Eluates and Compliment Only Positive DATs

[SMILLER]

Under what circumstances should a eluate be performed on a patient with a DAT that is negative for IGG but positive for compliment?

 

Scott

[DebbieL]

We do an eluate only IF the patient has been transfused in the last 3 months. I think the reasoning is it could be the early stages of the formation of a complement dependent antibody.  If it is only complement and the patient has not been transfused, we do not perform the eluate. We would put in a comment for BB eyes for future reference.

This was implemented by my previous supervisor and I have not changed it. As you can guess this scenario doesn't happen often because these people have always been transfused.

I will be interested in what others do.

[Malcolm Needs ☆]

I would do the same DebbieL.

[SMILLER]

So if the patient has been transfused recently, and ONLY the compliment is present in the DAT, you do an eluate?  (I get it that we all would do one if the Poly or IGG is positive regardless of the compliment)

Thanks, Scott

[goodchild]

Technical Manual, 18th edition, Chapter 17, DAT/Immune Hemolysis, page 428.

Test an eluate prepared from the DAT-positive red cells with reagent red cells to determine whether the coating protein has red cell antibody specificity. When the only coating protein is complement, the eluate is likely to be nonreactive. However, an eluate from the patient's red cells coated only with complement should be tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion. The eluate preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient's plasma.

[Malcolm Needs ☆]

27 minutes ago, SMILLER said:

So if the patient has been transfused recently, and ONLY the compliment is present in the DAT, you do an eluate?  (I get it that we all would do one if the Poly or IGG is positive regardless of the compliment)

Thanks, Scott

Yes, as there are times when the causative antibody is an IgM (such as anti-Vel), and, never forget, the titre of the anti-Vel may be VERY low, but the complEment system is an amplification system (i.e. one C1qrs complex will result in huge numbers of other activated complement molecules further down the line), but you can concentrate the eluate and be able to detect the antibody originally sensitising the red cells.  The antibody can also be a VERY weak IgG antibody (IgG1 or IgG3, or a mixture), and the same applies.

[SMILLER]

OK, this is where I would appreciate some explanation of what is going on at the serological level.  I was aware of this:

Technical Manual, 18th edition, Chapter 17, DAT/Immune Hemolysis, page 428.

Test an eluate prepared from the DAT-positive red cells with reagent red cells to determine whether the coating protein has red cell antibody specificity. When the only coating protein is complement, the eluate is likely to be nonreactive. However, an eluate from the patient's red cells coated only with complement should be tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion. The eluate preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient's plasma.

They seem to be saying on the one hand, if only compliment is detected, that the eluate is going to be likely non-reactive.  Then they mention that IgG may be present in the patient's serum in such small amounts that it may be non-detectable --- which seems besides the point -- we are not concerned here with a test that enhances what is in the patient's serum, but what is present on cells, and the DAT is already negative in spite of the serum.  Or will someone suggest that an eluate is going to be more reactive than the cells that the IgG is eluted from?

Indeed, in suspected transfusion reactions, the compliment portion of a DAT is significant without a positive IgG portion, as the offending antibody may no longer be present on the cells, leaving compliment behind.  But in this case, why look for something we already know is not there by performing an eluate?

And this from Malcolm: there are times when the causative antibody is an IgM

But what is the likelihood that you are goling to pick up an IgM antibody (significant or otherwise) with the anti-IgG reagents used for antibody detection?  And from an eluate no less, which I believe are notoriously weak anyway even if present?

Thanks, Scott

 

 

 

 

[Malcolm Needs ☆]

1 hour ago, SMILLER said:

Sorry Scott, it's getting a bit late in the UK.  I'll post tomorrow.

[Malcolm Needs ☆]

Hi Scott,

I'm back in the land of the living!

I think the key bit of your quote from the Technical Manual is:

"However. an eluate from the patient's red cells coated only with complement should be tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion.  The eluate preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient's plasma.".

which, incidentally, is essentially what I said in an earlier post.

The other important phrase from the quote from the Technical Manual is:

"When the only coating protein is complement, the eluate is likely to be nonreactive."

The word "likely" is very important here, as it means that the eluate is not definitely going to be nonreactive.

