Weak backtype reaction

[Veejay]

I'll contribute a little to this discussion too if I may. For ABO reverse grouping the column tests rely on haemagglutination already occurring in the upper part of the microtube. The Ag-Ab bonding is just happening via Brownian motion. Then during centrifugation these agglutinates get trapped within the gel or glass bead matrix.

In contrast, in a tube test the antibodies within the reverse grouping plasma / serum are physically forced to get close to the red cells and if the corresponding antigen is present the agglutination will occur. The technical challenge then is to shake the tube gently enough so as not to disrupt the agglutinates.

[rravkin@aol.com]

AMcCord,

Thank you for your detailed post. I appreciate the last pargraph specifically. If you don't mind explaining, I was wondering what additional work do you do to resolve a consistantly weak backtype? Thank you again.

I'm sorry, but I have to disagree that an adsorption process is easily feasible. There are many smaller facilities across this country that do not have the staffing, materials or the techs with the competency to perform adsorptions. There are many smaller facilities that don't even do antibody identification, much less adsorption, elution, enzyme treatments, etc. They send those tests to a reference facility. Their test menus are very basic and their budgets are not going to allow for extra reagents, training for testing and competency assessment materials for a test that would be done only rarely. Every time they had a patient that had a weak backtype, the crossmatch would have to be sent out, delaying transfusions by up to a day or more for rural facilities. That would make an adsorption requirement very burdensome. As to the rarity of a weak backtype, that depends on your patient population. We have a large percentage of elderly and oncology patients. I see a weak backtype or two (or three) almost every day and we do an average of only about 15 blood types daily.

Many of our transfusions are outpatients who are not prescheduled - from the time of order to checkout of the first unit is 90 minutes or less and that's the way management wants it for the benefit of the patients. Adding an adsorption step would require a delay of several hours, during which the patients would be hanging around waiting and waiting. Since most of the outpatients are Onc patients with low Hgbs who feel lousy and need the blood, that's not good patient care. What if that weak backtype was a patient in the ER with a 4.0 Hgb or a severe GI bleed? Can we tell the ordering physician, 'sorry, you're going to have to wait on that blood' or do we decide to exclude those patients from that extra step. We staff Blood Bank with 1 tech many days - if that tech has to do an adsoprtion every day or so, maybe even 2 or 3 on some days, that's going to delay the surgical crossmatches and the crossmatches on the transfuse orders, both inpatient and outpatient. The adsorption step would increase the cost of transfusion and I doubt that insurance companies would consider it a clinically necessary step, and Medicare is not going to be increasing reimbursements, so that means we'd eat the extra cost.

We don't ignore the problem of a weak backtype in terms of the ABO type. If we see a weak backtype, we increase the amount of plasma used for the backtype and the IS crossmatch. If the backtype still doesn't react (rare, thankfully), then we would have to do additional work to resolve the backtype issue and would perform an AHG crossmatch. We do not do additional work to try to detect alloantibodies. If the antibody screen is negative with solid phase or PeG, we accept the result.

There have been retrospective studies done with thousands of patients looking for adverse transfusion reactions post abbreviated crossmatch protocols (negative antibody screen/IS or electronic crossmatch only). There would have been a statistically significant number of patients with weak backtypes in those patient populations. I'm not aware of one of those studies showing an appreciable number of problems. If we were truly missing clinically significant antibodies by not using more aggressive methods to detect them, I think that it would have become apparent by now. I think we need to make an evidence based decision here. If there is evidence that more testing is beneficial, then I'm all for it. But if there is little or no evidence, then I think there is a line where we have to say that we have practised due diligence in our decisions and call it good.

Edited August 15, 2012 by rravkin@aol.com

[rravkin@aol.com]

Veejay,

Thank you for your post but I think that the tube test also relys on Brownian Motion to for the reaction to occur. We may not think of it this way bause it is an immediate spin procedure and therefore there is little delay between combining reactants and centrifugation. Here the centrifugation will concentrate the cells which have already adsorbed the anti A1 and /or B respectively. When we are working up a descrepancy that occurs in the backtype we increase centrifugation time, which may lend to your theory, but we also increase incubation times at RT, 37C, and 4C.

I'll contribute a little to this discussion too if I may. For ABO reverse grouping the column tests rely on haemagglutination already occurring in the upper part of the microtube. The Ag-Ab bonding is just happening via Brownian motion. Then during centrifugation these agglutinates get trapped within the gel or glass bead matrix.

In contrast, in a tube test the antibodies within the reverse grouping plasma / serum are physically forced to get close to the red cells and if the corresponding antigen is present the agglutination will occur. The technical challenge then is to shake the tube gently enough so as not to disrupt the agglutinates.

