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CAP today article about RHD genotyping


YorkshireExile

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I`ve just read a new article in CAP Today concerning the RHD genotyping of Rh Negative patients.

 

Link to the article is : (hope the link works)

 

http://www.captodayonline.com/groups-urge-phase-rhd-genotyping/

 

What do people think about this? Is anyone sending samples from Rh-negative patients for RHD genotyping as a routine?

Is anyone thinking about doing this? Do many labs do no further work-up on a Rh negative pregnant patient?

 

My lab does a Biorad Weak D confirmation test only on Rh Negative cord blood samples, not on routine antenatal patients. We use a biorad grouping card on our antenatal patients that will show a weak agglutination with anti-D if the D antigen is weak, so we would just say that the patient is actually weak D positive, but would treat the patient as Rh Negative with regards to transfusions or Rh immune globulin. Of course, we don`t know for sure if it is a weak D, or a variant D etc.

 

Am I wasting my Rh Negative blood stocks and Rh immune globulin as stated in the article? Should genotyping become routine?

 

 

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I would agree that for patients this is overkill.  If they are SO weak that they don't come up in gel (and I can only talk for the Biorad cards but they can usually detect down to about 600 D-sites per cell - and the anti-D that you are using, Yorkshire exile, will go down even further) then I would not want to transfuse D+ anyway. What would you actually do with the result?? For cord bloods, well - is a cord blood with so few D sites likely to immunise a Dneg mum?  Donors is another question altogether.  I can well see that soon donors will be fully genotyped and then D will just be another antigen amongst many.  Currently though, there are papers that showed that a number of Del donors was being missed each year.  I also believe that Switzerland, who has been routinely typing their donors for about 2 years, have also found a couple.  Very interesting, academically, but it has pushed up the price of a unit of blood quite a bit and there's no actual evidence that they have actually immunised anyone, as far as I know.  I just get a bit upset sometimes when we in the developed world go to extraordinary lengths to maybe reduce the already negligible risk down further when many countries in the developing world don't have the resources to do more than an ABD slide - if they have the blood in the first place.

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it is in our proceudre to send out a weak reacting (<2+) in tube anti-D if the patient is a woman of childbearing age.  They are treated as Rh negative and recieve Rh Immune Globulin until the testing comes back to confirm the patient's blood group.

FYI - we haven't yet had to send one out!

s

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We haven't implemented yet. We're still getting charge codes built into the system. For us another tricky part is trying to figure out how we'll address the serological results that interpret one way and the blood type interpretation that goes another, from a workflow perspective.

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Well, of course, there's a huge difference in the reactions you will get in tube and those you get in gel.  Normal tube (I am not talking by IAT here) is SO much less sensitive than gel, that the problem here is not that there is patient risk, but you will be wasting a huge amount of Dneg blood by not going on to do further testing. 

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I've sent out 3 whole patients in the last 2 years. None of them were a variant that's 'probably' going to make anti-D. We treat these patients as Rh negative and recommend RhoGAM, if applicable. I'm not too worried about the blood supply - 3 patients out of all the folks we typed w/o issue is not a large number, though in the big picture it could start adding up. My medical director is still feeling the need to be conservative and not take the risk - he wants to see more data in the US before we stop calling these folks Rh neg. The threat of malpractice lawsuits looms large. We also need to see much cheaper pricing for this type of testing before we use it routinely. At this point there is no 'added value' for the patient if we do it on everyone.

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Yes, I inquired at AABB why they specified 2+ at immediate spin when so many of us are not doing tube testing anymore. No answer.

Isn't it based on the UMich study? 1+ tubes/2+ gel were recommended for interpretation as Rh-neg/further investigation.

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It is great to see consensus from these six important groups involved with clinical transfusion medicine practice.  

 

Researchers are already working towards the point-of-care style platforms mentioned in the article, and I suspect it is not as far in the future as one might think.

 

I think genotyping should become routine.  Personally, I don't understand why we have been trying to approach RhD as simply positive or negative.  Transfusion medicine professionals, whether bench techs or physicians, are smart enough to handle the complexity of RhD in particular without putting blinders on and pretending D variants don't exist. (Here, I'm referring to the article where it says "some labs conduct the test for weak D serological testing for women when they’re patients, but then, no matter what the result is, report it as Rh-negative".) 