Particularly in the case of a very weak antibody, such as one that is derived from an anamnestic response, it is not unknown for almost all of the said antibody to be sensitizing the red cells, and for very little, if any, of this antibody to be free in the plasma/serum - certainly not by normal serological techniques.  However, if an eluate is performed, and the eluate is made with less eluting fluid than normal (normally it is a 1:1 ratio with the washed packed red cells, but that ratio can be changed to, for example, two volumes of washed packed red cells to one volume of eluting fluid), then essentially, the concentration of the antibody being eluted from the red cells is doubled.

Such findings were described in Sachs UJH, Roder L, Santoso S, Bein G.  Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661.

As I have said in other, earlier threads, although this paper was designed to look at DAT Negative cases of AIHA, the authors also actually describe many cases of a delayed haemolytic transfusion reaction (possibly delayed sereological transfusion reactions) detected by eluting antibodies from the red cells of the patient, even though the DAT was negative and there was no free antibody in the plasma/serum that was detected by routine serological techniques.

So, the evidence from this is that the eluate may well be more reactive that the plasma/serum in terms of being able to identify the presence of, and elucidation of the actual antibody specificity.

You go on to say:

"And this from Malcolm: there are times when the causative antibody is an IgM.

But what is the likelihood that you are going to pick up an IgM antibody (significant or otherwise) with the anti-IgG reagents used for antibody detection?  And from an eluate no less, which I believe are notoriously weak anyway even if present?"

What I was driving at here was the fact that, if there is clinical evidence of antibody-mediated haemolysis, particularly after a transfusion, every effort should be made to identify the specificity of the antibody, to ensure that the cognate antigen is not expressed on the red cells of any units that may subsequently be transfused.  This effort may include the use of a clotted sample, rather than a sample anticoagulated with EDTA, as, of course, the EDTA would chelate the calcium, manganese and magnesium ions that are required for maximal initiation of the classical complement cascade.  In such a case, a monospecific anti-C3d reagent, or even a monospecific anti-IgM reagent, rather than a monospecific anti-IgG reagent would be used.  It was in this way that one of the red cell immunohaematology reference laboratories of the NHSBT was able to show the presence of an IgM anti-Vel in the circulation of a patient who had undergone an acute (sadly fatal) haemolytic transfusion reaction, where no IgG anti-Vel could be detected in the plasma.

The bit about the "strength" of the eluate I have dealt with above.

I am acutely aware that the ability for the normal hospital blood transfusion laboratory to be able to carry out such tests is unlikely, as they would not carry the necessary rare and expensive reagents, but in a case where there is clinical evidence of antibody-mediated haemolysis, samples should be sent to a reference laboratory for full investigation before further transfusion is attempted, unless the physician decides that withholding further transfusion is more dangerous than a possible further transfusion reaction.

Sorry for the over-long post.

[SMILLER]

Thank you all for the responses.  By far the most useful part of this forum for me is the educational aspects available.

NB...

We are a trauma 2 medium-sized hospital in Michigan.  We typically have 4 techs for graveyard shift, one of which floats in and out of BB as necessary.  Like most labs of this type, this means that our most experienced (and best staffed) techs are NOT around in the wee hours -- we depend on the night techs to call for help (or in some cases, call IN help) when needed.

The other night we had a anemic patient from ER that had a positive gel screen, with what initially looked like an equivocal, but when running the rule-out cells, three K positives turned up reactive.  The techs were a little flustered.  The complication was that they had a positive AC on the gel panel and when they ran the DAT, it was positive (but only for compliment).  They then had to do a eluate (which was negative-hence all the questions on this thread) on top of all of the other bother.  The patient was not recently transfused.

We did the workup in tube in the morning and it was all just gel interference.  It seemed to me the eluate was an unnecessary distraction since the IgG part was negative and the patient had not been transfused.  Of course, at the time, they were suspecting that the patient had some kind of antibody (which they did not as we later determined).

This is not a situation one would see much in a reference lab, or a decently staffed large medical center for that matter.  With chronic staffing shortages in the US and all of the other issues that are going on in a hospital, I was wondering what the utility is of performing a procedure that would seem to have almost no clinical usefulness when it is finished.

Again, thanks for all you responses.  Malcolm, as usual, had a very clear and concise explanation of the important points regarding this issue.

Scott

 

 

 

[SMILLER]

NB2...

From the article referenced by Malcolm above:

Two major conclusions can be drawn from our study. The DAT must be interpreted in conjunction with clinical and other laboratory data to avoid erroneous conclusions; it can be expected to be false-negative in up to 3% of AIHA patients. Elution of RBC antibodies is a valid additional procedure to clarify whether autoantibodies are present in DAT-negative patients.

