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Positive antibody screen/ negative panel

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Is there a good explanation in a technical manual to better explain to my co workers how to handle this situation : positive antibody screen with a negative antibody panel

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there are three main reasons why this can happen:

1.  antibody against a low frequency antigen present on the screening cell but not on the panel

2.  Temperature difference between the screen and the panel.  Main culprit here is anti-M

3.  contamination

 

Are there lots of screens positive with this cell?  Yes, all in a batch - cell contaminated

                                                                             No, only this one, but neg on repeat - either 2 or 3

                                                                             Some - either 1 or 2

You can repeat the panel in IAT (I presume we are talking about an IAT technique here) at RT.  If it's an anti-M it will come out more strongly

If it's an anti-LFA then finding blood that is XM-compatible won't be a problem

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Thank you!

 

They were on the right track and antigen typed for M but they were still confused about why.

 

I'm not sure if we have a policy/procedure for doing panels at RT, I'll have to further look into that.

 

I'm a new BB supervisor and I"m trying to get as many reference charts, key things to remember and what not. My entire BB staff are floaters who don't get a lot of experience.

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If you're using gel cards, it could also be a gel card well problem. Enforce the visual inspection of each card before use and give people a rack to put the 'bad ones,' if they're uncomfortable of wasting reagents.

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Well quality guy.  If you've got 'bad ones' you should be looking into why they're bad - especially if it happens often.  It's usually due to disasters during transport or storage in a bad place.

Anna

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I've noticed that the Ortho screening (and abid ) cells seem to be extraordinarily sensitive.  If I use a different vendor's panel I may get a negative result (esp for anti-D due to residual RhIg).  I do keep an Ortho Panel around but tend to not use it as my primary panel, rather, I will use it to clarify rxs that only occur with the Ortho screening cells.

Edited by David Saikin

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Well quality guy.  If you've got 'bad ones' you should be looking into why they're bad - especially if it happens often.  It's usually due to disasters during transport or storage in a bad place.

Anna

I apologize if I came off as rude? I had no intention, I was simply responding to my experience. There's a number of factors involved and it should definitely be researched if a large volume are bad. Regardless, manufacturer instructions indicate they need to be visually inspected prior to pipetting.

Another one that we see rarely: if you're using Ortho gel system you might see an anti-E or anti-K that reacts in the antibody screen but not in the gel panel. Then if you switch to tube testing with PEG you can see a clear cut antibody. The manufacturer points out that Es and Ks can be missed with this test system.

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Sorry Quality Guy - I didn't think you sounded rude, and I didn't mean to be either - just to the point!  I see SO many cases of gel drying out or  supernatant in the reaction chamber because of poor storage or transport conditions...

Anna

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We're using an automated solid phase system and it has an similar issue sometimes, not always. Usually, the ab. screen is positive with both screening cells (usually 2+ or below) and the panel is totally negative on the same testing platform. As the LISS tube and manual solid phase methods are our backup methods, for the situation like this, we usually repeat the antibody screen with both backup methods (the same lot of screening cells is used for automated and manual solid phase tests, and different screening cells are used for LISS tube method) and if both (LISS tube and manual solid phase) are negative (majority of the cases), we usually think it's something wrong with the automated analyzer (report to the analyzer vendor) and result the antibody screen negative. Otherwise, if the manual solid phase ab. screen is positive and the LISS tube method is negative (plus the solid phase panel is negative), we suspect it's something particular with the lot of solid phase screening cells and  conclude "All common clinically significant antibody ruled out" and perform a full crossmatch if blood is required. If the repeat is positive with both the LISS tube and manual solid phase or only with the LISS tube, we set up a LISS tube panel for further investigation (So far, we haven't had this scenario, yet.).

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We are using Echos and prior to that we had a Galileo. There's an interesting phenomenon that can occur with automated solid phase that can cause a positive screen and then a negative panel. I have personally seen it happen at least twice. If there are bubbles/foam on the top of the specimen the instrument will pipet the bubbles/foam and this underpipetting of specimen can actually cause the absc to be look positive. When the panel is performed, the bubbles have already been removed and the instrument pipets the plasma correctly, the panel is negative. All of our techs are taught during training to inspect the specimen for bubbles/foam prior to placing on the instrument but sometimes it is a step that is overlooked when it's busy.

