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Using enzyme treatment to resolve non-specific results


SMILLER
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I have read an article from last year about a lab that routinely runs a gel enzyme panel when they get equivocal results (i.e. antibodies of undetermined significance, AUS's, non-specific reactions) on gel screens or panels.  About 25% of the time they identified significant allo-antibodies that otherwise would have been missed as they would  have been ruled-out on the regular panels.  Does anyone else do this?

https://academic.oup.com/labmed/article/48/1/24/2666003

Thanks, Scott

 

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We automatically performed a papain-IAT panel by DiaMed/BioRad gel technique, in addition to an IAT panel by DiaMed/BioRad gel technique on all samples, but that is in a Reference Laboratory.

I cannot remember ever detecting a clinically significant antibody by doing this, which we did not find by any other techniques (although, of course, we had access to a huge inventory of very rare red cells and antibodies).

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Right.  That's what I am wondering.  In the article I cited, they looked at about 100 specimens that they had equivocal results in gel.  Out of these, about 25 turned to have significant allo-antibodies that would have been  missed if the initial non-specific panel results were ignored as not significant.  Notably, some of those patients had things like anti-E where even homozygous cells were negative intiailly.

The thing is, no one goes any further with a negative gel screen (including this studies facility), so there is no way of knowing how many "negative" screens results are reported on patients who may have significant amounts of allo-antibody.  (Likewise, you have to consider how to detect those antigens that are destroyed by ficin!)

Scott

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The word 'significant' is interesting in this context.  In most of -Europe antibodies that are detected only in enzymes, including the enzyme-only anti-Es and -Cws would NOT be considered significant and most of the time would not be detected in the first place.  Nor would the numerous anti-Lea, -Leb and -P1 that you would pick up.  Always assuming that you are working with a sensitive IAT in the first place, of course.

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I think one would have to realise that VERY few examples of "enzyme-only" antibodies recorded in the literature that have proved to be clinically significant, which is why, as Anna says, in most countries, it is no longer a requirement to use enzyme-treated red cells in the antibody screen.

Of MUCH more importance are any clinically significant antibodies directed against antigens that are denatured by certain enzymes (for example, the Fya antigen being denatured by either of the two most commonly used proteolytic enzymes in transfusion laboratories; namely ficin and papain), and which may be "masked" by the antibody being described in this thread (i.e. antibodies of undetermined significance, AUS's, non-specific reactions).  Most clinically significant antibodies are either quite avid, or have a high titre, and so one way of ruling them out is to titrate the plasma until the known (but unidentified) antibody is either no longer detected, or is only detected very weakly, and then test it for the presence of clinically significant antibodies.  I know of some laboratories that do this, but it is certainly not a technique that I would recommend under any, but the most urgent circumstances.  If, for example, I had an anti-Inb in my circulation, in addition to the sort of antibody being described in this thread (seriously unlikely, as, not only am I of White European ethnicity, rather than of South Asian, Iranian or Arab ethnicity - and I also happen to know that I am In(a-b+), as my blood has been tested - by Joyce Poole, and I am quite prepared to believe her results!!!!!), and this clinically significant specificity was missed because of using the "titration technique", assuming that I survived, I would be straight down to my legal representative at the earliest opportunity!

Therefore, I would recommend that, in all cases, a sample is sent to the reference laboratory (and this opinion is NOT formed by the fact that I used to work in a reference laboratory, but is an honestly held view).

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So it makes me think about delayed hemolytic transfusion reactions.  If these "enzyme-ID'd-only" antibodies are due to a low titre, can we say that they are insignificant?  If a Jka antigen negative person has been exposed to Jka, and has a titre so low that it cannot be picked up with a regular gel screen, does that mean a DHT is unlikely?  Or, in spite of the initial low titre, can those nasty memory B-cells crank out enough anti-Jka in the hours or days to come to cause a DHT?

