About PeG

[Malcolm Needs ☆]

I don't think there is an easy way around this, without performing a "mini-panel" of, say, 4 to 6 red cells, and seeing if any of them are negtaive. You have to use a few, just in case there are alloantibodies underneath that just happen to react with antigens on the chosen cells (e.g. an anti-D and an anti-K, that may react with two different cells, and they just happen to be the two you have chosen). Ideally, you should chose the minimum number of cells that will cover all of the major antigens for both positivity and negativity, before going on to a full panel (if required).

In this way, it is the same as adsorbing with enzyme-treated red cells, as we do in my Lab.

[Liz]

Thank you. I see. So, if they all (4 or 6 cells with a variety) react and especially the same strength then that is my auto. We are trying this now with a great first sample: AC 3+, AbID all 3+ and Panel all 3+. I'll report back.

[Malcolm Needs ☆]

Well, yes, if you discount the fact that, if you are doing an auto-adsorption, there is the very remote, but not impossible, chance that the patient may have produced a genuine alloantibody directed against a high incidence antigen. I once had one that had an anti-Vel under an auto (NASTY)!

[Liz]

Partial results were good: we initially had a 3+ AbSc, 3+ panel and 3+ AC. I performed the commercial PeG auto adsorption with egaual volumes of Serum, PeG and patient Cells. 2 adsorptions and the Ab Sc was then negative (pooled cells) and AC was +2.

I shall repeat with non pooled cells and shall try with one adsorption. Moreover, the cells were not washed as Dave suggested.

I performed the AbSc with the adsorbed serum/PeG on the gel cards. This is a disadvantage because of the the serum/PeG to cells ratio. So I shall repeat by the tube method in order to have a 4:1 serum/PeG to cells ratio.

Am I on the right track?

[David Saikin]

It is almost impossible to absorb out autoab and use gel cards. Don't waste your absorbed plasma running an AC - they already have a +DAT. You can use the absorbing cells to perform an eluate (to id the autoab if you want to - I do). Like Malcolm advised - I run my screening cells with the absorbed plasma to see the effectiveness of the absorption. Enjoy . . .

[Liz]

Yes, I guess I learn the hard way. I am waiting for a new sample as I understand that the Abs are weakened if stored with Peg.

So, if I need to perform an Ab ID would that also be in tubes, and what would be the ratio of the volumes, 4 serum/PeG to 1 reagent cells? and at what dilution should the reagent cells be?

Lots of question .......

sorry.....

[Liz]

Follow up: New patient: Ab ID and Panel and AC all 2+. Performed one adsorption with PeG and the AbSc is negative. I love it. BUT: I used pooled cells so I am repeating, also used 2:1 Serum/PeG to reagent cells.

Question1: The reagent cells are diluted so is 2:1 ok?

Question 2: how do I validate my work?

(I was thinking of mixing in an anti-K, 1:1 from another patient, and testing for it after PeG adsorption, hmmm)

[Liz]

Important question to Peg adsorption users: how did you validate the PeG adsorption? pleeease.

And do you still use, order, WARM (zzap) for anything? if yes what?

Thank you.

[David Saikin]

Liz - couldn't send a message so here goes

I would validate PeG by running some abs that I know (I always have a bunch frozen for students). If you have found them using gel expect the reactions to be weaker (3+ in gel = 1+ in peg). If found using LISS, expect the rxs to be stronger. REactions in gel which are >= 2+ usually are neg with Peg. As I mentioned earlier, a way to increase sensitivity is to set up Peg in tubes (2 drops peg/2drops plasma and 1 drop 3% cells. Incubate at 37C for 10 min. Wash one time and add 50uL cells to IgG card and spin. you can validate that the same way (if you want to/I have not

The only thing I use WARM for is destroying Kell system ags. YOu can use it to remove autoab to type cells but I have never found it effective. Try it, you may be able to. Definitely get the ELUII kit (Hemobioscience now also has one, as well as a lectin kit - tell them I recommended them). I also buy gel diluent from them (half the price I pay from ORtho). YOu will need to do elutions for IgG + DATs. I don't think you need to give ag neg red cells when you find an auto with a specificity. The ag neg blood will be treated like just like the pts own. What I try to do with pts with autos is give Rh phenotype specific . . . I have found that if I do not they will invariably make abs to the Rh ags they lack. It really doesn't matter what red cells you give them, as long as they don't have any alloabs, the tranfused cells will have a shortened life span due to the auto

Dave

[Liz]

Thank you Dave, you have really put me out of my suffering with this information. (...... at least for the moment until I face another wall; hopefully not).:flirty:

[Liz]

