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txlabguy82

Anti-D Testing Mystery

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Hi All,

 

So I would like to present a scenario that happened to me and get your input.

 

I received a specimen from the ED for ABO/Rh testing on a young female (she had a miscarriage, which at the time I was not aware).  We use the BD Pink (EDTA) blood bank tubes for all of our blood bank testing, this particular sample was about a little more than 1/4 of the way full (yes not the best sample, learned my lesson with this case) - there were no visible clots in the tube or in the cell suspension I made for testing (testing was done fairly quickly since it was only an ABO/Rh and we use a STATSpin centrifuge so I had results out within 20mins of it being collected). 

 

These were my initial reactions at time of testing:

 

Anti-A:         0

Anti-B:         4+

Anti-D:       w1+

D Control:    0

A1 Cells:      4+

B Cells:        0

Du:               3+

Du Control:  0     CCC: 3+

 

So of course my interpretation was B Positive, which was reported. (We use the monoclonal Anti-D)

 

All of our samples are retested by another technologist if we have no previous history on the patient.  The samples sit at room temperature until they are tested and this was one was retested roughly 10 hours after my initial testing, these are the results:

 

Anti-A:         0

Anti-B:         4+

Anti-D:          0

D Control:    0

A1 Cells:      4+

B Cells:        0

Du:               0    CCC:  3+

Du Control:  0     CCC: 3+

 

Interpretation:  B Negative

It was tested 2 more times by two other techs later that morning and the report corrected and the patient had to be called back to get Rhogam injection. So of course and event report was initiated at that point for root cause analysis.  

 

I was approached 3 days later after knowing nothing about what happened and told that I "miss-typed" a patient sample.  After reviewing the work card I of course said No I didn't because I actually remember working on this particular sample due to the fact that the D got stronger at AHG phase.  I was extremely puzzled by the results and pulled the sample at this point had been in the refrigerator for 3 days and found that the same had numerous large clots and lots of visible small clots in my cell suspension (however none of the previous techs noted or expressed this to my director).  

 

I performed (3 days later) a forward and reverse typing (B Neg), DAT, and IAT.  The DAT (IgG and Poly) - Both negative (very sticky microscopically) and IAT in Gel was completely negative.  So at this point it was very perplexing as to what happened with the Anti-D.  Of course everyone my boss talked to said that this was impossible for the Anti-D to just disappear (I was not inferring that it disappeared to her) and I must have done something wrong, which really aggravates me to use the word "impossible" in medicine.  She said it was more logical that I mixed the control tube and the Anti-D tube at the AHG phase when I read the tubes which makes absolutely no sense to me (if the results were reversed from the results I got at immediate spin, then yes that would make sense and I would have questioned the results and started over).   

 

OK, so here is my theory as to be the possible cause for this particular scenario has to do with a sub-optimal sample that was in the process of clotting at the time of initial testing - however I can only assume this theory and I know that it didn't interfere with the Anti-A or Control on the forward type, but these are all different antiseras with different blends of antibodies/proteins, etc.. I am thinking that since I didn't see or detect clots when I tested the EDTA sample the first time it is possible that something during the clotting process potentially interfered with the Anti-D causing it to be a false positive. Since the specimen wasn't tested again until 10 hours later when the clotting process was definitely complete there is no way to prove this, unless someone where to have retested it immediately after I performed the first test.  We've seen cold autoantibodies disappear after sitting at room temperature due to autoadsorption, who is to say that since the clotting process was complete whatever was causing the interference may have been gone 10 hours later when the full clots formed.   My boss refuses to even think this is remotely a possible and I had to have made an error, she said we use to use plain red clotted tubes all the time for blood bank and never had any problems (don't think she is getting that these tubes have no additive and normally the clotting process was a lot faster in plain red tubes with no additive. Then again she also couldn't understand how a clot would get stronger overtime (I wanted to bang my head on the desk with that remark).  If I made an error or potentially made an error I would definitely own up to it, but in this instance her suggestion that I switched the test tubes around and reading the control as the patient makes no sense what so ever. 

 

Has anyone every encountered anything like this before and what is your thoughts on the potential reason for the various reactions between a 10 hour period?