Thanks, Scott

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Good point indeed.  Yes indeed, Kidd antibodies are renowned for causing delayed transfusion reactions via an anamnestic response, BUT, having been said, the most sensitive way to detect a Kidd antibody (if, indeed, it is not so weak that it cannot be detected by routine serological techniques) is by using red cells that are treated with proteolytic enzymes, and so, as I posted earlier, as long as enzyme-treated red cells are used in addition to the straightforward IAT in antibody identification, such an antibody specificity should be detected (with the earlier caveat).

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So in that case, the point of the article seems valid.  Perhaps enzyme-treated panels (and screens?) should be run routinely by transfusion services (hospitals), not just on those "problem specimens" sent to reference labs. It might be treasonable to run on patients known to be negative for things like Kell or Kidd antigens, and to have been transfused in the past?

Scott

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I see from where you are coming Scott, but you would have to perform a cost/benefit analysis on whether or not it is worth including an "enzyme screen" (and I doubt if things have changed since this was last done, because, if anything, indirect antiglobulin techniques, with untreated red cells, have become more and more sensitive) it is not worth it.

On the other hand, as far as panels are concerned, I have never understood why people would "drop" this test.  It is such a useful test, particularly when there is a mixture of antibody specificities.

One thing that is definitely TREASONABLE is having patients, commonly, who are either Ko or Jk;-3!!!!!  I TRUST you mean K- and/or Jk(a-)?  Come on my friend - you KNOW I am a pedant!!!!!  :haha::haha::haha::haha::haha:

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I suspect that routine use of enzyme-treated cells (in IAT) by "Non-reference Laboratory Staff" would cause more confusion than it would solve. Even the largest, most proficient hospital laboratory doesn't have high caliber serologists available on all shifts. I would suggest that tests with enzyme-treated cells be restricted to more difficult serological pictures, e.g., post-transfusion hemolysis without obvious cause (read "anti-Jka or anti-Jkb"), or for investigation of antibodies to high-incidence antigens.

I also suspect that many of the "enzyme-only" specificities have a major IgM component - notoriously difficult to detect by CAT (gel).

Just my two cents/pennies.☺

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The way I read the article; the lab has a policy of only running the ficin panel if they cannot clearly explain the reactivity, which seems like a good idea only if the ficin panel clarifies the reactivity. The article states that this occurred frequently enough that it was cost and time effective as fewer samples were sent to their reference lab.  I assume that if the ficin panel does not clarify the pattern more confusion results, as exlimey said, and they send the sample to the reference lab. So sounds a reasonable policy if the techs can interpret the results correctly.

The article also used an example of identifying an anti-C and anti-e in a sickle patient; most transfusion services (several threads on this site) match Rh and K for these patients.

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50 minutes ago, Ensis01 said:

The article also used an example of identifying an anti-C and anti-e in a sickle patient; most transfusion services (several threads on this site) match Rh and K for these patients.

I agree entirely with what you posted Ensis01, however, I am prepared to make a small wager that the anti-C and anti-e described in the article, found in a sickle patient, was actually an anti-hrB.  Classically, an anti-hrB mimics an anti-C+e (in the same way that anti-hrS classically mimics anti-ce/anti-f).  If I did win my wager, the patient would have a mutated RHce gene, and even if he/she had been transfused with Ro red cells from the start (and so probably matching the apparent Rh type), an anti-hrB would probably still have been formed (see, for example, Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff CM.  High prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from Rh-matched minority donors.  Blood 2013; 122: 1062-1071; doi: https://doi.org/10.1182/blood-2013-03-490623).

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18 hours ago, Malcolm Needs said:

I agree entirely with what you posted Ensis01, however, I am prepared to make a small wager that the anti-C and anti-e described in the article, found in a sickle patient, was actually an anti-hrB.  Classically, an anti-hrB mimics an anti-C+e (in the same way that anti-hrS classically mimics anti-ce/anti-f).  If I did win my wager, the patient would have a mutated RHce gene, and even if he/she had been transfused with Ro red cells from the start (and so probably matching the apparent Rh type), an anti-hrB would probably still have been formed (see, for example, Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff CM.  High prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from Rh-matched minority donors.  Blood 2013; 122: 1062-1071; doi: https://doi.org/10.1182/blood-2013-03-490623).

