Solid Phase Dependent Antibodies

[bloodybanker]

Hello to my fellow techs!  Glad to have found this group.

 

I have a strong blood bank background having worked in busy transfusion centers/hospitals, but am now working in a small hospital lab that has a very small workload.  The lead tech here doesn't even have a lot of experience.  About 3 years ago, right when I began working here, the blood bank moved from gel to solid phase.  The ECHO has been our primary method since then.  At the beginning, we did not know what was causing what we now call a solid phase dependent antibody (all cells positive in solid phase, neg in tube.)  My understanding is that it most likely an HLA antibody?  Can someone confirm this for me?  Reacting to RBC stroma that is not normally exposed on the cell surface?  (Never worked with solid phase before I came here.)

 

I'm not sure I agree with our lead tech on some issues.  I just do not think she graps all blood bank theory 100% (She thinks that a patient's ag type can change...)  My current concern with the solid phase dependent antibodies is the fact that she is telling me that "SPDA may be weakly positive on 1 cell also."  Meaning, one cell is weakly positive (2+ or less according to her) and the other 2 cells are negative.  If this same specimen is negative in tube and has a negative DAT, then it is a SPDA.  I want to know how one can tell if it is not really clinically significant.  The patient may be producing a new antibody that solid phase is picking up but tube (PeG or LISS) cannot.  (Because solid phase is more sensitive, right?)   If this is the case, and all antigens are rulled out due to nonspecific reactions, I think they need to be given solid phase, crossmatch compatible blood.

 

Can someone comment on this and let me know if I'm way off base?  Also, does anyone know of any scientific articles written concerning SPDAs and their source?  Thanks!  I'm so glad to have other blood bankers to bounce ideas off of!

[EDibble]

Great questions.

 

We have had the ECHO for many years now, and see this phenomenon fairly often.

 

We have a flowchart to determine the steps to take. This is the short version:

 

If the patient reacts on the ECHO in all screening cells, and they have no antibody history, we do an ECHO panel. There could be an antibody to a high frequency antigen, or a mixture of multiple antibodies causing all screening cells to react. If the panel is also all positive, we do a tube screen and Auto with PEG, our backup method.

 

If the tube screening and Auto are negative, end of story. Report screening as negative.

 

Even Immucor says they do not know why this happens with some patients, but the most recent theory is that these patients react with the "glue" that holds the rbc antigens to the walls of the sample wells.

 

I am very concerned about your coworker's thought process. You are absolutely correct that a newly developing antibody, or an old one with a low titer can react with the Capture method and be negative in tube. Just running tubes to "make the reaction go away" is very dangerous. Almost always, a patient with this non specific type reaction will react in all the wells of a capture strip.

 

Good luck,

 

Beth

[AMcCord]

The Echo is very sensitive, so you will see 'stuff' you wouldn't see with tube testing that is garbage. You will also see (and identify) weakly reactive antibodies that you can't pick up with tube testing. I've seen various explanations for the non-specific reactivity - possible HLA antigens, crypt antigens exposed when the red cells are bound to the test wells and reativity to reagents rather than red cell antigens - but there are no solid answers right now. There are fewer non-specific reactions currently, as compared to the past, so the last rework Immucor did to address the problem of non-specific reactions seems to have helped.

 

We handle the question of significant vs non-significant very much like Beth does. Run the Echo panels and see what you get. If all solid phase tested cells are not positive, try to rule out all clinically significant antibodies. Look for a pattern - are all the reactive cells positive for the same antigen? If there is no pattern, at this point we document a solid phase reactive antibody of undetermined specificity in our patient record. Next we run a tube/PeG screen and if it's all negative, then we report the screen as negative. I would most definitely not ignore a screen because one of three cells is weakly positive - you wouldn't do that with a manual screen would you? That's scary. If you have a weak anti-K for example, you've just ignored it. Solid phase crossmatch compatible blood for transfusion is recommended by the Immucor technical specialists I've talked to about this. Obviously you can't do this if all cells tested are reacting, but for the rest of your patients it's recommended.

[David Saikin]

There is a similar phenomenon in gel.

[tburl]

We have had the same issues and are looking at using gel as our backup instead of test tubes.  We have found in a small comparison so far that most are negative in gel but we have found true antibodies in gel that were negative in PEG and LISS and that gave no apparent pattern with solid phase even with homozygous cells that turned out to be Fy, E antibodies.  I have attached some literature comparing SP to GEL (one includes comparision to PEG).

