When To Perform an Eluate

[Brenda K Hutson]

So I have to admit, after 26 years, 6 Hospitals and having been a Reference Supervisor, I have never heard this before (so want to check it out with all of you). And I have to admit, the thought of being wrong on this all of these years is rather intimidating for me in writing this; but, better to eat my pride and do it right....

Because we are a relatively small (400 beds) Hospital and Generalists rotate through, we send complex antibody work-ups out to a Reference Lab (a place I used to work actually).

This past weekend, my staff sent them a work-up on a patient which appeared to be an Anti-e in the serum (though did not hit all cells) and a strongly positive DAT. The last transfusion of the patient was a couple of weeks ago. In calling the next day for results, I was told they did not perform the Eluate because it had been > 2 weeks since the transfusion and that at that point, any antibodies that one would obtain from the eluate, would also be present in the serum.

Well, this has always been my approach (and thought proces): If transfused in the past 3 months, I would perform an Elution (regardless of whether I had identifiable antibodies in the serum); at intervals established by the Institution. Wouldn't it depend on the titer of the antibody, and whether or not a lot of donor cells had already been destroyed, as to whether it would appear in the serum also?

Ok, there you have it....be gentle please...

Brenda Hutson, CLS(ASCP)SBB

[KKidd]

Brenda, I understand your confusion. The reference lab that our facility uses will do a complete workup if that's what we want. After all we're the customer! They will provide guidance. but have never refused.

[SMW]

You may want to take a look at the following articles, which were probably part of the basis for the policy:

Judd WJ, Barnes BA, Steiner EA, et al., The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited, Transfusion 1986;26:220-4 and also

Judd et al. The evaluation of a positive direct antiglobulin test in pretransfusion testing, Transfusion 1980;20:17-23.

...but basically, after 14 days you're unlikely to get any useful information from an eluate...

[L106]

We're about the same size as Brenda's institution and our staff are rotating generalists, but we do our own work-ups. If the Direct Antiglobulin Test is Positive, we perform the elution and identify the antibody in the eluate. (Simple rule.) In my mind, what you find in the eluate may or may not be exactly the same as what you find in the patient's plasma.

When you test the eluuate, you might find an alloantibody, you might find an autoantibody, you might find nothing (if the DAT was related to certain drugs or disease states.) ("Inquiring minds want to know......")

[Brenda K Hutson]

Brenda, I understand your confusion. The reference lab that our facility uses will do a complete workup if that's what we want. After all we're the customer! They will provide guidance. but have never refused.

Well, ours did it for us; however, I need to know for myself if in fact their information is accurate.

Thanks,

Brenda

[Brenda K Hutson]

We're about the same size as Brenda's institution and our staff are rotating generalists, but we do our own work-ups. If the Direct Antiglobulin Test is Positive, we perform the elution and identify the antibody in the eluate. (Simple rule.) In my mind, what you find in the eluate may or may not be exactly the same as what you find in the patient's plasma.

When you test the eluuate, you might find an alloantibody, you might find an autoantibody, you might find nothing (if the DAT was related to certain drugs or disease states.) ("Inquiring minds want to know......")

This isn't the emphasis of the question, but yes, it kind of depends on your staffing as to how much reference work you can do at your institution

1. # of people in dept. on each shift; are there enough for 1 to totally focus on a long work-up

2. Are Generalists comfortable with that complexity, and I can tell you that at my facility, most on PM and MN shifts are

not

3. And frequency of performing the tests so you can keep people proficient. Many of us here will perform the eluate,

but in this case, the timing was such that there were 3 people in the dept and the person doing the patient can

perform eluates, but was too swamped to take the time).

Brenda

[Malcolm Needs ☆]

We would perform an eluate on all cord samples with a positive DAT, to make sure that the antibody sensitising the cord cells is not another antibody in the mother's plasma that we hadn't idenified. For example, in group O mum, who is R1R1, K-, and in whose plasma we had identified anti-A, anti-B (+ anti-A,, anti-c, anti-E and anti-K, there could also be, for example, an antibody directed against a low incidence antigen that is actually causing the HDN.

Following transfusion, with a de novo DAT+, we would perform an eluate up to 1 month post-transfusion, even if an alloantibody is detected in the plasma, just in case there is a second antibody Lurking in the background that is not detected in the plasma.

