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Gel antibody panel and tube antibody panels


amym1586

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On 2/4/2016 at 4:24 PM, mollyredone said:

We use Ortho screening cells, 1 0.8% and one 3% and 2 0.8% panels and 1 3% panel.  We rarely do a 3% panel, but do 3% screens if there are gel issues.  If we use the 3% cells for selected cells, we convert them to 0.8% and run them in gel.

same at my place...we are level I trauma center...

 

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6 hours ago, Malcolm Needs said:

Dansket, my understanding, and please correct me if I am wrong galvania, is that the ratio of red cells to plasma, and the dilution of the red cells are both "sacrosanct", and that if you change either of these, you run the risk of getting false positive or false negative reactions?

Malcolm,

I'm not diluting the plasma prior to adding it to the reaction chamber.  For example, ABO plasma grouping test requires 50uL of plasma and 50uL of RRBC.  If you add 50uL of 0.8% RRBC, 50uL of test plasma and 50uL of saline, the ratio of plasma to rbc does not change.  Whether or not saline is added, there is 50uL of plasma and 50uL of 0.8% RRBC in the reaction chamber.

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3 hours ago, Dansket said:

Malcolm,

I'm not diluting the plasma prior to adding it to the reaction chamber.  For example, ABO plasma grouping test requires 50uL of plasma and 50uL of RRBC.  If you add 50uL of 0.8% RRBC, 50uL of test plasma and 50uL of saline, the ratio of plasma to rbc does not change.  Whether or not saline is added, there is 50uL of plasma and 50uL of 0.8% RRBC in the reaction chamber.

I'm not great at math, but, surely, if you are adding 50uL to 50uL of 0.8% red cells, and to 50uL of plasma, you are, at the very least halving the concentration of each.  That is 50uL of plasma becomes diluted by 25% of saline, and 0.8% of red cells are diluted by 50uL of saline, not to say that the 50uL of saline would be diluting both in a way?

I can't see it, but, as I say, I'm not great at math.

 

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2 hours ago, Malcolm Needs said:

I'm not great at math, but, surely, if you are adding 50uL to 50uL of 0.8% red cells, and to 50uL of plasma, you are, at the very least halving the concentration of each.  That is 50uL of plasma becomes diluted by 25% of saline, and 0.8% of red cells are diluted by 50uL of saline, not to say that the 50uL of saline would be diluting both in a way?

I can't see it, but, as I say, I'm not great at math.

 

Me, either.  Forget the math! The total volume in the reaction chamber is 150uL.  The number of antibody molecules present is the same, despite the addition of saline to the mixture.

I don't see this as any different than what is done in test tubes when 1-2 drops of albumin or a LISS additive solution are added to one drop of 3% cells and 2 drops of patient plasma.  What changes the serum/cell ratio is adding four (4) drops of serum to one (1) drop of cells in an attempt to enhance the reactivity of a weakly or non-reactive anti-A or anti-B in an ABO plasma grouping test.

Edited by Dansket
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No, it's more than just volume.

If you add normal saline (which is ionic - Na+, Cl-), this will, quite drastically, change the ionic strength in the reaction chamber from a low ionic to a high ionic situation, and I am sure, in the back of my mind anyway, that it this change in ionic strength, which alters the potential, that causes either the false positives or false negatives - but I can't remember which it is - neg or pos!!!!!!!!!!!!!!!!

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I agree with Michael when you add 50ul saline to 50ul plasma and 50ul cells your concentration is different then what is in package insert. I am sure you must have validated your procedure as you are adding saline and technically diluting your plasma.

when we do saline replacement in tube, we only use it to resolve discrepancy not to ID antibody!

DO you take same tube where you perform saline replacement and carry through all phases...

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1 hour ago, Eagle Eye said:
On ‎2‎/‎5‎/‎2016 at 6:12 AM, StevenB said:

Without tubes, your ability to perform any problem resolution is very limited.....extremely limited. Rouleaux is a classic example: See it with Gel testing and there is nothing you can do except ship it out to a reference lab if you don't have tube testing available.