 

Maybe with routine molecular testing available, we can change the way we think and talk about RhD and incorporate the true complexity of the situation into transfusion practice to improve patient treatment on both individual and systemic levels.  That said, routine molecular testing does not mean abandonment of serological testing.  Each has a niche and they complement each other well.

Edited by Kellar
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I agree that we should head this way, and obviously OB/Gyn physicians need more education on this issue. Most of them use other laboratories for their prenatal testing, and then when the patients arrive here to deliver, we get different results from the other Laboratory and then we are recommending RhIg based on weak D results (or not, based on our results with our methodology) and it is not taken very well.

I think the problem for us is that we do not have molecular testing available, so we send our samples out to the reference lab for Rh genotype testing. The results do not come back for 72 hours, so we are still giving the RhIg as the results are still pending. It will help for future pregnancies though, as long as the mom comes back to this hospital.

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 don't understand why we have been trying to approach RhD as simply positive or negative.  

 

But it is either positive or negative reactivity-wise, it is only degree of reaction that cause an issue. If a patient is clearly Rh negative in gel then I can't see the need in the expenditure - a 2+ reaction, yes, but a clear-cut 0?

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But it is either positive or negative reactivity-wise, it is only degree of reaction that cause an issue. If a patient is clearly Rh negative in gel then I can't see the need in the expenditure - a 2+ reaction, yes, but a clear-cut 0?

 

It is the weak reactions and discrepancies that I was referring to, which are the ones that the article proposes for routine molecular testing.  We have been trying to shove the proverbial square peg (D variants) into a round hole (positive vs. negative phenotype).  My point was that molecular testing provides another option, one that works well in conjunction with current methods.  There may come a day when molecular testing becomes superior to serology for all applications in transfusion medicine.  We aren't there yet, but that doesn't mean we should ignore molecular methods completely until then. 

Edited by Kellar
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I agree 100% with you there Kellar, but, as things stand, even a full RHD gene sequence will not definitively tell you whether or not an individual with a particular Weak D genotype (and, by inference, the phenotype) will, or will not be capable of producing an alloanti-D.

 

Obviously, if the amino acid residue substitution is predicted to be in one of the extracellular loops, we are looking at a probable Partial D, and, therefore, there is a very good chance that an alloanti-D can be produced, but even if the amino acid residue substitution is predicted to be in either the intramembranous area of the protein molecule or the cytosol area of the protein molecule, there is no guarantee that the individual will not produce an alloanti-D - witness such individuals as Weak D Type 53 who have produced an alloanti-D.

 

It's a proverbial "can of worms".

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My understanding of this issue is that when a "weak D" reaction (ie 2+ or less) is obtained the reaction needs to be investigated further to distinguish whether the patient had a subtype of D that is I, II, or III vs IV, V or VI.  Subtype I, II, III can be considered Rh +, would not need RhD immune globulin, and could receive D+ RBCs.  Subtypes IV, V, VI would.  If the donor is any other D variant then clinical judgment would need to prevail regarding the necessity for either RhD immune globulin or D+ blood. 

 

For me the issue is regarding the outcomes.  Would the patient outcomes be better, worse or the same after going through this exercise?  Right now it seems that there is another branch in the clinicians decision tree, and another place for an error.  I believe that one presentation suggested this process could free up about 50,000 Rh negative units annually in the US.

 

Regarding Molecular testing in general, I think that it is a wonderful tool.  As it pertains to this specific problem I am  not sure it is cost effective. 

Edited by Kip Kuttner
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so why stop at genotyping the weak D reactions, if we are talking patients?   there are many partial Ds out there, capable of producing anti-D, who give 4+ (i.e. normal strong) positive reactions with most anti-D reagents.  If we are really talking patient safety, it makes more sense to genotype the 'normal' D+ results....

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I've seen more sensitizations to other blood groups systems from a single transfusion than I have Rh(o)D sensitizations during the course of my career. It would be interesting if someone (namely one of the larger, prestigious institutions) did some kind of elaborate study to see the D sensitization rate among weak D individuals. It seems like spending an awful lot of $$$ for a very small gain - unless we are going to provide Rh, Kell, Duffy, Kidd, and Ss phenotype specific rbcs for all. As I intimated above, I've sensitized folks to other ags more than I have to the D - and usually with just one transfusion.

Let's see some hard data on this stuff.

Edited by David Saikin
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  • 4 weeks later...

