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ECHO D typing Discrepancy


BB1956
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I searched the forum and read some discrepancy info from back in 2013 and 2014 but most of those patients had history of transfusion.  Two days ago we had a prenatal T&S.  No blood transfusions involved.  We had no previous history of type or transfusions on file.  The sample was tested on the ECHO and clearly typed as D negative with no discrepancies or concerns with both Anti-D reagents.  Patient was reported as D negative.  The same day we receive a call from the patient's midwife stating the patient has a history of being B Positive.  We pulled the original sample and repeated testing on the ECHO.  The repeat testing resulted in ?  with at least one of the Anti-D reagents and resulted as NTD. (No type determined).  We proceeded to perform tube as well as ECHO weak D testing.  Patient was 2+ positive in tube with AHG phase using the exact same reagents used on the ECHO and 4+ positive with both Anti- D reagents ECHO weak D.  My question is why did the initial test not react?  If this was a particular variant etc., I could chalk it down to method or reagent but having one result on the same sample tested on the same day and then finding another result hours later when repeated does leave me wondering.  Any ideas??  I will say the sample was EDTA and was refrigerated for several hours prior to the repeat testing.

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I doubt if this is the answer, but just to put it into the "public domain" as it were, you are saying that the sample was an EDTA, kept at around 4oC, were the anti-D reagents also kept at that temperature, or were they brought to whatever room temperature may be in your laboratory?  The reason I ask is as follows:

Thorpe et al have reported that monoclonal anti-D molecules possess a V4-34 moiety, that is also present in anti-I and  anti-i.   As a result, if papain-treated D- red cells are tested with such antisera, or untreated D- red cells are tested with such antisera that have not been brought to room temperature, they may agglutinate.  This could result in D- red cells being mistyped as D+ - a particular danger in females of child-bearing potential.  Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellerberg MB, Natvig JB, Thompson KM.  Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.  Transfusion 1997; 37: 1111-1116.  Thorpe SJ, Ball C, Fox B, Thompson KM, Thorpe R, Bristow A.  Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal  anti-D: the same framework 1 residues are required for both activities.  Transfusion 2008; 48: 930-940.

As I say, I doubt if this is the answer, but just to put it out there for colleagues who do not know about this phenomenon.  It is MUCH more likely to be something else that, at the moment has escaped me (as my mind is still on zebras, rather than horses John C. Staley!!!!!!!!!!).

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I worked at a hospital that used the D4 and D5 on the ECHO and then Ortho Anti-D on the bench because there were instances of the same thing occurring.  If there was a ? for either D4/D5 on the ECHO or a physician reported a discrepancy we would run it in tube using the Ortho Anti-D.

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If the tube testing showed 2+ or less reactivity with anti-D reagents (same as used on Echo) I would suspect that the reason for the negative results on the Echo is that the reaction was shaken away during the resuspension step of the testing.

Remember that Echo has an algorithm that it follows for resuspension. Shake so many times, swirl so many times etc. This can cause weak reactions (their limitations and warnings state 1+ or less) to be interpreted as negative by the instrument.

Techs who are resuspending a button in a tube are visually looking for an end point and immediately stop shaking the tube when the cell button is resuspended whether that takes 5 shakes/swirls or 15 shakes/swirls......Echo can't read for an endpoint in that same way, it must follow it's algorithm. Additionally techs consciously or unconsciously will adjust the intensity of their shaking in response to what they are visualizing. A good way to demonstrate this is to take that same specimen and have a tech who doesn't know what the reactions have been resuspend it with their eyes closed. Tell them to shake it for say 15 seconds and then see what the reaction looks like. It's a fun experiment.

Having said all of that.....if you see this often you can always have your FSE come in and adjust the resuspension step.

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Thanks for some great ideas!  The suggestion that the EHCO is programmed to shake for a specified period which "shook off" the weak reaction producing the initial Rh negative result is very plausible however why then did it not repeat itself when we retested the sample?  Upon repeat after some time refrigerated the ECHO detected enough to provide us with the ?.  Call me crazy but I like reproducibility in my instrumentation! Instrumentation is great but I still say it is hard to beat an experienced Blood Banker on the bench.

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12 minutes ago, marvy1 said:

I have seen a Rh positive patient transfused 3 Rh negative units and subsequently tested on the Echo. Echo reacts negative with both Anti-D4 and D5. When taken to the tube, get a 2-3+ mf reactivity with Anti-D reagents.