I agree with Michael when you add 50ul saline to 50ul plasma and 50ul cells your concentration is different then what is in package insert. I am sure you must have validated your procedure as you are adding saline and technically diluting your plasma.

when we do saline replacement in tube, we only use it to resolve discrepancy not to ID antibody!

DO you take same tube where you perform saline replacement and carry through all phases...

My original post was in response to StevenB's post regarding to his perceived limitation of gel testing.

Regardless if the plasma is technically diluted 1:1, if it resolves the ABO discrepancy, I run with it!

In the past, I limited the use of the saline-replacement technique in standard tube testing to resolving ABO discrepancies detected in the immediate-spin ABO plasma grouping test.  I have not used saline-replacement for antibody identification.

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I can't talk for Ortho's gel but this is the situation with Bio-Rad's gel (ex DiaMed).

1.  You can't use saline.  If you do, you risk getting cells that 'hang in the gel and they can look like false positives

2.  You can double the volume of plasma but it won't usually give you better results.  However putting 150ul of 'mixture' in the reaction chamber is asking for trouble as you will overfill it and just get both a mess and carry-over.

3.  If you want to dilute out 3% cells you have to spin them down, remove all of the supernatant and reconstitute to 0.8% with either Diluent 2 or CellStab, preferably the latter.

4.  You CAN do pw in gel.  You put cards, plasma, cells pipette tips in your incubator.  You run your centrifuge next to the incubator, empty.  You use a prewarmed tip to pipette the cells into the card.  Then you use another pre-warmed tip to pipette the plasma on top of it.  You do both of these steps IN the incubator and you work FAST.  As soon as incubation is finished you place the cards immediately into the centrifuge that has been running empty to warm it up and you start it immediately.  Requires a good deal of practice!

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On Saturday, February 06, 2016 at 5:12 AM, Malcolm Needs said:

Dansket, my understanding, and please correct me if I am wrong galvania, is that the ratio of red cells to plasma, and the dilution of the red cells are both "sacrosanct", and that if you change either of these, you run the risk of getting false positive or false negative reactions?

I agree - if you add saline you are changing the ionic strength of the system and you also run the risk of diluting out an antibody, esp when the volumes used are so very small.

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Hello Everyone,

I am in the process of revising the policies and procedures.

Since past 3-4 years we are using gel cards and doing antibody screening and identification using BioRad gel cards and the corresponding cells.

I am not using the tube method for doing antibody identification .

I have tube method panel cells but I prefer using extended panel Plus 6 and papainized RBCs Panel to solve the problem.

I want advice that shall i continue having tube test for rare unresolved cases or shall I remove it .

regards, wajiha

 

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It would be nice to have the ability to perform tube testing for those rare cases of which you speak, but my only worry is, as they are rare, would you be able to guarantee competency when called upon to perform these tests.

It is slightly different for me, working in a Reference Laboratory, as, although such cases are rare within each of our hospitals, almost all of them pass through my laboratory, and so keeping up competency is extremely easy.

Tube testing in the wrong hands can be fraught with danger.

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On 2/12/2016 at 10:46 AM, Malcolm Needs said:

It would be nice to have the ability to perform tube testing for those rare cases of which you speak, but my only worry is, as they are rare, would you be able to guarantee competency when called upon to perform these tests.

It is slightly different for me, working in a Reference Laboratory, as, although such cases are rare within each of our hospitals, almost all of them pass through my laboratory, and so keeping up competency is extremely easy.

Tube testing in the wrong hands can be fraught with danger.

Dear Malcolm, This means that  i should take it as that it is better that I remove the tube testing from the routine procedure used by the technical staff and define it to be used in rare cases as non conformance by senior member or by me only. (actually in problematic cases I prefer to repeat the testing myself before interpretation of the test. a bit crazy !!! but better to be fussy and crazy than putting patient into problem)    

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Malcolm.  I believe the occasional use of tube testing to get around gel interference (especially when a pre-warm is necessary)  is relatively common at many hospitals in the US.  And we do, indeed, have to show that we have documented P&Ps and competencies for tube testing to regulators, just like we do for all the testing we do in the Lab.

 

Scott

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