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Anti-D Testing Mystery


txlabguy82

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PLEASE, PLEASE, PLEASE can you check how far pregnant this lady was.  I still think that the possibility that there were two populations of cells in this sample is the most likely explanation.......

 

 

If there were 2 populations of cells, and if the fetal cells were ABO incompatible with the maternal sample however D positive, the fetal cells could have been hemolyzed in vitro by the time the sample was retested 10 hours later.  Since the fetal cells were no longer intact, the repeat testing would have been on exclusively maternal cells and tested as D-negative. 

 

I realize your initial forward type did not detect/report the presence of mixed field agglutination.  With monoclonal ABO antibodies and using tube testing, it is often very difficult to detect mixed field reactions with small populations of other cells.  It seems the monoclonal ABO antibodies are so avid, the agglutinates seem to "trap" the other population of cells in the agglutinate rather than remaining free to be resuspended. This was demonstrated years ago when the AABB offered "damp" workshops where samples were provided for testing, registrants reported their results and results were tallied and presented to attendees.  During the time when the older polyclonal ABO reagents were still in use and the monoclonal reagents were just being introduced, the results indicated a significant difference in the detection of mixed field agglutination in the forward ABO testing between the groups using polyclonal reagents (MF detected) vs. monoclonal reagents (MF not detected).

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Very interesting SMW about the polyclonal vs. monoclonal reagents, thank you.

 

As for the initial, non-repeatable results: I would certainly think that rapid in vivo clearance could make a difference in what you would see in the original specimen vs. one drawn, say, 6 hours later. But how are those baby cells going to get hemolyzed in vitro using an EDTA sample that does not have any active complement?

 

Second, the scenario mentioned above sounds reasonable with a minor population of cells getting trapped in the agglutinates. But in this case the problem is how did those baby cells, which were plentiful enough to give a weak 1+, vanish from the specimen tube. They would have been the minor population, but also the ones involved in the agglutinates (with a polyclonal anti-A), not the ones not involved in the agglutinates, the opposite it seems of the AABB's scenario. They seem to have vanished from the specimen tube prior to testing, not during testing with monoclonals.

 

Many years ago we had a unit returned from the floor. The story was that they had started to hang it on the wrong patient, but then realized the error and never actually infused any. But some volume seemed to be unaccounted for. Trying to see if there were indeed any of the cells in the patient was complicated by the fact that the patient had recently received several other units. The donor unit was O, the patient A2. We typed the patient and all the units for a bunch of antigens and found that the unit in question was S-, where all the other units and the patient were S+. How to separate just those cells? Our reference lab suggested differential sedimentation: mix a dilute suspension of patient cells with anti-A (polyclonal), let sit for 15 minutes or so, during which the agglutinates would (hopefully) tumble down and the O cells remain in suspension. Then harvest the top half of so of the suspension. We repeated this several times, and ended up with two wispy cell buttons that typed S- and H+ (4+). So we seemed to have demonstrated that the patient did get a bit of the blood in question. Could that have happened with the patient we've been talking about as the tube sat. I think not, I think a tube of whole blood or packed cells would be a whole lot harder for the agglutinates of baby cells to work their way down.

 

Please forgive me all if I appear contentious, we're all just trying to puzzle out a mystery.

Edited by Dr. Pepper
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Your post has just made me think Phil (not a common practice - me thinking I mean, not your posts making me think!).

 

Foetal red cells cells are 22% larger than adult red cells.  They would,therefore, settle in the sample quicker than would adult red cells (although not a lot quicker).

 

If the red cells were taken from the top of the sample after it has settled, and the sample was not mixed, then these red cells are more likely to have been derived from the mother than the foetus.

 

Could this also be a contributory factor among a plethora of other contributory factors I wonder, or am talking complete garbage???!!!!!!!!!!!

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Your post has just made me think Phil (not a common practice - me thinking I mean, not your posts making me think!).

 

Foetal red cells cells are 22% larger than adult red cells.  They would,therefore, settle in the sample quicker than would adult red cells (although not a lot quicker).

 

If the red cells were taken from the top of the sample after it has settled, and the sample was not mixed, then these red cells are more likely to have been derived from the mother than the foetus.

 

Could this also be a contributory factor among a plethora of other contributory factors I wonder, or am talking complete garbage???!!!!!!!!!!!

 

Malcolm you are right - the same reason you are more likely to get a positive DCT on the analyser because it takes the cells from the bottom. All our analyser weak pos ones are repeated manually and are usually negative.