In a case of a patient with a known +DAT, we would be much more conservative, unless there was a sudden decrease in the time between the requirement for transfusion. In such cases we would perform an eluate, to see if an alloantibody could be identified that reacts more strongly than the auto-antibody.

BUT, if the above scenario occurs, and the auto-antibody in the eluate is really strong (and in very rare cases), we will perform alloadsorptions on the eluate to adsorb out the auto-antibody, to see if there is an alloantibody present too. It's a lot of work, but twice now we have discovered just such an alloantibody causing a transfusion reaction.

We would NOT expect one of our Hospital Blood Banks to do all this work!

:):)

[Brenda K Hutson]

We would perform an eluate on all cord samples with a positive DAT, to make sure that the antibody sensitising the cord cells is not another antibody in the mother's plasma that we hadn't idenified. For example, in group O mum, who is R1R1, K-, and in whose plasma we had identified anti-A, anti-B (+ anti-A,, anti-c, anti-E and anti-K, there could also be, for example, an antibody directed against a low incidence antigen that is actually causing the HDN.

Following transfusion, with a de novo DAT+, we would perform an eluate up to 1 month post-transfusion, even if an alloantibody is detected in the plasma, just in case there is a second antibody Lurking in the background that is not detected in the plasma.

In a case of a patient with a known +DAT, we would be much more conservative, unless there was a sudden decrease in the time between the requirement for transfusion. In such cases we would perform an eluate, to see if an alloantibody could be identified that reacts more strongly than the auto-antibody.

BUT, if the above scenario occurs, and the auto-antibody in the eluate is really strong (and in very rare cases), we will perform alloadsorptions on the eluate to adsorb out the auto-antibody, to see if there is an alloantibody present too. It's a lot of work, but twice now we have discovered just such an alloantibody causing a transfusion reaction.

We would NOT expect one of our Hospital Blood Banks to do all this work!

:):)

1. Are you saying you automatically do an eluate on ALL Cord Samples with a positive DAT? That would be a LOT of

eluates (not saying you are wrong; just commenting).

We only do them if:

a) Both Mom and Baby same ABO but Baby has POS DAT

Mom's Antibody Screen Positive (but even then, we would most likely just go by mom's antibodies initially rather

than looking for a Low Incidence to be lurking)

c) Regardless of whether Mom and Baby are ABO Compatible or not, and regardless of a NEG Antibody Screen on

Mom, if evidence of HDN in baby, we will look for a Low Incidence lurking out there somewhere (however, still

might do the search with mom's specimen rather than the Baby).

2. So you perform it 1 month after transfusion...that is the question...why the 2 weeks (that it appears may be coming

from John Judd)? My rationale has always been just that; that perhaps there is something coating the cells that is

not in the serum; however, it would appear that at least some people (John Judd et. al.) are saying that would not

be the case > 2 weeks.

3. I can honestly say I have never done an adsorption on an eluate.

Thanks for your input Malcolm!

Brenda

[Brenda K Hutson]

I decided to run this question past the Reference Lab Director for Dr. George Garratty; here was her point of view:

Hi Brenda,

I don’t know of a specific rule for not testing eluates in recently transfused patients, although the Medical Director’s statement is probably often true. There could be lots of circumstances, as you mention, where it might be a risky policy to follow… In any case, if you specifically ask them to test an eluate, I would think they could or would do it.

One other consideration; the Hospital we use for our Reference Lab (where I used to work), previously had a Reference Specialist there who trained for her SBB under John Judd; so given the reference in one of the responses above, that could be where they are coming from.

Brenda

[L106]

This isn't the emphasis of the question, but yes, it kind of depends on your staffing as to how much reference work you can do at your institution

Brenda

No, that wasn't my focus, either. My point was YES, we do an elution if we discover a Positive Direct Antiglobulin Test. (Our generalists staff don't have to try to weigh-in factors to make a decision about whether or not to do the elution.)

I am hoping that someone contributes more information about the two-week rationale that you are questioning. I don't remember ever hearing this concept, although it sounds like some of the most knowledgable professionals must have some evidence to suggest the two-week cut off. I'd like to hear more about it.

Donna

[Malcolm Needs ☆]

1. Are you saying you automatically do an eluate on ALL Cord Samples with a positive DAT? That would be a LOT of

eluates (not saying you are wrong; just commenting).