Cleveland Clinic_final.pdf

ECHOSP 240.doc

ECHOSP 263.doc

[Karrieb61]

As an about-to-be- new Echo user, this concerns me a bit. We tell our techs that we are converting to a "better" method (which I AM all in favor of BTW) but when we can't figure out a pattern, we switch back to tube and may or may not do a full crossmatch by solid phase? I anticipate some negative reaction by our techs (no pun intended) when the recommendation is made to switch back to tube and more or less ignore what they see on the Echo when there is no pattern to identify an antibody. Makes you wonder about the confidence level in the solid phase technology. Believe me, I am very excited about getting away from microscopic read tubes, and all the potential problems you can have with tube technology, primarily inconsistency in reading results, but how have you handled it when you've had to say "Oops, not working, lets use tube"?

[David Saikin]

Karrie

Remember you are going to a more sensitive method - you should anticipate losing some specificity. That is part of the deal whether you use solid phase or gel. This does not happen a great deal but you will need to have a process in place to deal with it when it does. More importantly, you and your Medical Director will have to determine at what point you will revert to a less sensitive methodology.

Enjoy

[BloodBankGuy]

Yeah very common issue, our hospital policy is very simialar with EDibble.  These also could be cold antibodies that are being picked up on the more sensitive ECHO and not in tube.  If we repeat with the tube panel, both PeG and LISS, and get a positive reaction in 37 or AHG phase, we will do a full panel with that potentiator, if it is just garbage (cold reactive non specific) we will do a cold panel and call it a cold. 

 

WIth using all methodologies solid phase is by far my most favorite to work with, with this situation being one of the only interferences.  Develop a flowchart around it and you will be good to go.  Just make sure you can also identify High Freq Ags as well with it.

[bloodybanker]

Thanks everyone for your responses!  I completely agree with all statements made.  I feel much better now.  I just need to talk to our lead tech about this.  That should be fun!

 

One other question.  What was going on with Immucor?  They were having manufacturing problems?  (Is that why their supply was so low back in Jan/Feb/Mar?)  We had what looked like a good bit of false positives on one certain cell, on one certain lot.  This happened on at least 2 different lot numbers to us.  Nothing recently though.  I assume that they have sorted it out.  Maybe.

 

Thanks so much for the input!!!

[bloodybanker]

tburl - Thanks especially for the documents!  I haven't gotten a chance to read them yet, but will sortly!

[BloodBankGuy]

Yeah, early this year they had a problem with their main manufacturing plant and it really cut down the amount of product they could produce.  For a while I believe everyone was on a strict, reduced shipmen of product that had everyone really worried, at least my hospital was.

 

Some of the product that came out, if your sales rep was open with the information as mine was, they would give us a heads up on some lots that were showing false positive reactions.  We had a couple lots that were effected which was not fun since we didnt really have our normal stock of product to switch to.  They seemed to get it all fixed and there hasn't been any issues that I am aware of recently.

 

If you do happen to get a lot that you are seeing a trend of a lot of positive reactions, call your service and they should be able to tell you if there have been complaints about a certain lot number.  And if so they will send you out a replacement lot.

[AMcCord]

BloodBank Guy is right. Immucor did have a problem with inconclusive results and did a root cause analysis to try and fix the problem. Things seem to be much improved - we are not getting very many inconclusive results currently. And also agree that if your are seeing problems that seem to be reagent related, report it to Technical support. They aren't going to investigate a problem unless they know they have one and it needs to come from more than one customer.

 

Remember, too, that no one method is going to always detect all antibodies. No one method is going to be free of inconclusive results. You need a good backup method for those times when your primary is giving you funky results. You just have to pick the method that is going to be the best fit for your patient population and your workload, understand the limitations of that method and your backup method, and develop a protocol for interpreting the results you get. We love an automated process because of our staffing levels. We have a patient population (elderly, oncology patients) that is frequently transfused, so we need a method that is sensitive to developing antibodies, without too many inconclusive results. Those facts are a big part of what guided our selection process for the methods we use. (And of course...we can't forget the money angle ...management expects a good deal.)

[cjs3811]

I work in a 240 bed hospital and some of the techs i work with will revert to tube if reactions in screen are 1+ i dont feel this is correct the iID panel should be done. What does everyone think of this situation? My fellow techs have alot more experience and feel these reactions are junk

[AMcCord]

I work in a 240 bed hospital and some of the techs i work with will revert to tube if reactions in screen are 1+ i dont feel this is correct the iID panel should be done. What does everyone think of this situation? My fellow techs have alot more experience and feel these reactions are junk

 

I would definitely run the panel - weak reactions are not all junk. Tube testing is taking a step down in sensitivity so obviously you are running the risk of ignoring a true positive. We've ID'd newly developing anti-Jka on screens/panels where the reaction strength was '?'.