Another cause of false positive can be using cold undermixed indicator cells. If a new bottle of indicator cells is placed on the instrument without allowing them to warm the cells may not resuspend completely prior to being pipetted. Since the instrument pipets from just below the surface of the reagent it's possible to not have the proper amount of cells. This also occurs when bottles are loaded without a stirball having been added to the bottle.

Just a couple of other ideas for troubleshooting.

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On ‎9‎/‎6‎/‎2015 at 2:12 AM, galvania said:

there are three main reasons why this can happen:

1.  antibody against a low frequency antigen present on the screening cell but not on the panel

2.  Temperature difference between the screen and the panel.  Main culprit here is anti-M

3.  contamination

 

Are there lots of screens positive with this cell?  Yes, all in a batch - cell contaminated

                                                                             No, only this one, but neg on repeat - either 2 or 3

                                                                             Some - either 1 or 2

You can repeat the panel in IAT (I presume we are talking about an IAT technique here) at RT.  If it's an anti-M it will come out more strongly

If it's an anti-LFA then finding blood that is XM-compatible won't be a problem

On number 2, you said there was a temperature difference between screen and panel. What did you mean by that? Both screen and panel were performed by Gel cards and incubated at 37 degrees C for 15 minutes.

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It depends a bit on how you work.  If you are working manually then it is quite common to pipette a whole series of tests .  In this time the cells can cool down enough for the anti-M to latch on, and once it's on it stays on.  On the other hand, panels tend to go up individually so the cells stay warmer

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5 hours ago, galvania said:

It depends a bit on how you work.  If you are working manually then it is quite common to pipette a whole series of tests .  In this time the cells can cool down enough for the anti-M to latch on, and once it's on it stays on.  On the other hand, panels tend to go up individually so the cells stay warmer

Thank you for replying. Actually everything was done by gel card, we don't use the tube method.  Patient was typed A Pos. The antibody screen was 1+ positive on Screening cell 1 and 3, negative on Screening cell 2.  But when we did the Antibody Identification panel and Auto control , they were all negative. We repeated everything and got the same results.  We redrew the patient for a new specimen and got the same results. 

We sent out the specimen for an Antibody Identification at a reference lab. The result we got back was Anti-M. 

I am just wondering, why didn't our panels picked this up? Why only the screening cells were positive? What was the reasoning behind this? The panel and all our reagents have passed QC and are not expired. There was no signs of contamination in our specimen or reagents. Thank you. 

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5 hours ago, galvania said:

It depends a bit on how you work.  If you are working manually then it is quite common to pipette a whole series of tests .  In this time the cells can cool down enough for the anti-M to latch on, and once it's on it stays on.  On the other hand, panels tend to go up individually so the cells stay warmer

I am sorry, but what do you mean by the "panels tend to go up individually"?  We just have 2 panels, and both these panels have about 20 cells. So about 40 cells of varying phenotypes. 

 

In your original post, you said the "main culprit here is Anti-M". You were right! How did you know exactly? 

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Because I've seen so many of them………….

But actually if your panel is not in the same buffer then pH or ionic strength could be the culprit rather than the temperature.

Important to stress that these cold anti-Ms (in that they dont react strictly at 37°C) have no clinical significance.

If it happens again, you can try the following:

1.  Repeat the panel, incubating for 15mins at RT (on a Coombs card).  The results will be stronger in this case.

AND 2.  Repeat the screen in the following way.  Warm the cells to 37°C  (best just to use a small aliquot - you dont want to 'cook' the whole bottle). And put the Coombs card  (foil still on) in the incubator for 15mins. Put the patient's plasma in the incubator. IN the incubator, pipette 50ul of cells into the appropriate wells followed by the patient's plasma.  Incubate for 15mins.  At the same time, start the centrifuge empty.  This warms the centrifuge up a bit.  After 15mins put the incubated card into the centrifuge which will have now stopped and centrifuge immediately.