I have been following this a little due the the fact that I came from a place that did Ortho Gel ABO to a place that we only do tube ABO.

 

I was going to switch us to gel and started validating the cards, but had a couple that would give me negative in the tube and then positive (3 or 4+ would never question if it was weak) in the gel card. I did the weak D in the tube and that was positive. This got me questioning and learning more about all this. I still dont quite understand thou!! At this point, with everything going on molecularly and the articles out there, I am just going to keep us at tube. We do not do the weak D normally. I called a hosptial close by to us to see if they had these patients on file, they use gel. One they did and they call that patient D positive. I was not able to send this for molecular testing due to my vito by management. I did however have 4 different manufacturer anti-D (collected from friends :) )and all tube with those were D negative.

 

 

I have wrote Ortho asking for a little explanation: They also refered me to an article I attached.

 

"Based on the monoclonal anti-D present in ID-MTS Gel and the handful of studies reported in the literature, the following seems to describe this reagent’s reactivity with uncommon Rh phenotypes:

  • Category VI partial D does not react
  • Weak D Type 2 is very reactive, may be 2+s in ID-MTS Gel vs neg, +/-, or 1+w in tube, depending on reagent anti-D used and other variables of traditional tube testing.

 

I’ve not seen sufficient data for any further characterizations of this reagent.

 

However, there are ample studies to suggest that any currently licensed anti-D reagent in the U.S. can give strong reactivity (≥ 2+) with some very rare partial D phenotypes that are at risk of immunization to the missing epitopes.    One anti-D reagent may even react up to 4+ with a low incidence antigen that is not a part of D.

 

This is certainly a current hot topic.  As molecular testing becomes more refined, we should be able to better understand and predict the behavior of the various reagent antisera, but for now, our knowledge is somewhat limited."

 

When I was working at U of Michigan, we decided to treat patients that reacted 1+ or less with anti-D by direct agglutination as though they were Rh Negative.   This was strictly tube testing back then.  Our concerns were not just that weak reactivity tends to increase the statistical potential for a partial D, but also because of concerns about fibrin, rouleaux, spontaneous agglutination from autoantibodies, overreading, under shaking, etc.   Gel certainly lessens some of these concerns, but it still may be prudent to be conservative and treat such patients as Rh Negative for the purposes of transfusion or Rh IG therapy."

 

http://www.aabb.org/annual-meeting/attend/2014/Pages/Using-Molecular-Methods-to-Resolve-Weak-D-Phenotypes.aspx

 

this link had some good info

Can anyone chime in on this? Do we need more standardiation of method(s)? Are the gel D positive results acceptable for the new recommened guidelines? Is the tube is too weak? My mind has went around and around on this!!

 

 

Immunohematoloty Vol 21 No 4 - Judd Westhoff 11-4-2005.pdf

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At a RAP session on this at the AABB meeting, it was clarified that Rh genotyping is being recommended for patients with child-bearing potential who react weakly in gel or in tube testing performed at immediate spin.  They are not recommending that we go back to doing D testing through the AHG phase on patients who test negative at IS in tube.  The genotyping test they recommend is somewhat limited (looks mostly for the type 1,2 & 3 weak Ds I think) and is supposed to cost about $250 as I recall.

 

They are aware of the other situations which may benefit from genotyping (like partial D III) but they wanted to start with the above definition as something that is recognizable at almost any lab.  Doing genotyping on these patients will help the labs doing the testing to ramp up their production so maybe with the economies of scale, the price of testing can come down.

 

I am not sure what impact the proposed new rules about Laboratory Defined Tests will have because apparently most of these genotyping methods fall under this FDA definition. The recommendation to do this testing is scheduled to go to the ACOG meeting next May or June for their approval so we may need to be ready with a plan by then.

 

One big concern I have is: the cost-effectiveness is dependent on the patient being genotyped only once and that record following her everywhere she goes.  We can't even keep patients straight in our own admissions database what with name changes and the like, how on earth would we deal with this for a patient who may switch labs, doctors, hospitals and even national regions?  And then there is the accuracy of specimen collection and labeling.  I guess we could solve that by making sure that we get a new specimen to send out for genotyping and making sure it still gives unusual typing results before we send it out.  That's less convenient for the patient.  This whole deal would work much better if we had a national health care number assigned to each patient and if doctor's charts had a place for lifelong information to be displayed where it might be noticed on later visits rather than lost in old lab results.

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