 

My rule is  ...  Before you dismiss weak reactions as junk - always run a panel to make sure you are not wrong. While that panel is running, repeat your screen with tube/PeG. Once you have the results of both, then you can make a decision about what you are seeing.

[cjs3811]

Thank you AMcCord that was initial reaction was due to sensitivity of the echo wasnt going to "rock the boat" since i am new to BB

[BloodBankGuy]

Yeah, with 1+ reactions you could just have a weakly reactive antibody that doesn't react in tube and that would be expected since solid phase is much more sensitive.  Which then comes into the question on "How sensitive is too sensitive?" because if we were still doing tube we wouldnt have seen it.

 

But times have changed and we do have a more sensitive method, so run the panel and if it follows a pattern you know.  There is still a change of sensitization if you dismiss a weak reacting antibody on echo and just transfuse any blood.

[mollyredone]

We also developed a flow chart to deal with gel anomalies.  One time someone did a gel screen that was iffy and then had a negative tube screen and did IS XM.  The patient came back days later with a delayed transfusion reaction and identifiable antibodies.  So now protocol is gel screen, if positive, gel panel.  If results are iffy, tube screen, but always gel XM.

[AMcCord]

Yeah, if you get it wrong, it might not be too hard to ID it next time when the titer is much higher

[carolyn swickard]

^{I always reccomend doing the ECHO panel too with any reactivity on the RS3 screen.  I think you should give the ECHO the chance to show you what it is trying to detect.  Then a procedure like the one mollyredone recommened for gel works well for solid phase too.  Solid phase crossmatches are good too, but I have the feeling that they are not quite as sensitive as the screen and panel though, because they are a fresh blood monolayer, not a manufactured layer of dry, lysed cells.  Doesn't hurt to do them though.} 

[JoyG]

We do the same as EDibble.  Also, if we do not get positive reactivity in all cells with solid phase as in your scenario of 1+ in one screening cell and negative in the others, we perform antibody identification to the extent possible using solid phase and other test methods as necessary (we have PEG and Gel backup).  If all clinically significant antibodies can be ruled out, we call that unexplained and must perform XMAHG.  We have had some of these antibodies turn into real clinically significant antibodies with subsequent draws.  Also, while Immucor does appear to have "fixed" the problem they were experiencing earlier in the year, we just reported a problem with the newest lot that appears to be acting in the same fashion.  Keeping our fingers crossed that it is not happening again!

[jalomahe]

Has anyone else come across this scenario?

Pt is 74 y.o. caucasian female. Pt has been pregnant but has no history of blood transfusion. Dx: Colon tumor

Echo Ready ID and Extend I show perfect Anti-c reacting 2-3+. DAT on Echo is negative. Patient is AB Negative C-c+E-e+.

Testing repeated with PeG and all cells negative except 1 cell that Immucor identified as Dantu+. Repeat DAT in tube also negative poly, IgG and C3d.

Since Anti-c in an Rh negative patient is exceedingly rare the specimen sent to Reference Lab and they report Rouleaux (we did not obseve this in our tube testing) and "Unidentified antibody to low frequency antigen" detected by both PeG and Gel. Tech asked and their cell(s) were not Dantu+.

We went back and checked using new specimen and had similar results. We performed crossmatch on Echo using 2 AB- c+ donor units and both units were compatible. 

I understand seeing non specific reactions or all cells reactive on Echo due to method specific issues but does anyone have any thoughts on why we would see such a clear-cut Anti-c on Echo panels but not demonstrable by other methods?

[Yanxia]

The patient is c pos and if she has anti-c, why the DAT is neg, have you do autocontrol with panel? If it is a real anti-c, the autocontrol should be pos.

I think we get the result from panel is the first step ,then we need to do adsorption and elution test to confirm the antibodies, this is the why we need to adsorption elution test.

[jalomahe]

The suspicion is that the patient might be a c variant, again this would be very rare. The results we are seeing repeat consistently with multiple specimens drawn on different days. We have contacted Immucor and are sending them specimens to investigate.

[Yanxia]

you say you performed crossmatch on Echo using 2 AB- c+ donor units and both units were compatible. 

I remenber some antibodies reacted with the preservation of panel cells looks like anti-c, I am not familiar with the tech you use ,this is just a suggestion.

Edited August 29, 2014 by shily

[Kattiebugs_42]

Question concerning the ECHO: repeating panels? I feel that repeating a panel should not be done because really Why? What are your thoughts?

Next question along side that, repeating the 3 cell antibody screens performed on the ECHO when only unequivocals (?) are demonstrating i.e. reeming the sample, re-spinning, rerunning on different ECHO and if unequivocals are gone report as negative??

 

=) Thanks!!!

Edited June 16, 2019 by Kattiebugs_42