[AMcCord]

The additional work we would do for a weak backtype would be:

A. Try increasing the amount of plasma added for the backtype (and the IS crossmatch) - 4 drops instead of 2. If that fails..

B. Incubate tubes which have 4 drops plasma at RT for 30 minutes, with an O cell (use an antibody screen cell or panel cell) as a

negative control plus an auto control. Cold agglutinins can cause false positives, so interpret with caution (this is where the O

cell and auto control comes in, of course). If that fails...

C. Incubate the tubes from plan B at 6-10 C for 30 minutes. False positives due to cold agglutinins will be more likely, so again,

interpret with caution. If that fails....

D. Perform adsorption/elution and test eluate against A, B and O cells.

And/Or treat A, B, O and patient cells with ficin, then add 2 drops plasma. Enzyme treated cells react well with anti-A and anti-B.

Again....interpret with caution.

If we got to step D, we would transfuse type O red cells until the problem is resolved. With CAP proficiency testing requirements what they are, I would send probably send step D to our reference lab. If we did perform the adsorption/elution or enzyme testing, I would send a sample to reference to confirm our results.

We occasionally get to step B and rarely to step C. I can't remember the last time we had to go farther than that (and I'm thankful!).

[Mabel Adams]

The ABO discrepancies that we are unable to resolve are usually post bone marrow transplants that got an ABO mismatched transplant. Their new marrow will never make antibodies to the ABO antigens they had previously due to tolerance, no matter how many enhancing techniques we try. Although these patients may be on immunosuppressants that is not causing the weak (actually absent) backtype. We know this because other bone marrow transplant patients that we know are equally immunosuppressed have perfectly normal back types. I am still not convinced that with all of the complexity of the immune system we can be sure that someone not making a strong back type is also not making IgG allo-antibodies. Even if they weren't, I think they would not destroy transfused cells very easily.

[David Saikin]

John,

Certainly the enhanced technique can be used for other patients but I would think that this would be a judgement call and not a routine occurence.

And if David hadn't got a valid ABO result he would be performing a further workup to investigate an apparent ABO discrepancy not simply an extended crossmatch.

The only thing I would do is try to enhance a "backtype" with either rt/cold temp or enzyme pretreatment. I would not, however, use those techniques on my xm . . . we would just perform the ahgxm. I think you are making a mountain out of a molehill. There are many causes of reduced abo abs - disease, age to name a few. There are ways to enhance the abo's - that's all I would try. I leave the absc study to the standard SOP.

[Eagle Eye]

same as David.

[StevenB]

Be thankful you have a negative antibody screen and move on...

[rravkin@aol.com]

David,

Thank you for your honest post. You are right, it is entirely possible that a mountain is potentially being made of a mole-hill but when the backtype enhancements are tried and do not enhance the reaction and the patient does not fall into the usual category of an immune-suppressed patient and there is no responsible history attainable for this patient, do we just kiss it up to God and hope for the best or should we be aa little more proactive?

The only thing I would do is try to enhance a "backtype" with either rt/cold temp or enzyme pretreatment. I would not, however, use those techniques on my xm . . . we would just perform the ahgxm. I think you are making a mountain out of a molehill. There are many causes of reduced abo abs - disease, age to name a few. There are ways to enhance the abo's - that's all I would try. I leave the absc study to the standard SOP.

[John Eggington]

David,

Thank you for your honest post. You are right, it is entirely possible that a mountain is potentially being made of a mole-hill but when the backtype enhancements are tried and do not enhance the reaction and the patient does not fall into the usual category of an immune-suppressed patient and there is no responsible history attainable for this patient, do we just kiss it up to God and hope for the best or should we be aa little more proactive?

This is not quite how the thread started. I think that most of us responded with respect to what we might 'routinely' do when we encountered a 'weak backgroup'. If we come across scenarios that do not 'add up', Im sure we all have a 'bag of tricks' we go to to further the investigation. I also bet all of our 'bags' are different!

[janet]

My two cents about worrying about what we might be missing:

-- all antibody screen methods have there good and bad qualities (eg. you may pick up an autoantibody in gel but when using less sensitive tube IAT the auto is gone and you still hope you have the sensitivity to pick up an alloantibody possibly underneath that auto

-- one method may be better for one antibody than another (eg. LISS and Anti-K .... I have suspected an anti-K in gel, increased the incubation time to 40 minutes and had those weak? reactions a 1+ eg. Anti-Jka in gel not detected by reference lab using PEG)

-- I recall "before my time" when screens were set up at 4, 20 and 37 degrees - looking for ANY possible antibody (in time it was realized these were a waste of resource for the rare time they might be significant)

-- progression to I.S. and now Computer Xmatch - we realize there may be units we would have had incompatible by IAT testing are being transfused but are more patients reacting since we went to these time/resource saving methods??