We only do them if:

a) Both Mom and Baby same ABO but Baby has POS DAT

Mom's Antibody Screen Positive (but even then, we would most likely just go by mom's antibodies initially rather

than looking for a Low Incidence to be lurking)

c) Regardless of whether Mom and Baby are ABO Compatible or not, and regardless of a NEG Antibody Screen on

Mom, if evidence of HDN in baby, we will look for a Low Incidence lurking out there somewhere (however, still

might do the search with mom's specimen rather than the Baby).

2. So you perform it 1 month after transfusion...that is the question...why the 2 weeks (that it appears may be coming

from John Judd)? My rationale has always been just that; that perhaps there is something coating the cells that is

not in the serum; however, it would appear that at least some people (John Judd et. al.) are saying that would not

be the case > 2 weeks.

3. I can honestly say I have never done an adsorption on an eluate.

Thanks for your input Malcolm!

Brenda

I can see why you would be more than a little aghast at my answer Brenda (particularly concerning the cord bloods), but, as a Reference Laboratory, we only get the odd one or two sent to us, and these are usually listed as for investigation of HDNF. As a result, we are sort of "duty bound" to throw everything at them.

I prefer the 1 month time limit. George Garratty and his co-workers wrote a paper concerning auto-adsorptions some time ago, and why they should not be performed on samples from patients who had been transfused within the previous three months. One of the reasons was that the transfused red cells remaining in the circulation could adsorb out a "new" alloantibody, even though there would be very few transfused red cells left. This suggests that the majority of the "new" antibody would be coating the transfused red cells, rather than be free in the plasma (although I am not suggesting that there would be no free "new" antibody in the plasma). These days, however, the elution kits are excellent, making the removal of any antibody much more efficient, and, as a result, the identification of a "new" antibody in the eluate is probably more sensitive than just examining the plasma.

I see that you say that you have never performed an adsorption on an eluate. My advice is, DON'T, unless there is a very good reason, you have loads of spare time on your hands and you decide that you do not want to get out more! It is something that we would only do in extremis.

:D:D:D:D

[jlemmons]

The problem I have with the two week cut off is that there are some of these antibodies who just don't read the textbooks and they take longer to emerge, Jka, Jkb, Duffy. I really like the 3 month time period myself. We also don't do an eluate unless the DAT is 2+ or great just because we have not found it useful. That is really experience in our institution talking.

[Yanxia]

Malcolm, why the limite is one month not three months, because we know the transfused cells may exist in the circulation more than three months . Thanks in advance for your explanation.

[Malcolm Needs ☆]

Hi Yanxia,

Good question!

Sorry, but the totally honest answer is, I don't know.

It was a decision made several years ago by the Red Cell Immunohaematology Section of the NHSBT (before I joined) and is does not seem to be 100% logical.

I will go back and ask at the next meeting of all the reference Managers (but that will not happen for some time).

Perhaps the decision needs to be re-visited, but there may be a logical answer of which I am unaware.

[jcdayaz]

So I have to admit, after 26 years, 6 Hospitals and having been a Reference Supervisor, I have never heard this before (so want to check it out with all of you). And I have to admit, the thought of being wrong on this all of these years is rather intimidating for me in writing this; but, better to eat my pride and do it right....

Because we are a relatively small (400 beds) Hospital and Generalists rotate through, we send complex antibody work-ups out to a Reference Lab (a place I used to work actually).

This past weekend, my staff sent them a work-up on a patient which appeared to be an Anti-e in the serum (though did not hit all cells) and a strongly positive DAT. The last transfusion of the patient was a couple of weeks ago. In calling the next day for results, I was told they did not perform the Eluate because it had been > 2 weeks since the transfusion and that at that point, any antibodies that one would obtain from the eluate, would also be present in the serum.

Well, this has always been my approach (and thought proces): If transfused in the past 3 months, I would perform an Elution (regardless of whether I had identifiable antibodies in the serum); at intervals established by the Institution. Wouldn't it depend on the titer of the antibody, and whether or not a lot of donor cells had already been destroyed, as to whether it would appear in the serum also?

Ok, there you have it....be gentle please...

Brenda Hutson, CLS(ASCP)SBB

Oh No! You have a reference lab going on two weeks instead of the 3 month standard? What is that? It is the standard thinking that red cells survive for 3 months.

