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Anti-D Testing Mystery


txlabguy82

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Oh auntie D - that sounds complicated to me!  And you can only use whole blood for the forward group if the box insert for the reagetns allows you to.  AND - if you have a very low haematocrit, you can end up with very weak suspensions and miss weaker reactions.  I'm sorry, but I would really have to disagree with you.  Suspensions made from packed cells always for me (exception - slide technique where the reagent specifically requires whole blood)

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So if the baby was only 4 weeks gestation, then the foetal cells must have been maternal - has HPLC been done to see if she has HPFH?

I think you're confused here auntie-D.  I think we have established that there were no foetal cells - so just one population of red cells (maternal).  Are you mixing this post up with another one??

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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)

 

The anti-sera are manufactured in vials by Immucor so we use the vial dropper - we do not put pipettes into the vials that is really asking for cross contamination. All of the reagents have vial droppers - the only thing except of the screen cells since we do these in Gel card, but there was no Antibody Screen ordered initially.  The only control was the antisera with the antibody © tube, reagent QC is ran once per day only.  

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I think you're confused here auntie-D.  I think we have established that there were no foetal cells - so just one population of red cells (maternal).  Are you mixing this post up with another one??

 

Yep I sure am!

 

OP - Anti-D can go away (rare but it happens). We have a patient who is male and has a recently developed anti-D that is no longer expressed. It is more likely that the scenarios to have happened that others have mentioned.

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OP - Anti-D can go away (rare but it happens). We have a patient who is male and has a recently developed anti-D that is no longer expressed. It is more likely that the scenarios to have happened that others have mentioned.

 

Did you perform a DAT and, more importantly, an eluate, and are you ABSOLUTELY certain it was an anti-D, and not a mimicking auto-anti-LW, which can often be transient?

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Back in the days of serum-only blood banking, if we had to send EDTA specimens to the reference lab they would defibrinate them because I think EDTA tubes can eventually clot after being stored several days.  I don't think that you would get a clot mixed up with agglutination when reading.  They look rather different.  I don't think clots would get stronger in the IAT D test, especially after all of that washing.  Fibrinogen levels rise a lot in pregnancy so her specimen might be more prone to clotting on storage.  I don't know how early in pregnancy that happens and she was awfully early.

 

Quotient's anti-D blend (Alba) has that tendency to react with the i/I moiety.  We see it only on cord samples, probably because there is more i available.  When we find one that is weakly positive at IS and take it through IAT, it goes to negative.

 

Baby cells are bigger but less dense (more retics) so they rise to the top of a spun sample just like in the reticulocyte separation technique.

 

I vote for something getting into that tube with the anti-D that you didn't intend.  It could even have got in there before you touched it.  Let's say someone got out tubes to do some testing but later changed their mind and put them back.  While they were out they got a splash of a group O or A patient's plasma in the one that you later chose for your D typing tube.  Now that tube had a bit of anti-B in it plus the anti-D that you added.  The anti-B would react with the patient's B neg cells and look like a weakly positive D type.  Because you put the same tubes in to incubate for the IAT D typing, this carried over. Because it was a patient's sample it could even have some IgG anti-B in it that could react better at AHG.  I have a long habit of starting with new drops of cells and antisera in new tubes when I do a weak D test--that way I can make absolutely sure that I really put anti-D in the tube in case that is why the IS D type was negative.  Another possible idea would be that a splash from your reagent anti-B got in the D tube.  You'd have to test whether that would get stronger at AHG since it likely contains no IgG anti-B.

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Makes sense Mabel, and could explain the jump in reactivity from 1+w (IS) to 3+ (IAT), although that seems to be a pretty big jump. I had been considering a bit of D+ RBC from a different patient in the RBC suspension but I think txlalabguy82 only does one patient at a time.

And txlalabguy, please don't hate blood bank! I think you need to compartmentalize all the issues running around in there (I do know that can be hard) and just concentrate on the job at hand. Blood bank, chemistry, UA, heme, it's no different - keep focused on what you're immediately doing, and let the other guys out of the boxes when it's time for them.

Edited by Dr. Pepper
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this does not actually reply to anything, but I noticed another glaring error in the hospital procedures (not in what you did, txlabguy82).  This lady came in with a miscarriage, so the doctors wanted to know whether to give anti-D - WITHOUT ordering an antibody screen.  And so the lab did not do one.  In my books this is dangerous practice.  If she had already had an anti-D she would not have needed Prophylax. 

