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Weak D testing in the presence of a positive DAT?


SusieQ132

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Our facility is evaluating making a change to our process for Weak D testing for patients with a positive DAT.  For years, if we were required to do a Weak D, but the patient had a positive DAT, we used to cancel the Weak D as invalid.  Another hospital in our system mentioned that they tended to perform the Weak D, but then only cancel as invalid if the Weak D is positive.  We are thinking about changing to this process, as we now have to result many babies as "Rh Unknown" and give their mothers Rhogam.  

Per our Anti-D's package insert:  "Red blood cells coated with alloantibodies or autoantibodies of the same or similar specificity as the reagent (i.e. cells that are DAT positive) may give weak reactions. This is due to decreased availability of antigen sites because of antigen blocking or steric hinderance. In extreme cases false-negative results may occur." 

I'm worried about the "extreme cases" where a false negative could occur, but I cannot see this being common.  Also, would you think that if the cells were coated with that much antibody, that we would see any other odd reactivity in the ABO/Rh?  What do other facilities do?

Thank you in advance,

Susan

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You are talking about patients?  The biggest problem as I see it is that you will get a false positive result due to the positive DAT and then transfuse with D+ blood.  Either you go to a lot of time and effort and strip off the antibodies from the red cells as Malcolm said - or, probably a lot easier - and safer - treat them as D-negative

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2 minutes ago, galvania said:

You are talking about patients?  The biggest problem as I see it is that you will get a false positive result due to the positive DAT and then transfuse with D+ blood.  Either you go to a lot of time and effort and strip off the antibodies from the red cells as Malcolm said - or, probably a lot easier - and safer - treat them as D-negative

Yes.  I should be more specific in that the main patient population where this is an issue is for neonatal patients where the mother needs to be evaluated for Rhogam eligibility.  Where treating the neonate as D negative is not enough. 

1 hour ago, Malcolm Needs said:

We would treat the red cells with chloroquine diphosphate.

My question for this is when would I know to do this treatment?  (Or send it out to have this performed, since we wouldn't have the resources to perform it in-house.  :))  Do you think I would know the Weak D was falsely negative due to other odd reactivity in the ABO/Rh typing, etc.?  Or would I unknowingly result a neonate as Rh negative, and say that the mother is not eligible for Rhogam, only to later find out the mother developed Anti-D? 

Just trying to think about the risk of performing the Weak D in the presence of a positive DAT.  I know it's more likely to cause a false positive, which is why we were thinking about performing it even if the DAT is positive, and only cancelling it as invalid if the Weak D is positive.  But I don't want to cause patient harm by reporting it if it could be a false negative reaction. 

Hope this all makes sense.  Trying to get all my thoughts into words is hard today!  ;)

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52 minutes ago, SusieQ132 said:

Just trying to think about the risk of performing the Weak D in the presence of a positive DAT.  I know it's more likely to cause a false positive, which is why we were thinking about performing it even if the DAT is positive, and only cancelling it as invalid if the Weak D is positive.  But I don't want to cause patient harm by reporting it if it could be a false negative reaction. 

Hope this all makes sense.  Trying to get all my thoughts into words is hard today!  ;)

No, it all makes perfect sense, but, if you don't have access to CDP yourself (and are used to using it, as it can, in itself, weaken the expression of Rh antigens), I would send samples to the Reference Laboratory, and if their TRT is too long, give the anti-D immunogobulin anyway, on the grounds that, even though it is a human-derived blood product, it is still probably safer (even if not required) than the risk of the mother making an immune anti-D.

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I have seen weakly positive DAT cells taken through AHG for a weak D test end up negative in both the D test and the control.  I have always thought that this was due to a mild heat elution during the incubation removing the coating antibody (which we have usually expected was ABO antibody, not anti-D, because mom and baby are ABO incompatible and mom is not known to be sensitized to D).  Would anyone be concerned that this is a false negative weak D test as defined in the insert mentioned initially?

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In my experience good monoclonal anti-D reagents will pick up all but the weakest of D antigen variants (by which I mean all normal D+ and all D variants with a reasonable number of D antigens on their surface).  The positive DAT will not interfere with them unless it's caused by clinically significant cold haemagglutinin disease.  So only the very very weak D variants will come up anyway with the weak D test in an IAT but not in direct testing.  So if you did not do the weak D testing you might miss a few cases.  Those few would have to nonetheless be able to stimulate mum into making an anti-D.

I would be very interested to know exactly how many cases you see where the routine testing (using a sensitive method and good quality monoclonal anti-D reagents) is negative and the Weak D testing was positive (with a negative DAT).

I wonder if it's more or less than the number of D variant mums you miss because they group as normal D+ ...........

 

Also, for the anti-D to give you a false negative result because all the antigen binding sites are already loaded, your DAT would have to be VERY strong - and mum would have a known (hopefully), very high titre, antibody.  So you would have to carry out an eluate anyway on baby's cells and find for example that mum has a high-titre anti-D and you can elute anti-D from the baby's apparently D-negative red cells.  That's when you know that baby really is D+ and all the D sites are saturated. I've seen this twice in 'real life'.