 

Here's a zebra for you? Could the baby sample that was tested have been from the wrong baby, and the baby was in fact rh neg? The rh neg foetal cells would persist...

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Very interesting SMW about the polyclonal vs. monoclonal reagents, thank you.

 

As for the initial, non-repeatable results: I would certainly think that rapid in vivo clearance could make a difference in what you would see in the original specimen vs. one drawn, say, 6 hours later. But how are those baby cells going to get hemolyzed in vitro using an EDTA sample that does not have any active complement?

 

Second, the scenario mentioned above sounds reasonable with a minor population of cells getting trapped in the agglutinates. But in this case the problem is how did those baby cells, which were plentiful enough to give a weak 1+, vanish from the specimen tube. They would have been the minor population, but also the ones involved in the agglutinates (with a polyclonal anti-A), not the ones not involved in the agglutinates, the opposite it seems of the AABB's scenario. They seem to have vanished from the specimen tube prior to testing, not during testing with monoclonals.

 

 

Some ABO antibodies are very potent and can result in in vitro hemolysis even when using plasma so I don't feel the EDTA sample necessarily precludes the possibility of in vitro hemolysis occurring. 

 

For the testing scenario presented, it was only the ABO antibodies that appeared to trap the minor population of cells.  This did not occur with the anti-D reagents in use, perhaps they were not as avid.  This was reported by many different labs/staff with many different reagents. So based on previous experiences it seems it could be possible that when the initial testing was performed there was a mixed population of cells that were not detected with the avid ABO reagents since the minor population was trapped in the agglutinates, however the minor population was detected with the less avid anti-D reagent.  At a later testing period of the same sample, all of the fetal cells could have been hemolyzed in vitro in the collection tube (not the tube actually mixed with the monoclonal antibody and used for testing), and would no longer be detectable with the anti-D reagent. 

 

Unfortunately it is not possible to re-create the "initial" sample in the same time frames to test this theory. however it seems to be a potential explanation to this mystery among others.

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Here's a zebra for you? Could the baby sample that was tested have been from the wrong baby, and the baby was in fact rh neg? The rh neg foetal cells would persist...

 

As I read the initial scenario presented, there was a miscarriage so I didn't think a fetal sample was available or tested?  I believe the speculation is that it could have been fetal cells that were D-positive resulting in the initial weak positive testing result.

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Seems like we rarely have a debate like this on these forums, nice to see some back and forth for once.

 

I hope txlabguy82 doesn't take this as being personally critical, but some of these explanations call forth Occam's razor - some sort of sample/reagent error that wasn't recognized at the time/misremembered.

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I hope he doesn't too.  After all, there are an awful lot of "brains" here on this site, and we can't agree!.........or, at least, none of us have come up with a definitive answer.  Actually, far from being critical, I would like to thank him for bringing this to our attention.  The discussion has been, and, I think, will contine to be, exceedingly interesting.

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PLEASE, PLEASE, PLEASE can you check how far pregnant this lady was.  I still think that the possibility that there were two populations of cells in this sample is the most likely explanation.......

and txlabguy 21 - don't beat yourself up about this one.  You tested the sample according to your SOP, and you got the result that you got.  For me, this just indicates that there is a lesson to be learnt - that your SOP needs tightening up.

 

She was only about 4 weeks I believe, not very far along.  I'm trying not to beat myself up, but the individual that oversees blood bank doesn't really care for me and I always feel I have this big target on my back.  Of course she also thinks that she knows everything there is to know about blood bank.  I also just found out the AABB standard says not to perform weak D testing, so if I didn't do that weak D I would have called it Negative. I wonder why we are still performing weak D testing then, I plan to find out. 

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Monoclonal anti-D is manufactured anti-D (particularly if it contains IgG and IgM, as it would be a blend).

 

Yes, all of the tests were performed at 4oC - but that is the point!  If the reagents were taken straight from the fridge, for an immediate spin test, they would still be close to 4oC when the test was performed.  As I said in an earlier post, if the IgM part of the blend had sensitised the I and/or i antigens on the red cells at this point, and the test was (gently) centrifuged, more and more of the antibody would agglutinate these red cells (which is the whole point of the centrifugation step - to bring the red cells into closer proximity with one another).  What I then went on to say, though, was that I did not, and still do not know, how quickly the antibody (in the shape of the V4-34 moiety) would dissociate from the red cells when the tests are then taken to the Weak D test - but I also mentioned that clinically insignificant anti-M, that does not react strictly at 37oC, can often be detected by IAT at the end of just such a technique, unless the reactants are only introduced to one another at 37oC, and washed with pre-warmed saline.  In the routine Weak D test, I doubt if the saline used for washing the test would be pre-warmed, and yet the centrifugation step would be at a much higher speed than would be used for the initial test.  If the antibody has not fully dissociated, these steps would enhance the reaction caused by the V4-34 moiety, and so the stronger reeaction could, quite possibly, be detected.

