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Cgore

When is Rhesus D positive in test tubes?

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While this seems a fairly obvious answer, we’ve had issues lately. Two weeks ago, we had a cord sample give a ‘?’Reaction in Rh D in the newborn card. As per our sop, this was rerun and came up negative and to be sure, the bms washed the cells and checked the group in test tubes.  It was weakly positive in one clone of Rh D, only detectable under the microscope. This was later confirmed as weak D by the reference lab and the mother was dosed appropriately with Anti-D. 

Historically, we’ve always checked negative test tube  Rh D under the microscope and this rarely caused problems. Since this incident though, we’ve had problems with weak (+/-) reactions which are confirmed as negative by the reference lab. Anti sera instructions state observation is macroscopic which would overrule our historic habit of checking microscopically BUT how else do you detect weak Rh D? If we had followed the macroscopic only rule, we would have missed the weak Rh-D and the mother could have been sensitised. Is weak Rh D in a foetus less likely  to cause sensitisation?

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There are one or two comments I would make, which you may find pedantic, but, that notwithstanding, they are true.  There is, and never has been, a blood group system named Rhesus.  Rhesus was an ancient king of Thrace.  The correct name for the blood group system is Rh.  So, it is the Rh Blood Group System, the two genes involved are RHD and RHCE, the carrier proteins are RhD and RHCcEe, but the antigen of which you are talking is just plain D, and not Rh D.

There is a very good reason why the instructions that come with the anti-D state that the tests should be read macroscopically.  Thorpe et al, in two papers, 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 papain-treated D- red cells are tested with such antisera, or untreated D- red cells are tested with such antisera that have not been brought to room temperature, they may agglutinate.  This could result in D- red cells being mistyped as D+ - a particular danger in females of child-bearing potential, and babies (Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellerberg 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, and 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).

In addition, if you do not follow the manufacturer's instruction, and something goes wrong, under UK (and EU) Law, you could well be liable, as described by Bob Doughty some 30 years ago now (Doughty RW.  Product liability in the medical laboratory.  Medical Laboratory Sciences 1989; 46: 68-71.

In the case that you describe, do you know the Weak D type of the baby (it may well be Weak D Type 2, which can be particularly weak) and, at any time, did you test the mother's red cells to see if she was also a Weak D of the same type?  If she was, then it is highly likely that anti-D immunoglobulin would not have been required anyway, although I am aware that this is not part of the BSH Guideline (White J, Qureshi H, Massey E, Needs M, Byrne G, Daniels G, Allard S and British Committee for Standards in Haematology.  Guidelines for blood grouping and red cell antibody testing in pregnancy.  Transfusion Medicine 2016; 26: 246-263.  doi: 10.1111/tme.12299).

My honest advice is that you do not read the tests under a microscope.  If you have ANY doubt, give the mother anti-D immunoglobulin anyway - it is not that expensive, and has a good safety record in terms of TTI.

.

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Thank you so much for your input. Our concern was merely regarding the mother being avoidably sensitised by us missing a weak reaction. Our tubes and anti sera are at room temp (replaced daily) so I don’t think it’s a reaction to the cold but that is a good point for future reference. 

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Sorry, in response to your question, the mother had a clear meg reaction on the cards so we didn’t test her further. The cord blood tested 0.5, rerun was neg and the second rerun was a query which is why we went for a tube group.

Edited by Cgore
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On 2/13/2019 at 4:18 PM, Cgore said:

BUT how else do you detect weak Rh D? If we had followed the macroscopic only rule, we would have missed the weak Rh-D and the mother could have been sensitised. Is weak Rh D in a foetus less likely  to cause sensitisation?

I have a question about the "newborn card".  I am not familiar with this card, so forgive me if this is the case.  Does this card contain IgG, and is it incubated at 37 prior to centrifugation?  And when you perform the testing in tube, do you incubate and carry it through AHG phase?  This is how we detect weak D.

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If you are seeing a lot of weak results in your anti-D well that subsequently turn out to be negative, I suspect the reason to be one of manipulation rather than anything serological.  I would guess that NONE of these babies are group O.  I am guessing that you are seeing carryover from your anti-AB.  It can happen that if cards are stored somewhere where condensation can take place then drops of antiserum can condense into the reaction chamber and 'jump' into the next well or even next 2 wells when you remove the aluminium.  This can cause false positive results.  I suggest you check the cards before pipetting in them and see if there are any signs of these drops.  Don't use the cards if there are and look for another place to store them

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