Antibody Evaluation

[Muhammad Awwal]

Hi👋

I am solving case study of patient with AIHA (consistent with lab findings). DAT is +ve in IgG only. Antibody screen and panel is pan reactive in AHG (& enzyme-treated RBC) . After auto-adsorption, defined reaction was appeared in AHG but still panreaction in enzyme-treated RBCs. 

My question is, is the reaction of enzyme-treated RBCs with auto-adsorbed plasma still due to incompletely adsorbed auto-antibody? Is it IgM, since some reactions are absent in AHG? How can this be resolved, please? 

I have searched the internet without success, and I found this forum during my search.

Thank you 👋🙏

[Malcolm Needs ☆]

Well Muhammad, my immediate question is, does it matter whether the antibody is IgG or IgM, but the reaction is most certainly due to incomplete adsorption?

That having been said, you could treat the plasma with 0.01 M dithiothreitol (DTT), which would disrupt the di-sulphide bonds that hold an IgM molecule together, via the J-chain, which will prevent the molecule forming agglutinates with the red cells, but it really doesn't matter whether the antibody is IgG or IgM (or, indeed, a mixture of the two), if the reaction is by enzyme-only.  Indeed, within NHSBT, we do not bother to test adsorbed plasma with enzyme-treated red cells after adsorption.

[Muhammad Awwal]

Thank you, Malcolm.

[exlimey]

I agree with Malcolm - the IgG/IgM nature of the antibody is not relevant, and I would avoid tests with enzyme-treated cells in patients with confirmed WAIHA, especially after adsorption procedures. I know that some workers also avoid PEG when testing adsorbed serum in these cases, opting instead for LISS or even saline antiglobulin tests on the adsorbed serum.

[Muhammad Awwal]

Thank you, exlimey.

[Lucky Jack]

On 8/16/2018 at 7:14 AM, Malcolm Needs said:

That having been said, you could treat the plasma with 0.01 M dithiothreitol (DTT), which would disrupt the di-sulphide bonds that hold an IgM molecule together, via the J-chain, which will prevent the molecule forming agglutinates with the red cells, but it really doesn't matter whether the antibody is IgG or IgM (or, indeed, a mixture of the two), if the reaction is by enzyme-only.  Indeed, within NHSBT, we do not bother to test adsorbed plasma with enzyme-treated red cells after adsorption.

I have a question regarding DTT treatment of plasma - I understand that if there is reactivity persistent in the saline control and absent in the DTT treated plasma, it indicates that reactivity is caused by an IgM antibody. What steps, if any, can be taken to show there wasn't also an IgG antibody that was diluted to the point of undetectability in both the DTT treated plasma and the saline control plasma dilution? Or am I just thinking about this wrong?

[Malcolm Needs ☆]

5 hours ago, Lucky Jack said:

I have a question regarding DTT treatment of plasma - I understand that if there is reactivity persistent in the saline control and absent in the DTT treated plasma, it indicates that reactivity is caused by an IgM antibody. What steps, if any, can be taken to show there wasn't also an IgG antibody that was diluted to the point of undetectability in both the DTT treated plasma and the saline control plasma dilution? Or am I just thinking about this wrong?

No, you are not thinking about this wrong, but, if there is an IgG antibody present that is diluted out at one in two, it is not going to be of any clinical significance in that particular episode of transfusion, and in particular as it is a "warm" auto-antibody, as most "warm" auto-antibodies are directed against a very high prevalence antigen (such as Rh17 or Rh18), even though the antibodies tends to mimic specificities such as anti-E or anti-e, and so you will be unable to provide the required D--/D-- or Rh_null red cells that would be the only red cells properly compatible.

[Muhammad Awwal]

What is a 'saline control plasma' please?

[Malcolm Needs ☆]

39 minutes ago, Muhammad Awwal said:

What is a 'saline control plasma' please?

Sorry Muhammad, where is that posted?

[Muhammad Awwal]

Hi Malcolm,

6 hours ago, Lucky Jack said:

in both the DTT treated plasma and the saline control plasma dilution?

 

[Malcolm Needs ☆]

Ah right.  Well the plasma is treated with 0.01M DTT by making a 1 to 1 dilution of the plasma in the DTT (there is a bit more to it than that, but, essentially that is what it is).  Then to make sure that the negative results are not just due to the diluting effect of the DTT, you have to dilute some of the untreated plasma 1 to 1 in saline and test that as well to make sure that the antibody can still be detected.

[Mabel Adams]

So if both your saline control and your DTT treated tests are negative, then you can conclude that you have diluted out the antibody in your neat plasma, whether it was IgG or IgM, right?  The DTT treated test might be negative just because of the dilution, not because it was IgM.  I bet this saline control is usually positive so the problem is seldom encountered.  Maybe a problem when working up a fairly weak anti-M in pregnancy?

[Malcolm Needs ☆]

8 hours ago, Mabel Adams said:

Maybe a problem when working up a fairly weak anti-M in pregnancy?

Well, it is and it isn't, because if the anti-M is that weak, it is definitely NOT going to cause clinically significant HDFN; in fact, it is unlikely to cause HDFN of any sort.