This should negativise the screen results.  This is about the nearest you can get to doing a gel test at strictly 37°C

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On 4/29/2020 at 9:29 AM, galvania said:

Because I've seen so many of them………….

But actually if your panel is not in the same buffer then pH or ionic strength could be the culprit rather than the temperature.

Important to stress that these cold anti-Ms (in that they dont react strictly at 37°C) have no clinical significance.

If it happens again, you can try the following:

1.  Repeat the panel, incubating for 15mins at RT (on a Coombs card).  The results will be stronger in this case.

AND 2.  Repeat the screen in the following way.  Warm the cells to 37°C  (best just to use a small aliquot - you dont want to 'cook' the whole bottle). And put the Coombs card  (foil still on) in the incubator for 15mins. Put the patient's plasma in the incubator. IN the incubator, pipette 50ul of cells into the appropriate wells followed by the patient's plasma.  Incubate for 15mins.  At the same time, start the centrifuge empty.  This warms the centrifuge up a bit.  After 15mins put the incubated card into the centrifuge which will have now stopped and centrifuge immediately.

This should negativise the screen results.  This is about the nearest you can get to doing a gel test at strictly 37°C

Thank you very much.  This have been very helpful. I am sorry, I just have another question. You said you have seen so many of them.  What was the cause for majority of those cases you encountered?  What was the typical/common reason? 

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In my case, temperature, as the screening cells and panel were both from the same manufacturer, therefore the same buffer system

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On 4/20/2020 at 7:41 AM, galvania said:

It depends a bit on how you work.  If you are working manually then it is quite common to pipette a whole series of tests .  In this time the cells can cool down enough for the anti-M to latch on, and once it's on it stays on.  On the other hand, panels tend to go up individually so the cells stay warmer

I am sorry for so many questions. You said the panels tend "to go up individually". What do you mean by that "go up individually"? Can you elaborate more on that? Individually, like by cell#? Thank you . 

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sorry for the late reply.  

What I meant is that one tends to put up a batch of antibody screens.  In the time taken to put up say 12 screens the cells can cool down enough for a cold anti-M in the plasma to latch on.  On the other hand, panels tend to go up one at a time (1 patient at a time) so cells have less chance of cooling down. (Always assuming the screening cells and the pane are from the same manufacturer and being tested in the same method)

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On 4/30/2020 at 11:31 AM, diplomatic_scarf said:

Thank you very much.  This have been very helpful. I am sorry, I just have another question. You said you have seen so many of them.  What was the cause for majority of those cases you encountered?  What was the typical/common reason? 

We, too, encounter Anti-M often using Gel.  Apparently, Gel is just acidic enough to enhance this pest.  

Because the enhancement is more about pH than temperature, whenever we suspect Anti-M, we revert to other methods to help determine it's 'etiology'.  Prewarming Gel does not change the pH issue so we don't do that. We can't trust that negative results in prewarmed Gel are truly due to a cold Anti-M or that the positive results aren't due solely to the acidity.  (Besides, if testing is done using a Blood Analyzer (e.g. Vision, Eflexis), the cards are pre-warmed anyway.)

So, we test the plasma using Prewarmed Tube Testing (e.g. PEG which has similar sensitivity for most antibodies sans the low pH).  If positive with that, it's a Warm Anti-M. 

We also test at Room Temperature using Pooled Screening Cells.  If positive, then it's a cold Anti-M. 

If negative with both Prewarmed Tube and RT Pooled O Cells, it's 'Anti-M (Detected using Gel Only)' ... just due to the pH drop, no clinical significance. 

Likewise, positive with both, it's an Anti-M with a broad thermal amplitude.

Do what you like with the results.

PS.  Testing positive with Anti-M reagent does not mean the patient can't make Anti-M (Patient may be Mg Pos).  Someone please correct/confirm that or am I working on old reagent data?  (Like we don't see Anti-T or Aquired-B anymore because the reagents are 'purer'.)

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