 

Thanks,

-TxLabGuy82

Edited by txlabguy82

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Just one quick question?  If you are using tubes (and I kind of got the impression that you were), did you allow the anti-D reagents to come to room temperature before you tested the sample, or did you take them straight from the fridge and test the red cells?

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Another possibility - how far pregnant was she when she had the miscarriage?  It might be that there was actually a mixture of D- maternal blood and D+ foetal blood in the sample you had.  Of course, mixed fields are not always easy to see in tubes.  Then you could be back in the discussion we had a few weeks ago on this forum about where you sample from in a sample that has a mixture in it.

Having said that, I would never be happy about calling a young woman with a 1+w reaction in a single anti-D D+ without carrying out further tests (and I don't mean an IAT anti-D test) and if this were not possible I would have treated her as Dneg  (When in doubt.......)

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Was your initial observation W1+  a macroscopic or microscopic reading?   Your reading of 3+ for the Weak D (Du) test is not ambiguous.  Is it usual to immediately put the anti-D and D control tubes into 37C incubator for the Weak D test, or are there other steps taken first?  I am also wondering about the anti-D manufacturers direction insert indications for performing a Weak D test and for microscopic readings?

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Just one quick question?  If you are using tubes (and I kind of got the impression that you were), did you allow the anti-D reagents to come to room temperature before you tested the sample, or did you take them straight from the fridge and test the red cells?

 

Now that I think about it I want to say I used them cold, which I really never do - but for some reason I used them cold this time.  Maybe I was thinking just an ABO/RH and STAT. However, reviewing the package insert doesn't state the reagent should come to room temperature. 

Edited by txlabguy82

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Was your initial observation W1+  a macroscopic or microscopic reading?   Your reading of 3+ for the Weak D (Du) test is not ambiguous.  Is it usual to immediately put the anti-D and D control tubes into 37C incubator for the Weak D test, or are there other steps taken first?  I am also wondering about the anti-D manufacturers direction insert indications for performing a Weak D test and for microscopic readings?

The observation was macroscopic, if negative/weak we put them right to 37 for 15 minutes, wash 4 times, and IgG added. The package insert doesn't really tell you at what grade you should consider the patient to be Rh Positive. Of course there is an unwritten policy of 2+ or weaker call it negative. 

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Another possibility - how far pregnant was she when she had the miscarriage?  It might be that there was actually a mixture of D- maternal blood and D+ foetal blood in the sample you had.  Of course, mixed fields are not always easy to see in tubes.  Then you could be back in the discussion we had a few weeks ago on this forum about where you sample from in a sample that has a mixture in it.

Having said that, I would never be happy about calling a young woman with a 1+w reaction in a single anti-D D+ without carrying out further tests (and I don't mean an IAT anti-D test) and if this were not possible I would have treated her as Dneg  (When in doubt.......)

 

Could you please further explain what you mean with further testing, what tests exactly?

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Now that I think about it I want to say I used them cold, which I really never do - but for some reason I used them cold this time.  Maybe I was thinking just an ABO/RH and STAT. However, reviewing the package insert doesn't state the reagent should come to room temperature. 

 

Well, the reason I asked is because Thorpe et al1,2 have reported that monoclonal anti-D molecules possess a V4-34 moiety that is also present in anti-I and anti-i.  As a result, if these reagents are used straight from the fridge, rather than allowing them to come to room temperature, there is a chance of a false positive, as this moiety will bind to the I and/or i antigens expressed on the red cells, and so D Negative red cells can be mis-grouped as D Positive.

 

I do not know how long it would take for the antibody, under these circumstances, to dissociate from the I and/or i antigens at 37oC, but, certainly, in the case of a cold reacting anti-M (one that does not react strictly at 37oC), it takes longer than the incubation time, as can be seen if the reactants are not allowed to come to 37oC before they are mixed (hence a clinically insignificant anti-M can appear to be active at 37oC).

 

I am not, for one minute, saying that this is the answer to the problem, but I am saying that it could be a contributory factor, particularly if there was a substantial amount of the foetal red cells (which would be, essentially, I-, i+) in the maternal circulation, and, hence, in the sample you tested.

 

1.  Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellberg MB, Natvig JB, Thompson KM.  Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.  Transfusion 1997; 37: 1111-1116.