[Mabel Adams]

Wise John Judd once advised me to do titers on anti-M antibodies without concern whether it was IgM or IgG.  We didn't do DTT testing there. Once the titers reached 16 we could send it out then for further testing to delineate IgG vs. IgM .  We had to report it carefully and clearly so it was clear what we were measuring. No titer ever exceeded 16 so we saved a lot of money on send-out tests.  Now we do pre-warmed testing on them and only pursue those that react by that technique.  Still haven't seen one with a high titer.

[Tabbie]

On 8/22/2018 at 9:30 AM, Malcolm Needs said:

No, you are not thinking about this wrong, but, if there is an IgG antibody present that is diluted out at one in two, it is not going to be of any clinical significance in that particular episode of transfusion, and in particular as it is a "warm" auto-antibody, as most "warm" auto-antibodies are directed against a very high prevalence antigen (such as Rh17 or Rh18), even though the antibodies tends to mimic specificities such as anti-E or anti-e, and so you will be unable to provide the required D--/D-- or Rh_null red cells that would be the only red cells properly compatible.

Hi Malcom

When you say mimic does that mean that the patients phenotype would not be E+ or e+ which would be the case if it was just an auto anti-E (wondering about how sometimes phenotype is not the same as genotype). Or would you not know until you performed adsorption/elution with D—/D— to confirm the Rh17.

Thanks

Edited August 28, 2018 by Tabbie
Adding

[Malcolm Needs ☆]

Not quite Tabbie - although it is difficult to explain - but I will do my best (but the explanation may not be perfect).

Rh_null individuals are incredibly rare throughout the world, and I really mean INCREDIBLY rare.  I have worked most (although not all) of my professional life in Blood Group Serology (partly at the International Blood Group Reference Laboratory, and partly at NHSBT-Tooting RCI Laboratory).  Because of the fact that the IBGRL is a World Health Organisation (international) establishment, they got/get samples of rare blood from all around the world.  Because NHSBT-Tooting RCI Laboratory covers hospitals that have an incredible mix of patient ethnicity, we also used to get a lot of VERY rare antibodies/antigens.  I think that I can say that, in 43 years in this environment, I have only seen two, maybe three families that included Rh_null individuals; that is how rare they are.

These individuals can (and do) make an antibody named anti-Rh29.  This antibody, in essence, reacts with all red cells apart from those of other individuals who have the Rh_null phenotype.  On the other hand, some individuals who have warm auto-immune haemolytic anaemia make an antibody that is very similar, if not (to all intents and purposes) identical to anti-Rh29.

Similarly, D--/D--, D^../D^.., D--/D^.., D--/Rh_null and D^../Rh_null individuals can make antibodies that look like a combination of anti-C, anti-c, anti-E and anti-e that cannot be separated (it is, needless to say, a bit - a lot - more complicated than that).  This antibody is termed anti-Rh17.  This antibody is, once again, not straightforward (as you might expect by now!).  This antibody is THE single most common specificity found in cases of WAIHA.  HOWEVER, just in case you thought things were getting easier, they are not!  The auto-anti-Rh17 you find in individuals with warm AIHA often (more often than not actually), reacts more readily with, for example E Positive red cells (or e Positive red cells, C Positive red cells or c Positive red cells) than with E Negative red cells (or e Negative red cells, C Negative red cells or c Negative red cells), irrespective of the fact that the patient is E Positive (e Positive, C Positive or c Positive).  You can tell that it is NOT anti-E (or any of the other specificities) by the fact that the antibody can be adsorbed out by E Negative red cells (it make take more adsorptions than with E Positive red cells, but you can still do it) - and, again, the same applies for other mimicking red cell antibodies.

These days, with the ability to use monoclonal Rh grouping antibodies, it is normally quite easy to tell if the antibody  is an auto- or an allo-antibody, even if the red cells are DAT Positive, because it is (usually) quite easy to sort out the patient's own Rh type.  As a result, it is VERY rare that an RCI Laboratory would bother to do too much work on it - basically, it would be a waste of time, reagents and money.

Turning to your other question, concerning how the genotype can be different from the phenotype, this situation is surprisingly much more common than you make think.

Some 68% of Black individuals are (phenotypically) Fy(a-b-), and yet, genotypically, most of these individuals are FYB/FYB, which begs the question, why are the red cells not typing as Fy(a-b+)?  Well, the answer is that there is another gene, called the GATA-1 gene that has to be present (and present in an "unmutated" form) for the antigen to be expressed on the red cell.  A large percentage of Black individuals have a mutation (and mostly as a homozygous gene mutation) that prevents the expression of the Duffy antigens on red cells, even though the blood group antigen genes are present on the chromosomes (chromosome 1 in this case, as it happens).  This is by no way the only time that a genotype does not match the phenotype.  There are loads of others (for example, Partial D Type III has gene mutations that can allow the individual to make a genuine allo-anti-D - and pretty strong anti-D at that - but, phenotypically, the person is a straightforward D Positive individual).  The same applies with e variant mutations, such as hr^B and hr^S.

I hope all this helps, but, if not, get back to me (or others on this wonderful site).

[Tabbie]

Your a star Malcolm as ever complex but clear and reference to the GATA-1 gene to compare scenarios, putting it into perspective.

Much appreciated