 

During the very short time in my professional life, when I worked in a haematology laboratory, rather than a blood transfusion or blood group serology laboratory, we occasionally received an EDTA sample that would not go through the Coulter Counter properly because of cold agglutinins.  We used to put these into a 37oC incubator for a minimum of an hour, to allow the cold agglutinin to dissociate from the red cells, prior to putting the sample through the machine again.  I was told by the Senior Chief Technician that the time of one hour had been chosen because experiments had shown that anything much shorter did not allow enough time for the dissociation of the antibody/antigen complex, and we would get rubbish results a second time.  Even then, on occasions, the removal of the sample from 37oC to ambient temperature was enough for the antibody to go back onto the red cells.  This incubation time of at elast an hour was, I would suggest, much longer than the incubation time used these days for an IAT.

 

I do not think the testing temperature really had anything to do with it, because the Anti-A is also a monoclonal blend and it was completely negative, I wondered this too but then researched that the Anti-A we use is monoclonal as well.  Also I wonder why they do not state in the package insert that is must warm to room temperature before use, as most any other kit in the laboratory does state. 

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Seems like we rarely have a debate like this on these forums, nice to see some back and forth for once.

 

I hope txlabguy82 doesn't take this as being personally critical, but some of these explanations call forth Occam's razor - some sort of sample/reagent error that wasn't recognized at the time/misremembered.

 

Hi, not at all. This was actually the only sample I had all evening oddly and I always look at the actual label on each anti-sera vial before I use them for testing, I don't rely on if the reagent is in its correct spot in the rack - never have. I also label my test tubes the same since my clinical rotations. 

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I do not think the testing temperature really had anything to do with it, because the Anti-A is also a monoclonal blend and it was completely negative, I wondered this too but then researched that the Anti-A we use is monoclonal as well.  Also I wonder why they do not state in the package insert that is must warm to room temperature before use, as most any other kit in the laboratory does state. 

 

Sorry txlabguy82, but as the V4-34 moiety is in the variable region of the monoclonal anti-D reagents, the fact that the monoclonal anti-A reagent was completely negative is not relevant in this case as, of course, the variable regions of the different specificities will be different.

 

That having been said, I am still NOT saying that was the reason myself either; I am just saying that it could be a contributory factor.

 

I'm really enjoying this discussion!

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Hi Malcolm

I've tried to add a quote but it's not working.  Going back to your post above about foetal cells being about 22% larger than maternal cells and would therefore settle more quickly - this is what I have been trying to get across from the beginning when I keep going on about 2 populations of cells. 

So we now know mum was only about 4 weeks pregnant.  So unless it was a very bloody miscarriage I now think that this theory is less likely, as I doubt (but would be happy to be proved wrong) that there would be enough foetal cells present

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Sorry txlabguy82, but as the V4-34 moiety is in the variable region of the monoclonal anti-D reagents, the fact that the monoclonal anti-A reagent was completely negative is not relevant in this case as, of course, the variable regions of the different specificities will be different.

 

That having been said, I am still NOT saying that was the reason myself either; I am just saying that it could be a contributory factor.

 

I'm really enjoying this discussion!

 

Hi Malcolm, 

 

It is a great discussion and I like reading everyone's responses and opinions....  I was also thinking that it could have possibly been a cold, but the individual over our blood bank and my director apparently say there is no way it was a cold.  As I was trying to tell her that I've seen cold autos autoadsorp plenty of times at room temp, but they will not listen to anything I have to say. This is the reason I am doing this forum as well as contact some other experts, because them just telling me you made an error in typing and writing me up for it to have the reason to close the event report has me a bit upset at this point. 

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right - so now that my theory no. 1 has been ruled out by the age of the foetus, to get to theory no. 2, I need to know from you, txlabguy, EXACTLY how you pipetted the reagents and samples, from the moment you got the sample to the moment you read the IAT D test. - then I will probably have more questions too!