 

2.  Thorpe SJ, Ball C, Fox B, Thompson KM, Thorpe R, Bristow A.  Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both activities.  Transfusion 2008; 48: 930-940.

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Could you please further explain what you mean with further testing, what tests exactly?

Well, as a minimum, test with several different clones of anti-D but preferably test with molecular biology.  But, as I said before, this patient should have been treated as Dneg.  A 1+w is very weak indeed, regardless of what you got in the IAT phase.  For future cases, it might be a good idea to check this type of reaction under the microscope, for a mixed field agglutination - which would indicate that the mother's blood also contained a large amount of foetal cells.

About the point - at which strength do you treat as D+, my answer to that would be, it depends on how you are testing and who you are testing.  You have to bear in mind that a 2+ in tubes and a 2+ in gel do not represent the same 'true' strength, as gel is much more sensitive than tubes. In gel, anything less than a good 3+ should be treated with suspiscion of being a variant - either a weak or a partial.  does it matter?  Well, if it's a donor, they would all be treated as D+, no problem.  A man - also no problem, unless he's going to be regularly transfused for the rest of his life.  For 'old' women (I can say that - I am a dinosaur) - also, no problem.  However, for women with child-bearing potential, you have to be much more careful.  You need at all times to do your very best to prevent her from forming an anti-D which will cause horrible problems in her next pregnancy.  So anything less that a normal straightforward positive (4+ or 5+ depending on your scoring system) should be treated with utmost caution and fully investigating before deciding that it is safe to treat her as a D+.  And while waiting for those results, she should be treated as a Dneg.

It's honest of you to own up to using the reagents straight out of the fridge.  Reagents should never be used cold - and as Malcolm has said, that MIGHT be the reason for this discrepancy. whether it is or is not the reason, I hope there is a lesson here for everybody.  But could I ask again about how far pregnant this lady was?

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Regardless, I think it's odd that the patient control was negative when apparently you did have a "false positive" at AHG for some reason.  You sure you used anti-D?

 

Scott

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Smiller - I don't think it's fair to say that the original result was a 'false' positive.  Until the actual reason for the discrepancy is known, then both results should simply remain as discrepant.

Another lesson to learn from this case - It is not good practice to give out a blood group on a previously unknown patient  when just one group has been done.  The best scenario is two separate samples.  If that really is not possible, then at least the 1 sample should be tested twice, from 2 separate cell suspensions and preferably with two sets of different reagents (i.e. 2 different cell lines)

Cases like this one really help to understand why blood banking sometimes has such strict rules about things.

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There seems to be intense focus on explaining the W1+ reaction on immediate-spin with monoclonal anti-D while ignoring the original Weak D test result of 3+.  Given that repeat testing produced no agglutination in the immediate-spin test and in the Weak D test, I have to wonder if something other than anti-D was added to the D tube?

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But if the monoclonal anti-D was used straight out of the fridge, and was an IgM, that would also explain the 3+ Weak D Test for two reasons.  Firstly, the centrifugation wwould be a lot stronger, and secondly, you shouldn't be doing Weak D test (like this, anyway) using those reagents.

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But the repeat testing by other techs using same reagents was negative in both D and Weak D testing?  I believe monoclonal anti-D in US contain both IgM and IgG components.

Edited by Dansket

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Colors of my Ortho reagents (for example): anti-AHG: green, anti-A: blue, anti-B: yellow, anti-D: clear, anti-AB: also clear. 

 

I would not be able to tell a tube with a drop of anti-AB from a tube with anti-D with these reagents.  Not sure but it seems like accidently running a weak D test with anti-AB on a A patient instead of anti-D might result in a "false" positive interpretation. 

 

Scott

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Malcolm,

 

Are you suggesting that failure to allow an anti-D reagent antiserum to warm to room temperature prior to testing could result in a false-positive (3+ reaction) in the Weak D test done by the indirect antiglobulin test method?

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We do not use Anti-AB in our facility and I always check the actual label of each vial before using it.  I never trust that someone put the reagent vial where it is supposed to go in it's labeled spot.  If I followed our SOP as it is written then I did nothing wrong in calling this Rh - Positive. I have never worked anywhere that required special testing for patients that are weak D positive.  Yes, I am glad I am getting a lot of feed back with great information.  