That is a very good point marvy1.  A few of my colleagues have noted this phenomenon.  As I understand it, it is all to do with where the probe of the automation samples the red cells, as opposed to where the human "tends" to sample the red cells - the former from the top, the latter from the bottom.

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23 hours ago, BB1956 said:

Thanks for some great ideas!  The suggestion that the EHCO is programmed to shake for a specified period which "shook off" the weak reaction producing the initial Rh negative result is very plausible however why then did it not repeat itself when we retested the sample?  Upon repeat after some time refrigerated the ECHO detected enough to provide us with the ?.  Call me crazy but I like reproducibility in my instrumentation! Instrumentation is great but I still say it is hard to beat an experienced Blood Banker on the bench.

Without seeing the well images and/or the reaction strengths the Echo uses for grading reactions I can't say. Did the "0" result look at all grainy? Since the reaction strength for a "0" (Neg) is 0-2 and for a "?" is 3-9 perhaps your patient might have fallen at the upper end of "0" and the lower end of "?". I like reproducibility in instrumentation also but you will get variation at the low/high ends of cut-off ranges in all instruments. Would you have been as concerned if the difference was between a 1+ and 2+ reaction? Also remember that Immucor recommends reviewing all reactions prior to reporting your results just to make sure that it's not reporting a very weak reaction as negative. I'm not saying that happened in your case but it has been reported to happen. And remember, if the instruments were perfect all of the time they wouldn't need the techs LOL!

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I have the initial EHCO printout which does not look grainy for the individual reactions but I will go back and check the reaction strength on the instrument for both runs to see how that compares.  You could be absolutely correct.  There has to be a break point somewhere and it could be that these samples just might have fallen into those areas. I do have techs review the reactions before reporting and I don't see anything on the printout that would have caused much alarm.   Unfortunately it is difficult to explain all of this to a physician or mid-wife to get them to understand why you "mistyped" their patient.

It is good to keep in mind  the probe sampling position in discrepancies with transfused patients. Should this occur again, it will be one of my considerations.   In this case my patient had not been transfused.  We learn  much from each other and I do appreciate this forum.

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Remember also that this patient could be a variant or weak D. Those three anti-D reagents use different clones so you may see different reactivity depending on the antigen variant. The previous result that the midwife was talking about could have been from entirely different reagents or the lab reporting the original results was reporting any/all reactivity as Rh positive instead of following the current suggested guidelines for reporting Rh types. We report anyone who does not react >2+ by tube testing with all three of the anti-D reagents we use as Rh negative. In the case of an OB patient molecular testing is the only good way to resolve the puzzle.

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I would also go for the Variant D idea.  On the Echo, you had once a neg and once a ? result.  Well, the ? could be due to something other than a true reaction; or it might be that the particular D variant is detected by that clone but that the number of antigen binding sites is at the absolute limit of detection so that tiny differences in pipetting (say, in the length of time the cells were in contact with the antiserum, the exact cell suspension) could explain a difference between neg and ?.  If the tube testing is done with a different clone, no problem at all in explaining that difference. She should of course be treated as Dneg until the molecular biology sorts it out.  (Maybe she was B+ as a donor???

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  • 2 weeks later...

We reported the patient as a weak D positive patient.  Due to cost cutting we no longer have a different Anti D sera on site.  We use the same reagents on the ECHO and for Tube typing. That is why I was concerned when we were able to pick up the D in tube testing using the same lot number of Anti-D typing sera and we did not even see a ? on the original ECHO run.  We are a community hospital and most of the time there are no problems.  Cost vs Benefit indicates the few patients that have typing problems are more cost effective to send out for resolution.  Gone are the days we can do our own work ups I'm afraid.  In this case it was safe to call the patient Rh negative for transfusion purposes.  Since she is prenatal the physician can order molecular studies to get a more accurate picture.  Thanks for all of your help.  Some great ideas!

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We had discrepant results recently when Echo results were compared to Ortho gel results when a sister hospital was in the process of switching from the Echo to the Vision.  This doesn't answer why you got different results on the same specimen, but may explain why the patient was positive on a previous specimen.  We have seen a number of weak or partial D patients test differently on the Echo vs. gel.

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