I recall a quote from a while back "medicine is not a perfect science and if you expect it to be you will be disappointed....we do the best we can with the resources we have" (or something like that).

[rravkin@aol.com]

Thank you John.

I guess I wanted to know the details of the "Bag of Tricks."

This is not quite how the thread started. I think that most of us responded with respect to what we might 'routinely' do when we encountered a 'weak backgroup'. If we come across scenarios that do not 'add up', Im sure we all have a 'bag of tricks' we go to to further the investigation. I also bet all of our 'bags' are different!

[John Eggington]

Thank you John.

I guess I wanted to know the details of the "Bag of Tricks."

Well now, that would be telling...!

[rravkin@aol.com]

Well John, thank you for sharing with us your insight. This is why we all joined PathLab Talk so that we can benefit from the wealth of knowledge and experience from interesting and insightful professionals like yourself.

Well now, that would be telling...!

[John Eggington]

Well John, thank you for sharing with us your insight. This is why we all joined PathLab Talk so that we can benefit from the wealth of knowledge and experience from interesting and insightful professionals like yourself.

Joke, for goodness sake!

[rravkin@aol.com]

Oh dear, John, don't get your nickers in a bunch!

Joke, for goodness sake!

[Abdulhameed Al-Attas]

NO further tests for the Abs.Screen,and by the way,This is a strange way of thinking! But the ABO discrepancy must be resolved by increasing the plasma ratio,4°c incubation or enzyme enhancement.

The immediate spin crossmatch is meant to detect ABO incompatibility. It can also detect cold reactive (clinically insignificant) antibodies that react at room temperature (RT).

If the patient's expected ABO antibodies are not reactive or weak at immediate spin,donor units should be ABO confirmed prior to testing with this method.

Edited August 27, 2012 by Abdulhameed Al-Attas

[John Eggington]

Oh dear, John, don't get your nickers in a bunch!

Well, as they obviously are 'bunched', I suppose I should actually say what I might do in this situation;

Firstly, 'weak' is fairly subjective. Variables might include the technique/technology you are using, how your automation is set up, who's performing the test, etc., etc., we all know them. So I'll define 'weak' as 'repeatedly unable to interpret ABO grouping by first line test and subsequent testin by alternate technique(s)' (I'm assuming the first line test will be some form of automated system).

More often than not our 'weak' results are resolved by applying the alternate technique. In my case this will be a 'tube' ABO group. I've worked with people who have variously advised the use of enzyme treated grouping cells, increasing the volume of patient serum/plasma, and lowering the incubation temperature, as additional ways to enhance reactivity.

So, the alternate technique gives 'strong' reactions; is the backgroup still 'weak' enough to require an enhanced antibody screen? We'll each have our own take on that! If it is decided that the alternative technique is still 'weak', and an enhanced antibody investigation is deemed necessary, my approach would be to use alternative techniques, rather than adjust the routine one. For example, you could apply a range of techniques in combination with one another; enzyme IAT, tube IAT (good for detecting weak anti-Fya and anti-Fyb, I find), reducing the temperature, selecting test cell phenotypes you think may offer more sensitivity than the screen profile (e.g., including a K+k- cell, if the screen is K+k+). Some of these techniques may enhance 'insignificant' antibodies but that is always the 'price' for applying enhancements. If these techniques failed to show anything of 'significance' I'd stop, relatively comfortable in the knowledge that if my routine technique (most of which seem almost too sensitive, as it is) and my enhanced combination had failed to find anything, then it's unlikely that anything of immediate significance is present.

Having said this, I would not do an 'enhanced work', on a patient with a 'weak' backgroup, unless the patients condition suggested that an undetectable, clinically significant, red cell antibody was thought to be present, and causing problems.

[rravkin@aol.com]

Thank you John, I greatly appreciate this post.

Well, as they obviously are 'bunched', I suppose I should actually say what I might do in this situation;

Firstly, 'weak' is fairly subjective. Variables might include the technique/technology you are using, how your automation is set up, who's performing the test, etc., etc., we all know them. So I'll define 'weak' as 'repeatedly unable to interpret ABO grouping by first line test and subsequent testin by alternate technique(s)' (I'm assuming the first line test will be some form of automated system).

More often than not our 'weak' results are resolved by applying the alternate technique. In my case this will be a 'tube' ABO group. I've worked with people who have variously advised the use of enzyme treated grouping cells, increasing the volume of patient serum/plasma, and lowering the incubation temperature, as additional ways to enhance reactivity.

