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This is a very silly question, but I think I have confused myself. Why is it that when Rh phenotyping, we do not require incubation step, but when looking for antibodies, we need incubation to detect Rh antibodies? 


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The Rh typing reagents are designed to react that way. These days, the reagents are monoclonal, IgM in nature and give direct agglutination in a very short amount of time (similar to anti-A and other ABO reagents). Centrifugation is also usually part of the process.

Antibodies to Rh antigens in patients (or donors) are typically IgG and require incubation and an antiglobulin phase. Most manual tube testing systems these days also use a potentiator to enhance reactivity and/or reduce incubation times.

In the "bad-old-days", Rh typing reagents were human-source, IgG in nature and usually required incubation and an antiglobulin phase.

Edited by exlimey
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I agree entirely with exlimey, except to say that even today's monoclonal antibodies need a potentiator.  Many of them include a small amount of bovine albumin in the reagent bottle.

I don't, however, agree with you Jermin.

The reason being is that there is no such thing as a silly or daft question.  The only silly or daft question is the one you (anyone) don't ask, because, if that question is not asked. the person who doesn't know the answer will never know the answer.  Sadly, there are numerous examples of silly or daft answers!

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A further question.

I saw a senior BMS putting a manual tube grouping in the fridge for ABO typing. I know that there will probably have a better reaction in reverse group, but according to the senior BMS, the patient appears to have a weak D, and the BMS wanted to see if the reaction will be enhanced, but in forward grouping. I was confused since the books mentioned Rh bonding being hydrophobic and therefore warm-reacting. 

There was no enhanced reaction, but I was wondering if putting the test in the fridge would indeed cause an enhanced reaction for D, or be it on ABO, on forward grouping? Would it be due to IgM monoclonal antibody being used?


Edited by Jermin
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The reaction with anti-D may well be enhanced - but not for the right reasons!

Thorpe et al1, 2 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, and you will get a false positive - which is the very last thing you want.

1.  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.

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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