment Share on other sites More sharing options...
Liz Posted July 1, 2011 Author Share Posted July 1, 2011 I am back.By: Use of polyethylene glycol for performing autologous adsorptionsJoan Cid, Xavier Ortín, Asunción Pinacho, Rafael Parra, Enric Contreras, and Enric Elies, TRANSFUSION 2005;45:694-697.we use:1 volume of patient’s RBCs, 1 volume of plasma, and 1 volume of PEG (Immucor). This mixture is incubated for 15 minutes at at 37 C and then centrifuged for 7 minutes at 1000g.We harvest the adsorbed plasma-PEG mixture and test four drops of this mixture with one drop of reagent RBCs, incubated for 15 minutes at 37 C and proceed to the antiglobulin test with anti-immunoglobulin G (IgG). Questions:Is this right?What if I use Polyspecific AHG (IgG + C)? would I be defeating the purpose and if so why?Thank you.Liz Link to comment Share on other sites More sharing options...
Liz Posted July 1, 2011 Author Share Posted July 1, 2011 From Dave's ref 1999 Transfusion, it is the same: No washing. Equal volumes of untreated allogeneic RBCs, serum, and PEG were incubated at 37°C for 15 minutes. Four drops of the harvested serum-PEG mixture (to account for the dilution of the serum by the PEG) were added to 1 drop of 3- to 5-percent RBCs and incubated for 15 minutes at 37°C before the antiglobulin test. When subsequent adsorptions were needed to remove antibody, additional PEG was not added.Question: I can use atuologous cells instead of allo, right? ( of course unless there has been Tx and preg in the last 3 months.) Link to comment Share on other sites More sharing options...
Liz Posted July 5, 2011 Author Share Posted July 5, 2011 Please tell me how to check the adsorbed serum for complete adsorance of the auto antibodies.If I add the patient's cells and perform the AHG test, they are already sensitized so it will be positive in both cases.....Thank you Link to comment Share on other sites More sharing options...
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