Liz - couldn't send a message so here goes

I would validate PeG by running some abs that I know (I always have a bunch frozen for students). If you have found them using gel expect the reactions to be weaker (3+ in gel = 1+ in peg). If found using LISS, expect the rxs to be stronger. REactions in gel which are >= 2+ usually are neg with Peg. As I mentioned earlier, a way to increase sensitivity is to set up Peg in tubes (2 drops peg/2drops plasma and 1 drop 3% cells. Incubate at 37C for 10 min. Wash one time and add 50uL cells to IgG card and spin. you can validate that the same way (if you want to/I have not

The only thing I use WARM for is destroying Kell system ags. YOu can use it to remove autoab to type cells but I have never found it effective. Try it, you may be able to. Definitely get the ELUII kit (Hemobioscience now also has one, as well as a lectin kit - tell them I recommended them). I also buy gel diluent from them (half the price I pay from ORtho). YOu will need to do elutions for IgG + DATs. I don't think you need to give ag neg red cells when you find an auto with a specificity. The ag neg blood will be treated like just like the pts own. What I try to do with pts with autos is give Rh phenotype specific . . . I have found that if I do not they will invariably make abs to the Rh ags they lack. It really doesn't matter what red cells you give them, as long as they don't have any alloabs, the tranfused cells will have a shortened life span due to the auto

Dave

Dear Dave,

To clarify, and please excuse my ignorance or if I sound thick:

Running serum with known Abs: I would need 1:1 volume with the autos and allos, and then do the PeG adsorption, or just apply PeG to the Allos with ... with what RBCs? (remember this is stored serum for students).

By "2 drops peg/2drops plasma" you mean 4 drops of the PeG adsorbed serum, right?

My reagent cells are .8% so I have to get the 3%... just talking to myself here .

You lost me (again) at "Wash one time and add 50uL cells to IgG card and spin". What test is this?

"You will need to do elutions for IgG + DATs" why? what do I do then?? You said above that its useless to know the specificity of the RBC ags, so why use the Elu kit?

Why do you do with your cells after you have destroyed the Kell ags with WARM ???

Its nice that you take my intelligence for granted its there but not yet for PeG.

Thank you for accepting abuse

Liz

[David Saikin]

I wouldn't go nuts trying to validate PEG - it is an enhancement solution and is widely used. For ab work it will be weaker than gel but stronger than LISS. When there is autoab I stop using gel, as I cannot absorb all the ab out. I switch to PeG and then am able to obtain useful results. Do you have any 3% panels? If not you should get at least one ( I like BioRads and Medions - always a Kpa+ and Jsa+ with Biorad/not always Jsa+ with Medion).

Let's not get too confused. When I talk about adding pegylated cells to the IgG gel card I am talking about an antibody id NOT an autoabsorption. This is useful if you have a bunch of weak rx in gel - but I use it very sparingly.

As far a validating a PeG absorption . . . how have you validated any other absorption? Do you need to? If you have a panagglutinin in the serum and you can absorb and elute it using Peg autoabsorption I would say it is validated.

Why do you want to do an elution on your autoabsorbed cells? I don't know . . . I like to see if what I am finding in the plasma is what is on the pt red cells. It is not useless to know the specirficity of the autoab . . . it is just useless to give ag negative red cells if you find specificity.

WARMed red cells . . . in the past I have had a pt with anti-K and I presumed an anti-Dia. The only cells I had that were Dia+ were also K+. I WARMED those cells and was able to prove anti-

Dia. I also ran my reagent anti-K vs the WARMED cells to prove that I had destroyed the K ag. I actually have a pt with anti-Kpb . . . I can WARM my panel to see if there might be any other underlying alloabs (which would not be destroyed by WARMing).

[Mabel Adams]

I learned recently that the PEG in Europe is designed to use 4 drops PEG to 2 drops plasma whereas most in the US uses 2 drops PEG to 2 drops plasma. At least that is how the Quotient rep explained it. This is for testing, not adsorptions but it might mean that the PEG reagent is formulated differently. At least Quotient had to resubmit to the FDA to use the 2 drop method here. I guess that could just be a different way of using the same reagent, but it might be good to be sure since we are passing advice/instructions across oceans these days.

[jcdayaz]

WOW! And the PEG adsorption discussion goes on ..... When are these antibodies going to start reading the rule books? haha!

I had a patient just last week with a fairly impressively strong warm auto (3-4+)--I did 3 successive PEG adsorptions and couldn't get rid of it. The testing had started on the previous 2nd shift, continued on the third shift and then was "gifted" to me the next morning. After I spent close to 7 hours on it, I finally gave up and sent it to our reference lab. They start with PEG adsorptions also. They had to convert to the WARM adsorption procedure. Does anyone have any information on when/why the PEG adsorption doesn't work?

Thanks in advance.

[Liz]

Thank you Dave (the thanks button stopped working).

[BBKTech]

This is something that I would also like to know.

From the technical manual: " to check for completeness of adsorption, test the adsorbed serum against the red cells used for the adsorption. If positive, repeat..."

If the patient has a + DAT and their cells are already sensitized, won't this always be positive?

Am I missing something?

Thank you.