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Do you have any information regarding V4-34 moiety you are talking about?

 

Only the two references that I originally posted, I'm afraid.

 

I'm sorry, but I must agree with the Director and the Lead Technician that it is unlikely to be a "cold" auto, because were it to be a strong enough "cold" auto to cause agglutination in your Rh testing, even if this had been adsorbed out on storage, there would have been frank agglutination in the sample after it had been stored at 4oC, as the auto could only have been adsorbed onto the lady's own red cells (and, don't forget, it is very rare for a "cold" auto to be anything but an agglutinating IgM).

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Only the two references that I originally posted, I'm afraid.

 

I'm sorry, but I must agree with the Director and the Lead Technician that it is unlikely to be a "cold" auto, because were it to be a strong enough "cold" auto to cause agglutination in your Rh testing, even if this had been adsorbed out on storage, there would have been frank agglutination in the sample after it had been stored at 4oC, as the auto could only have been adsorbed onto the lady's own red cells (and, don't forget, it is very rare for a "cold" auto to be anything but an agglutinating IgM).

 

I am by no means even suggesting it was a cold - was just using it as a reference since you were referring to the moeity being associated with colds at least that is my take.  I still haven't heard anyone's opinion on the sample quality issue that I feel could have been the possible associating factor in this case, not saying it was but one has to take this into consideration.  

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right - so now that my theory no. 1 has been ruled out by the age of the foetus, to get to theory no. 2, I need to know from you, txlabguy, EXACTLY how you pipetted the reagents and samples, from the moment you got the sample to the moment you read the IAT D test. - then I will probably have more questions too!

 

Hi,

 

So this is how I pipette every sample been doing it the same way since I was taught by a very experienced blood banker.

 

I always label my tubes as: A, B, D, C, α, β along with the patient initials on each tube.  I always pipette 2 drops of plasma into each of the reverse type tubes first, then I remove the pack cells and make my cell suspension.  I then pipette 1 drop of each Anti-Sera into each test tube and then pipette the 1 drop of each reverse cell into each tube as labeled.  I then pipette my cells suspension 1 drop into each A, B, D, and C test tubes - then in the centrifuge for 15 secs.  

 

Once I read all the tubes I placed the Anti-D and Anti-C directly into the incubator for 15 minutes, washed 4 times, added 2 drops of IgG to each tube and then centrifuged for 15 seconds - read reaction. 

 

I've now learned that AABB has changed the standard that Weak D testing no longer needs to be performed and will be bringing this up to my director, as well as, the fact that our procedure doesn't state at what strength a Rh should be considered a reportable Positive. 

 

I really hate working blood bank mainly because I have so much on my mind since I am the laboratory supervisor and have tons of projects that need to be done always running through my head.  

Edited by txlabguy82
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Dear txlabguy82.

Many thanks for this.  Can I come back to   I always pipette 2 drops of plasma into each of the reverse type tubes first, then I remove the pack cells and make my cell suspension. I then pipette 1 drop of each Anti-Sera into each test tube and then pipette the 1 drop of each reverse cell into each tube as labeled. I then pipette my cells suspension 1 drop into each A, B, D, and C test tubes - then in the centrifuge for 15 secs.

Can you tell me in which order you pipette the antisera, and what type of pipette - and do you change the pipette tips or wash inbetween and how exactly you make the suspension.  What do you use to add the 2 drops of anti-IgG to the washed tubes?  Did you have a pos and neg control in the centrifuge at the same time as you washed these tests?

(Sorry if I'm being really nitpicking about the details, but I'm honestly trying tohelp you find the source of the discrepancy)

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I always label my tubes as: A, B, D, C, α, β along with the patient initials on each tube.  I always pipette 2 drops of plasma into each of the reverse type tubes first, then I remove the pack cells and make my cell suspension.  I then pipette 1 drop of each Anti-Sera into each test tube and then pipette the 1 drop of each reverse cell into each tube as labeled.  I then pipette my cells suspension 1 drop into each A, B, D, and C test tubes - then in the centrifuge for 15 secs.  

 

Just for future reference - it is considered good practice to do the forward and reverse groups off different 'spins'. We have an SOP that we use whole blood for the forward group (and half the diluent), then spin and use the spun plasma for the reverse group. If there was a discrepancy on the forward group then the sample is now spun and ready to go for a second check.

 

I find, doing it this way also saves that crucial 5 minutes as you don't have to wait for the sample to spin to get a provisional group.

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