 

And txlabguy82, I'm still waiting for the answer to my last question.  If you don't want to post here, you can always send me a private message

Anna

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this does not actually reply to anything, but I noticed another glaring error in the hospital procedures (not in what you did, txlabguy82).  This lady came in with a miscarriage, so the doctors wanted to know whether to give anti-D - WITHOUT ordering an antibody screen.  And so the lab did not do one.  In my books this is dangerous practice.  If she had already had an anti-D she would not have needed Prophylax. 

 

And txlabguy82, I'm still waiting for the answer to my last question.  If you don't want to post here, you can always send me a private message

Anna

There is a question confused me, why it is dangerous of giving double doses of anti-D Prophylax. I know it is a waste :)

Do you mean there is a  risk of infection? Thanks

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Wow, can't remember the last time I read a 4 page thread from start to finish! 

 

Txlabguy82 this is definitely one of those "s....tuff happens" occasions.   Personally I see nothing in your process that would concern me,  Actually there is a fair amount to commend you for.  As far as this possibly being a sample problem, I think not.  I've used short sample EDTA tubes far more often than I care to admit.  I guess, just to join in on the questions, could the sample have agglutinated as opposed to clotted?  Just a random thought and not real sure where it's going but it did come to mind as I was reading. 

When I was supervising blood banks and transfusion services I never got overly excited about the random one time occurrences, especially those where no real cause could be pin pointed.  The corporate Transfusion QA group hated it when I would remind them that as long as humans were involved in a process there would be the occasional human error.  Now, whether or not this particular case was human error will probably never be determined.  Bottom line; the patient received her dose of RhIG because the process discovered a problem.  Why the problem occurred will, most likely never be truly determined so don't let this eat at you over much.  My recommendation, find employment as a dedicated bloodbanker and enjoy the rest of you career.  :whew:

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so let me present my theory number 2 - carry over.

Carry over can and does occur quite regularly for a number of reasons. I'm not going to go into all of them! I think it is important to bear in mind that commercial ABO antisera are extremely concentrated.  You can dilute them 1000-fold and still get a reaction.  When you pipette 100ul, or drop 2 drops into a tube, it really does not take very much to fall into the next tube.  You would not see 5ul.  You may not even be aware that it jumped into the next tube.  The most common reason this happens is that a tiny bit of antiserum has dried on the very small hole at the base of the dropper  - so when you squeeze to dispense a minute amount deviates. 

This patient was group B.  Presumably this would have been pipetted in the order Anti-A  -  anti-B  - anti-D  -  ctl.  so if a tiny amount of anti-B got into the tube with the anti-D, you would get a weak pos, which could get stronger in IAT with time, bearing in mind that monoclonal antibodies, and I presume these were monoclonal antibodies, otherwise in will come theory no. 3, are only designed to be used as per stated method and might do all sorts of unexpected things otherwise.  For those using repeater pipettes, the other big danger is antiserum, or patient sample, sucking up into the barrel. 

So, back to my theory.  The other possibility for carry over in this case is that the drop stayed at the top of the tube, and when the patient cells were being pipetted, came into contact with the anti-B and then transferred to the anti-D.....

(I'm not saying Malcolm's theory is wrong, by the way, just offering alternatives)

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So, txlabguy - what about the sample itself?  What is that pipetted with.  And in what order do you pipette the sample into the tubes?  and what about the cells for the reverse group.  Do they also have droppers?  And what is their Rh group?

 

Ok let me just go through my routine process of pipetting reagent and sample.

 

First of course I label my tubes - there is at least 1 tech that doesn't which no one seems to care about which scares me!

 

Second steps:

  1. Pipette 2 drops of patient plasma into the two reverse cell test tube - discard pipette, sometimes I will go ahead and make my cell suspension at this time as well. 
  2. I always open the bottle of antisera away from the tubes, due to sometimes being dried up stuff on the bottle (I rarely see this being a bad problem, but on a side of caution I never open it over my test tubes).  I pipette 1 drop of each anti-sera (all come with droppers) and 1 drop of reverse cell (come with droppers).  Reverse cells are always RhD negative I can't remember if that was a question I saw somewhere or it would be pointless in doing a reverse if they weren't negative for the D antigen.  
  3. Drops are always free-falling as I was taught they should never touch the sides of the test tube or be allowed to run down the side of the test tube to the bottom.  
  4. When I pipette my cell suspension they are also free-falling never allowed to touch the test tubes.  