So, the alternate technique gives 'strong' reactions; is the backgroup still 'weak' enough to require an enhanced antibody screen? We'll each have our own take on that! If it is decided that the alternative technique is still 'weak', and an enhanced antibody investigation is deemed necessary, my approach would be to use alternative techniques, rather than adjust the routine one. For example, you could apply a range of techniques in combination with one another; enzyme IAT, tube IAT (good for detecting weak anti-Fya and anti-Fyb, I find), reducing the temperature, selecting test cell phenotypes you think may offer more sensitivity than the screen profile (e.g., including a K+k- cell, if the screen is K+k+). Some of these techniques may enhance 'insignificant' antibodies but that is always the 'price' for applying enhancements. If these techniques failed to show anything of 'significance' I'd stop, relatively comfortable in the knowledge that if my routine technique (most of which seem almost too sensitive, as it is) and my enhanced combination had failed to find anything, then it's unlikely that anything of immediate significance is present.

Having said this, I would not do an 'enhanced work', on a patient with a 'weak' backgroup, unless the patients condition suggested that an undetectable, clinically significant, red cell antibody was thought to be present, and causing problems.

[rravkin@aol.com]

To all who posted here,

I just wanted to thank you all for your input. Although it was somewhat of a rough ride I walk away with a much better understanding of what we do and the limitations that exist in our techniques. I only was trying to inspire the kind of post that I eventually got from John Eggington. I in no way was trying to speak of changing our whole routine or instrututing any significant change there of and I really do not know how we managed down that path in the first place as I did state earlier in this thread. As far as the " strange way of thinking" from Abdulhameed, if you are refering to the fact that the backtype performed on the ProVue is not incubated and the ABSC is I can see where you would think this logic to be strange given that the incubation phase of the ABSC would greatly enhance reactivity over the virtual imediate spin of the backtype on the same instrument. It would be nice to further explain such remarks in the future as they may be misunderstood. I have done some resarch on Delayed Hemolytic Transfusion Reactions since starting this thread and I come to find that they can be every bit as severe as an Acute Reaction and therefore every bit as detrimental to our patients. I think it is important to maintain a back-up technique that is available if and when needed.

Thank you all again.

[Malcolm Needs ☆]

Sorry to a "Johnny come lately" to this thread, but I took my eye off the ball whilst I was on holiday.

I can't say that we are overly bothered by a weak back type in my own Reference Laboratory, as long as we are told of a diagnosis that may point to a reason (e.g. the patient is immunosuppressed following stem cell transplant); mind you, it would be nice to have any diagnosis written on our request forms some of the time!!!!!! That does not mean that we will not test at 4oC and/or use enzyme-treated reverse cells to try to demonstrate the missing anti-A and/or anti-B (using group O cells as a negative control - a MUST).

These days, however, from the point-of-view of grouping the patient, I would rather rely on the specificity and avidity of the modern monoclonal grouping reagents in the forward group, than getting a corresponding reverse group.

Turning to atypical alloantibodies, I must agree with many people who have contributed to this thread in saying that, if the screen is clear, using your normal techniques and technologies, I would not go to any great lengths in trying to enhance for any antibody that may or may not be present. I think that we often go far too far in trying to detect very weak antibodies (even with a nod of honour to those antibodies, such as anti-Jka, that can cause anamnestic responses), as the vast majority, even if they are boosted by a transfusion, will not cause a classic delayed haemolytic transfusion reaction, where there are clinically significant sequalae, but may just cause the transfused red cells to be removed from the circulation a little quicker than may be expected; if you like, something between a classic delayed haemolytic transfusion reaction and a delayed serological transfusion reaction, where there are no clinically significant sequalae whatsoever.

One must remember that many patients who are transfused leave hospital and are not seen again for a long time, and then come back several years (sometimes) later. There MUST have been some who have produced atypical alloantibodies following their initial transfusion, which we do not know about, because they have left hospital, who come back for their second visit requiring a transfusion, and we cannot demonstrate any atypical alloantibodies as being present at all. Such patients are eligible for electronic issue of blood, but we do not see a huge number of such patients (if any) keeling over following transfusion of antigen positive blood.

Just my (very late) thoughts.

[rravkin@aol.com]

I just wanted to post a correction to my most previous post where I state that the ABO/Rh typing card is performed as an immediate spin reading. It is actually spun the same time that the ABSC card is spun by program and therefore it incubates for approximately 10 mins at RT, close enough to the 15min RT inc performed with the ABO discrepancy workup. The fact that this card incubates at room temp for 10min would help increase reactivity forward and back and therefore lend to the idea that if the backtype comes up weakly reactive then, dare I claim that, it is possible that a false neg ABSC may occur. This is based on the idea that a patient with a healthy immune system will readily produce ABO antibodies incuring a strong reaction with our testing method and if that reaction is weak then one possibility is that the concentration of ABO antibodies is decreased do to lack of production and therefore any allogeneic antibody production would also be diminished such that it will not be detected with our ABSC method. Just some food for thought; lets not get crazy now.

Edited September 4, 2012 by rravkin@aol.com