I have seen multiple presentations of Fya and Jka that were NOT present in the antibody screen but were proven in the elution.

[adiescast]

We do an elution on any patient that had a negative pretransfusion DAT and a positive post transfusion DAT. I've not had one present more that one month post transfusion, so I actually do not have a separate rule to address that. We do an eluate on an initial positive DAT also. We do not perform an eluate if the anti-IgG is negative unless there is a specific request for it.

Having said that, I should mention that we do not routinely perform DATs as part of the Type and screen or crossmatch batteries. So if a patient presents with a positive DAT who was transfused > 14 days previous, we would not know if the pre-transfusion DAT was positive (because the sample would be gone).

[Marilyn Plett]

In 2002 we looked at the positive DATs in transfused patients. Of 192 samples for antibody identification and elution, only one sample showed antibody that could only be identified in the eluate. I don't remember the time frames of each sample, but the experience certainly showed us that we were doing a lot of eluates costing a lot of money and tech time but leading to very little gain for patients. Certainly, I agree that are situations in which an elution might be helpful, but the one month post-transfusion limit sounds like a reasonable compromise to me.

[Malcolm Needs ☆]

For those of you who have not yet read it, there is a new paper on this subject that strongly agrees with some of the posts above, and suggests that individuals like me can be far more conservative when it comes to performing elutions. It is,

Yazer MH, Triulzi DJ. The role of the elution in antibody investigations. Transfusion 2009; 49: 2395-2399.

It is a very interesting and thought-provoking paper.

:D:D:D:D

[jcdayaz]

We only perform elutions on at least 2+ DATs. It works for us and we have never encountered any problems of which I am aware.

[Joanne P. Scannell]

Just so happens we've been collecting data so we can see if it is safe to cut down on the number of elutions we prepare/test.

Currently, we prepare/test an elution only if the patient has been transfused during the past 3 months (using that old rule ... an rbc can survive 120 days ... IF it is healthy and left alone! What were we thinking?)

For the 971 elutions we reviewed, only 4 of them yielded a positive eluate with the 'offending' antibody 'missing' in the plasma ... and all of those were within the 14 day window. (We are using MTS for our Antibody Screens, btw.)

To support the converse: We had 3 inconclusive eluates but were able to define the antibody using the plasma and 38 cases where the results were the same whether using eluate or plasma.

So, guess what I am going to be presenting! Cut it down to 14 days as the literature and a lot of you are suggesting in here.

Thanks for the support!

[Malcolm Needs ☆]

More power to your elbow.

[GilTphoto]

We only perform elutions on at least 2+ DATs. It works for us and we have never encountered any problems of which I am aware.

What does the strength of the DAT have to do with whether you perform an elution or not?

I have personally eluted antibodies with 4+ reactions in the eluate, when the DAT was only microscopically positive.

[jcdayaz]

What does the strength of the DAT have to do with whether you perform an elution or not?

I have personally eluted antibodies with 4+ reactions in the eluate, when the DAT was only microscopically positive.

Wow! Were they clinically significant antibodies?

Scary.

They (I say "they" because it was prior to my arrival at this institution) were able to prove via multiple clinical trials that most reactions less than 2+ were insignificant or drug-related.

We do gel testing. What method do you use? Gel is extremely sensitive. It might be that your microscopic reaction would be 2+ in gel. I certainly don't know that last statement for sure. Just a guess.

[GilTphoto]

Yes, they were clinically significant. One was Jk(a), another anti-c.

We routinely incubate elutions for 30 minutes, tube method Gamma Elu-Kit.

In drug related pos DAT, the elution is either 100% positive or negative.

In fact the Technical Manual states that antibodies can be eluted even with a negative DAT (forgot where it said that - maybe in HDN section)

[Mabel Adams]

I just read the thread about providing blood to patients with Abs to low-frequency antigens where all those posting support taking the risk of not antigen typing units--even if an anti-Kpa is no longer reacting with Kpa pos cells (odds of transfusion reaction about 1:50--although probably delayed). Yet, here we have many that can't bear to give up doing eluates for those with transfusions from 3 months prior (from the above data a risk of less than one in a thousand). Either they are not the same people posting or we don't have very good tools for determining risk accurately across various issues we deal with. The old Blood Bank "gut feeling" really isn't very accurate, I fear.