OK playing devils advocate for fun: the anti-C and anti-e were identified as allo-antibodies, as the article reported that the patient lacked the antigens, so phenotype R2R2. If the policy was to give patient's Rh and K matched RBC, then exposure to HrB (and HrS) would have been avoided (I can not figure out the frequencies of RBC that are e neg and HrB pos and Jeff Daniels was making my head spin, again).

Blood 2013; 122: 1062-1071: I like these types of paper as they empirically, and definitively show the complexity of transfusion medicine and in this case of the Rh system, which reminds me how much I have to learn. Correct me if I am wrong please, but it seems that the Bioarray assay shows genetic mutations but does not identify some antigens, for example it does not seem to separate the e antigen mosaic and identify HrB and HrS, which the newer assays can. So my question is how many cases of alloimmunization in sickle cell disease the paper describes over the 13 year period would have been avoided if our current knowledge of the Rh system was utilized? Ask the same question in 5 years time!

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22 minutes ago, Ensis01 said:

OK playing devils advocate for fun: the anti-C and anti-e were identified as allo-antibodies, as the article reported that the patient lacked the antigens, so phenotype R2R2. If the policy was to give patient's Rh and K matched RBC, then exposure to HrB (and HrS) would have been avoided (I can not figure out the frequencies of RBC that are e neg and HrB pos and Jeff Daniels was making my head spin, again).

Blood 2013; 122: 1062-1071: I like these types of paper as they empirically, and definitively show the complexity of transfusion medicine and in this case of the Rh system, which reminds me how much I have to learn. Correct me if I am wrong please, but it seems that the Bioarray assay shows genetic mutations but does not identify some antigens, for example it does not seem to separate the e antigen mosaic and identify HrB and HrS, which the newer assays can. So my question is how many cases of alloimmunization in sickle cell disease the paper describes over the 13 year period would have been avoided if our current knowledge of the Rh system was utilized? Ask the same question in 5 years time!

Three things I would say about this.

Firstly, I would have lost my money, as I had not read that the patient was C-, e-.  Lesson for me; read articles properly before commenting!

Secondly, I was most certainly NOT talking about either the HrB or the HrS antigens and antibodies.  These antibodies are seriously clinically significant.  I was talking about hrB and hrS antigens and their antibodies.  These antibodies are much less clinically significant, and usually (not always, but usually) only result in very mild, delayed haemolytic transfusion reactions.  It is somewhat typical of the nomenclature of antigens and antibodies (before the ISBT grabbed it by the throat and sorted it out a bit) that there should be antigens and antibodies so similarly named that such a mistake is easily, and commonly made - so I don't blame you!

Thirdly, as for the paper, you would have to ask the authors, but I did hear Connie Westoff give a splendid lecture on the subject at the retirement symposium of Geoff Daniels and Marion Scott, and she implied that the problem was not so much being unable to detect the mutations in the RH genes of the patients, but more identifying matching mutations in the donor population; that is certainly the case in the UK.

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  • 6 months later...
On 5/21/2018 at 5:17 AM, Malcolm Needs said:

We automatically performed a papain-IAT panel by DiaMed/BioRad gel technique, in addition to an IAT panel by DiaMed/BioRad gel technique on all samples, but that is in a Reference Laboratory.

I cannot remember ever detecting a clinically significant antibody by doing this, which we did not find by any other techniques (although, of course, we had access to a huge inventory of very rare red cells and antibodies).

Can you tell me where to buy Glycine soja lectin to prove the effectiveness of enzyme treated red cells. One company did not sell this product kit anymore. 

Doc1.pdf

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