Basically saying that around 3% of negative DAT reports are false negative even though the patient has AIHA.  I wonder how many physicians would consider ordering an eluate after seeing a negative DAT?

Scott

[Sandy L]

We would not routinely perform eluates if the IgG DAT was negative but if we suspecting a delayed hemolytic transfusion reaction we would definitely consider doing this testing.  I have been able to elute IgG antibodies from DAT negative samples on several occasions.  Initially I thought this phenomenon was unique to the eluting of anti-A or –B from cord samples.  I think we’ve all experienced this, with mom group O and negative antibody screen, infant group A or B and negative DAT.  MD suspects ABO HDN and requests elution.  Generally you will be able to elute the maternal IgG ABO antibody despite the negative DAT.  As a med tech student we were always taught that this related to the way the infant’s ABO antigen developed, less branched structures.  Over the years we have seen that this happens with other antibodies too.  In several cases where mom had a known significant antibody, and infant DAT was negative but infant was positive for the antigen to which the antibody was directed.  This has happened with anti-c, anti-Jka and most recently with anti-Ge3. Most of these eluates reacted 2+ to 3+ despite the negative DAT.  Also think about Delayed Transfusion Reaction Investigations that you may have performed where the DAT may be weakly positive but the antibody in the eluate reacting quite strongly.  Another observation: Just recently I was preparing competency unknown samples for eluate testing by coating cells with various patient antibodies.  Some of the samples had DAT’s that were negative or only weakly positive yet the antibody was recovered in the eluates, again reacting 2+ to 4+.  And this was not even increasing the concentration of red cells as Malcolm suggested.  If you think about it, the process of preparing the eluate really does have a “concentrating” effect.  The eluate is prepared from a large mass of red cells where the packed red cell volume to eluate volume is nearly equal.  That eluate is then tested against a very diluted concentration of reagent red cells, e.g. 3-4%, compared to the very MUCH larger quantity of cells that it came from, thus you are greatly increasing the sensitivity of the test.

[John Eggington]

The strength of a DAT can depend on where you take your red cell sample from (of the centrifuged patient simple); sampling from the 'bottom' can give a stronger reaction. With 'minor' populations of DAT pos cells I sometimes fin a tube DAT is better to see a pos population, using an inverted microscope (with a stage adapted to receive the test tube), but I am prone to 'old school' techniques!

[goodchild]

Recent scenario we had, I thought was relevant:

Patient with newly positive antibody screen, ordered for transfusion of two RBCs. Had been admitted for about a month; now diagnosed with numerous medical problems that were previously unknown to the patient. Patient was transfused a total of six RBCs with the last transfusion five days prior.

The bench technologist wasn't able to determine a specificity with plasma testing but the autocontrol was positive. (Recent grad, a more seasoned technologist might have suspected the specificity based on pattern of positivity.) DAT was positive with polyspecific AHG and anti-C3. Our procedure indicates to proceed with elution in this case if the patient was transfused in the last 45 days. Eluate testing clearly identified anti-Jk^a.

[Mabel Adams]

Our policy is to do eluates if the patient with the pos DAT was transfused in prior 14 days (or longer if there is evidence of hemolysis or not getting the expected rise in Hgb).  I believe the 14 day rule was provided us by our reference lab before I came here.  It is nice not having to do eluates for transfusions 3 months back but, now you have got me to thinking, does anyone else use such a short time-frame for requiring eluates?

[Malcolm Needs ☆]

We still do eluates up to three months after a transfusion, but ONLY if there are good reasons, such as a symptomatic patient.

[jalomahe]

Our reference lab recommended doing the eluate if patient transfused within 3 weeks.

[macarton]

We would do an elution if the patient was recently transfused.

[Rerun]

We follow the 14 day rule, too, if the patient was transfused.

[Mabel Adams]

We sometimes do them out more than 14 days based on our judgment but our policy requires that they be done if the transfusion was within the past 14 days.

[carolyn swickard]

This might be useful too.  I remember reading about a "super DAT" test on this forum, but have not been able to find it again.  Does anyone remember how to do it?  (I think it involved an AHG phase.)  Concentrating the DAT - which I believe this procedure did - might be a good test in some of these situations (symptoms present but the DAT looks negative). 

[dragonlady97213]

We recently went from 2 weeks out to 3 weeks from transfusion for requiring an elution.  Anything further out is at the discretion of the tech doing the workup; usually if we see an increase in the strength of the DAT or evidence of hemolysis if there hasn't been in the past.