 

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On ‎8‎/‎20‎/‎2018 at 8:04 AM, SusieQ132 said:

Our facility is evaluating making a change to our process for Weak D testing for patients with a positive DAT.  For years, if we were required to do a Weak D, but the patient had a positive DAT, we used to cancel the Weak D as invalid.  Another hospital in our system mentioned that they tended to perform the Weak D, but then only cancel as invalid if the Weak D is positive.  We are thinking about changing to this process, as we now have to result many babies as "Rh Unknown" and give their mothers Rhogam.  

Per our Anti-D's package insert:  "Red blood cells coated with alloantibodies or autoantibodies of the same or similar specificity as the reagent (i.e. cells that are DAT positive) may give weak reactions. This is due to decreased availability of antigen sites because of antigen blocking or steric hinderance. In extreme cases false-negative results may occur." 

I'm worried about the "extreme cases" where a false negative could occur, but I cannot see this being common.  Also, would you think that if the cells were coated with that much antibody, that we would see any other odd reactivity in the ABO/Rh?  What do other facilities do?

Thank you in advance,

Susan

I have never seen an "extreme case" of antigen blocking resulting in a negative test and I would think that the baby would have a lot of other serological issues that might alert you that this might be going on.   Our facility allows newborn weak D testing when the DAT is positive and most of the time the weak D is negative.   If you have staff that can understand this process then it might be a good idea. 

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I've seen one baby with D antigen sites blocked by Mom's anti-D. Of course the DAT was 4+++++. It almost didn't need centrifuged in order to see agglutination.

We perform the weak D testing. If the Rh control is positive, the weak D test is reported as invalid. If the control is negative, we report the test. There aren't a large percentage of samples that have a positive control.

 

Edited by AMcCord
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On ‎8‎/‎21‎/‎2018 at 6:26 AM, galvania said:

In my experience good monoclonal anti-D reagents will pick up all but the weakest of D antigen variants (by which I mean all normal D+ and all D variants with a reasonable number of D antigens on their surface).  The positive DAT will not interfere with them unless it's caused by clinically significant cold haemagglutinin disease.  So only the very very weak D variants will come up anyway with the weak D test in an IAT but not in direct testing.  So if you did not do the weak D testing you might miss a few cases.  Those few would have to nonetheless be able to stimulate mum into making an anti-D.

I would be very interested to know exactly how many cases you see where the routine testing (using a sensitive method and good quality monoclonal anti-D reagents) is negative and the Weak D testing was positive (with a negative DAT).

We find that Immucor's monoclonal blend anti-D (Gammaclone, I think) does not detect all type 1 & 2 weak D cells at IS.  This is not a particularly bad thing when typing moms and recipients--less helpful for cord blood.  It picks up these as well as category VI partial D at AHG testing.  Our Quotient (Alba) ant-D blend picks up most of these type 1 & 2 weak D cells at IS but won't pick up partial VI until the AHG test phase.  Ortho MTS gel reacts similarly to the Quotient anti-D.  I assume a partial D VI baby could theoretically sensitize a mom but that it is highly unlikely.  I think the type 1 & 2 weak D cells might be more likely to sensitize.  Please correct me if I am mistaken. 

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Hi All

So if a patient who is a D Variant (weak/partial) that can produce Anti-D is subsequently immunised with D positive RBC. The auto and DAT would be negative usually ?  However anti-D causes extravascular haemolysis so DAT should be positive. Is anti-D produced from a D-Variant any different in strength/avidity. Realise that pos DAT can occur for other reasons AIHA etc. 

Can anyone explain the mechanism of the anti-D anomaly? Or a link to an article ? 

Thanks

Edited by Tabbie
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The anti-D produced by a D Variant individual is slightly different to the anti-D produced by a true D Negative individual.  For example, if we look at the attached cartoon of the amino acid residues in Partial DVI Type 1 (based on a slide by Dr Geoff Daniels), the black circles represent amino acids encoded by exons derived from RHD genes, whilst the gold circles represent amino acids encoded by exons derived from RHCE genes (see also the exon map at the bottom of the cartoon)  However, the anti-D produced by such an individual will only be produced against the D epitopes missing from the Partial D Type 1 molecule, rather than the entire D molecule.  Such an "anti-D" would, of course, react with a "normal" D antigen, as the epitopes missing from the Partial DVI Type 1 would be present in the molecule of the "normal" D (and would, of course, also react with any other D Variant that expresses these epitopes).  This can also be proved by the fact that the "anti-D" produced by one individual with the Partial DVI Type 1 variant will not react with the red cells of another individual with exactly the same Partial DVI Type 1 variant.

The same applies with all other variants (i.e. the "anti-D" produced will only react against the D epitopes "missing" from the molecule).  What must also be remembered is that changes in amino acid residues may also affect the quaternary structure of the molecule as a whole, particularly if, for example, an amino acid with an acidic side chain, replaces one that has a basic or neutral side chain (or vice versa), which could have the effect of causing the entire molecule to distort, meaning that such an individual can make an "anti-D" against D epitopes that are apparently present, but are, in fact missing, because of the affect of this distortion.

As a result of all this, the "anti-D" produced is a true allo-antibody, rather than an auto-antibody, and this would explain why the DAT would be negative, as would the "auto".

I hope this rather complicated explanation helps a little bit!

Partial DVI Type 1.pptx

Edited by Malcolm Needs
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