I will be happy if they ever decide to get a ProVue like we've been talking about.  We had a similar incident the other day a tech got a negative IS and a +/- at AHG and called the patient Rh - Positive, but apparently nothing was said to him because I definitely wouldn't have called the Rh Positive being a +/-.  I've also learned that AABB really are pushing for not doing weak D testing and actually doing genotyping to see if the patient has the genotype(s) that could cause the formation of Anti-D. It just has been frustrating me that depending on who you are influences it being blown up into a big deal or not. 

 

I need to see if there are references out there that state how strong of a reaction with Anti-D should one consider a patient Rh Positive at IS or even AHG. 

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Wow, can't remember the last time I read a 4 page thread from start to finish! 

 

Txlabguy82 this is definitely one of those "s....tuff happens" occasions.   Personally I see nothing in your process that would concern me,  Actually there is a fair amount to commend you for.  As far as this possibly being a sample problem, I think not.  I've used short sample EDTA tubes far more often than I care to admit.  I guess, just to join in on the questions, could the sample have agglutinated as opposed to clotted?  Just a random thought and not real sure where it's going but it did come to mind as I was reading. 

When I was supervising blood banks and transfusion services I never got overly excited about the random one time occurrences, especially those where no real cause could be pin pointed.  The corporate Transfusion QA group hated it when I would remind them that as long as humans were involved in a process there would be the occasional human error.  Now, whether or not this particular case was human error will probably never be determined.  Bottom line; the patient received her dose of RhIG because the process discovered a problem.  Why the problem occurred will, most likely never be truly determined so don't let this eat at you over much.  My recommendation, find employment as a dedicated bloodbanker and enjoy the rest of you career.  :whew:

 

I've actually stepped out of blood bank for the time being and kind of glad - when you have a big bull's eye on your back, because the blood banker doesn't like you makes things very unnerving and I just can't work like that on top of all my other responsibilities in the laboratory. 

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Anti-D neg at IS and +/- at IAT and called D+.  I'm sorry, in my book, that is not only scary and dangerous but downright irresponsible.  I agree - start using gel - any gel - and an analyser - then it's so much easier.  And stop doing IAT Ds on patients.  For patients you want to see as few antigens and as many antibodies as possible; for donors as many antigens, and only the strong antibodies.  So an IAT D is relevant for donors not for patients.  For a patient, if in doubt, treat as neg.  For a donor, if in doubt treat as pos.  'In doubt' means anything other than a clear pos or a clear neg, unless you've taken steps to differentiate between a Dweak and a partial D.  Even MORE important in the States, because while >70% of D variants are Weak D Type 1, 2 or 3 in Europeans, amongst people of African origin, this is definitely not the case.  Rant over  - for now!

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And another piece of advice (everyone must be sick of me by now - sorry!)

Weak results in ABO and D testing are quite unusual, except for the reverse group.  So if you get a weak result, then repeat from scratch.  More weak results are due to technical errors than 'true'.  And that goes for tubes AND gel.

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See what you all think of this as a recommended policy for transfusion services:  Take D typing through AHG phase only on babies (who can be thought of as little donors to their moms) and to resolve problems like why a fetal screen is coming up wonky or why an autologous unit is labeled Rh pos.  Then, the new approach will be to send for Rh genotyping any young female who reacts 2+ or weaker in gel (tube reagents are a bit weaker, but similar policies for them).  For males and older females who react weakly, it is reasonable to call them Rh positive (if they lack anti-D), but we should do repeat testing to make sure it wasn't an error and probably testing by another method or with another anti-D the first time you work them up. I would turn these out with a comment that their Rh type is weak or atypical so they won't be so surprised if they get different results from different labs.  If someone is going to be chronically transfused they also could benefit from genotyping and being treated as D negative until those results are back.  Some of this will be hard to get through our computer truth tables since they aren't set up to look at age and gender. :(

 

I am in the process of figuring out how we are going to start offering Rh genotyping to our prenatals etc. and what our cut-offs will be for that so I am open to tweaks on the above approach.