 

I am still perplexed about the no clots in the sample when I tested it, to several clots in the tube when I was asked told about it 3 days later. My concern is also with specimen quality or some other possible interference of course I am then one being thrown under the bus as I made a definite mistake, because there it is the easiest way to close the event report and move on. I am by no means saying I couldn't ever make mistakes - we are all human. 

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Malcolm,

 

Are you suggesting that failure to allow an anti-D reagent antiserum to warm to room temperature prior to testing could result in a false-positive (3+ reaction) in the Weak D test done by the indirect antiglobulin test method?

 

 

 

I do not know how long it would take for the antibody, under these circumstances, to dissociate from the I and/or i antigens at 37oC, but, certainly, in the case of a cold reacting anti-M (one that does not react strictly at 37oC), it takes longer than the incubation time, as can be seen if the reactants are not allowed to come to 37oC before they are mixed (hence a clinically insignificant anti-M can appear to be active at 37oC).

 

I am not, for one minute, saying that this is the answer to the problem, but I am saying that it could be a contributory factor, particularly if there was a substantial amount of the foetal red cells (which would be, essentially, I-, i+) in the maternal circulation, and, hence, in the sample you tested.

 

1.  Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellberg MB, Natvig JB, Thompson KM.  Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.  Transfusion 1997; 37: 1111-1116.

 

2.  Thorpe SJ, Ball C, Fox B, Thompson KM, Thorpe R, Bristow A.  Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both activities.  Transfusion 2008; 48: 930-940.

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After reading Malcolm's reference #2,  ​I don't see any mention of the use of manufactured reagent anti-D in this study.  My impression is that all the hemagglutination testing in this study was done at 4C.  Given that US anti-D antisera for tube test contains both IgG anti-D and IgM anti-D components, it seems a stretch to attribute the 3+ reaction in the Weak D test to the use of cold antiserum. 

 

Has txlabguy82 contacted the vendor of his anti-D antiserum?  What do they say?

Edited by Dansket

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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.

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After reading Malcolm's reference #2,  ​I don't see any mention of the use of manufactured reagent anti-D in this study.  My impression is that all the hemagglutination testing in this study was done at 4C.  Given that US anti-D antisera for tube test contains both IgG anti-D and IgM anti-D components, it seems a stretch to attribute the 3+ reaction in the Weak D test to the use of cold antiserum. 

 

Has txlabguy82 contacted the vendor of his anti-D antiserum?  What do they say?

 

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.

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They did not use anti-D antisera manufactured for Rh typing in test tube in this study, by that I mean ORTHO anti-D, Immucor Anti-D, BioTest Anti-D, etc.  At this point, we do not know if txlabguy82's anti-D typing antiserum contained the cell lines mentioned in your references.

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They did not use anti-D antisera manufactured for Rh typing in test tube in this study, by that I mean ORTHO anti-D, Immucor Anti-D, BioTest Anti-D, etc.  At this point, we do not know if txlabguy82's anti-D typing antiserum contained the cell lines mentioned in your references.

 

Oh, right.

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A few thoughts (always a dangerous thing when it happens to me):

- You could put Malcolm's theory to the test and try repeating the testing with reagents and, for that matter, the patient specimen, straight from the fridge.

- If Anna's theory about 2 cell populations is correct, why weren't the reactions repeatable when retested several hours later? The baby's cells should have still been there.

- Speaking of 2 cell populations, was another D+ patient being tested at the same time? Could some of his cells have gotten into the RBC suspension that was mostly D-? Years ago we had three patients in a row who had positive antibody screens. They all had anti-D. They were all D- but the auto controls were all positive. The tech had aliquotted the sera off of three clots sitting side by side into second tubes in the rack for the testing, then added her typing reagents to the testing tubes. Trouble was, she apparently added a drop of anti-D to each of the three serum aliquot tubes instead of the tubes for the anti-D testing! Point is, maybe here the wrong stuff got in the wrong tubes. 

- The initial testing sure looks like a weak D though.

 

Who really killed the Kennedys? Sometimes we will never know the answers. I'll stop thinking for now.

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