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I like this generally Mabel, but I would start off by using 2 different anti-D reagents in the first place; and I don't know about doing IAT Ds on babies.  So many have pos DATs, you would get a lot of false positive results.  And gel is really VERY sensitive.  I have seen weak Ds with as few as 200 D-sites per cell coming up weak positive.  And there is another possibility before sending off for genotyping.  Several companies provide kits of monoclonal antisera (no names, no advertising) that work a bit like an antibody panel, except that this time you are looking for epitopes on the D antigen.  It's not foolproof, but will act as a good guide.  And in gel, I would actually consider anything less than a strong 3 as suspicious. 

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We got in the partial D kit that is available here and have used it on some of these odd cases.  They all appear to be Type 1 or 2 weak D which isn't a big surprise.  It is a lot of time and reagent to run it--something like 12 antisera all taken through AHG.  Running controls also burns through the reagent fairly quickly.  I will have to look at what billing options I have for it if I am going to use it on all of these funky D prenatal patients before deciding whether to send them out for genotyping.

 

Using 2 different anti-D reagents seems to be standard elsewhere but not in the US.  We do keep a couple for problem resolution. I am not sure which 2 would be our best choices. The Immucor tube anti-D that we use is almost always negative at IS with these cells that are 1-2+ in gel and with Quotient (alba) anti-D.  How comparable are the 2 clones frequently used "over there"?  Do they intentionally react with different epitopes are are they both blends?

 

It is standard in the US to do the "weak D" test on initially D neg babies of D neg moms.  We should find positive DATs on the D control test that is run through AHG also.  Besides that, we have a policy of doing DATs on any of these babies who is D positive so we should catch any strange interfering DAT that caused a D positive test without causing a positive D control.  We are doing tube DATs at this point but we will be automating next year so will likely start doing gel DATs, which in my limited experience are more sensitive so we may start picking up more positives. We will also begin doing gel blood types routinely, so yes, I am sure we will pick up many of the babies with weak Ds in gel rather than having to do weak D testing in tube. How immunogenic is D VI in a baby for a D neg mom?  That is the main thing the tube weak D test is good for detecting and both IS tube and gel intentionally don't detect it.

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  1. Pipette 2 drops of patient plasma into the two reverse cell test tube - discard pipette, sometimes I will go ahead and make my cell suspension at this time as well. 

 

Again - please don't do this. Forward and reverse groups should be off two separate aliquots to reduce the risk of lab generated WBIT (if the wrong patient is accidentally selected there will be a descrepancy between the forward and reverse groups if there is an ABO incompatibility).

 

There are two 'correct' approaches (IMO and also has been policy in all the labs I have worked) 

 

1 - Do your forward group on whole blood with half the amount of dilutent (not ideal for patients with a low haematocrit but useful in emergency situations - though I have never seen a weak forward group due to prozone effect), then spin the sample and do the reverse group.

 

2 - spin the sample and set up your reverse group, then spin the sample again and set up your forward group. The 'waiting' time whilst the sample is respinning is a mini incubation period and will lessen the chance of you getting really weak back groups.

 

The chances are you aren't going to kill a patient due to ABO incompatibility (assuming they have a historic group), but what you could do is delay the provision of blood by crossmatching the wrong group and having to start again. But then you can't guarantee an AGH crossmatch to pick up ABO incompatibility...

Edited by Auntie-D
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Again - please don't do this. Forward and reverse groups should be off two separate aliquots to reduce the risk of lab generated WBIT (if the wrong patient is accidentally selected there will be a descrepancy between the forward and reverse groups if there is an ABO incompatibility).

 

There are two 'correct' approaches (IMO and also has been policy in all the labs I have worked) 

 

1 - Do your forward group on whole blood with half the amount of dilutent (not ideal for patients with a low haematocrit but useful in emergency situations - though I have never seen a weak forward group due to prozone effect), then spin the sample and do the reverse group.

 

2 - spin the sample and set up your reverse group, then spin the sample again and set up your forward group. The 'waiting' time whilst the sample is respinning is a mini incubation period and will lessen the chance of you getting really weak back groups.

 

The chances are you aren't going to kill a patient due to ABO incompatibility (assuming they have a historic group), but what you could do is delay the provision of blood by crossmatching the wrong group and having to start again. But then you can't guarantee an AGH crossmatch to pick up ABO incompatibility...

 

Maybe it is just too early in the morning and I haven't had enough coffee, but I am not understanding what you're trying to accomplish here. What do you mean by

"lab generated" wrong blood in tube and how does spinning the sample twice help prevent that?

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