We have been having some problems with our Eluate testing with GEL.  Trying to see if some of you have also encountered problems, and what solutions you may have come up with.

The problem is that too often (maybe 30-40% of time ??), all 3 Screening Cells come up Positive with the Eluate.  If we do a Panel, everything is positive.  If we repeat it in Tube, often times (maybe 90%), it is Negative.  I honestly believe that at least some of those are Warm Autos being picked up in GEL but not Tube.  But I suspect that a fair number of them are related to technique and are not truly reflective of antibodies.  Variables I have considered:

1. Final centrifuge- we have switched to doing a hard spin X2; but that has not resolved all of it (may have decreased # of Positive, but hasn't solved extent of problem)
2. We do not just make our stopping point when the Eluate starts to look blue; we actually measure the pH.  But therein might lie another problem; the differences in the shades from 1 pH to another, is so subtle....so I wonder if sometimes people are interpretting them erroneously.
3. Even if we were to make our stopping point when it is blue, and not use the pH strips....my experience tells me that there even seems to be a difference sometimes in what people consider "blue."

Anyway, would love input from anyone else who has experienced this and come up with the culprit, and/or, anyone who just has other suggestions.  

 

Thanks,

Brenda Hutson

I run my eluates in gel.  99% of the time they are either all positive or all negative.  Occasionally I do get specificity and that is when I expect it (delayed TrxN or the CAP survey).  Are your eluate pos/tube neg rxs 2+ or less?  I have found that 2+ rx in gel are invariably negative in the tube. If your last washes are negative I don't see how what you are finding is technique related.  I just go with the eluate when the tube turns blue - as do my techs who do eluates.  I don't have to know what the pH is because the procedure defines the blue color as the end point.  If you added too much of the buffering reagent I would expect you would get a negative rx - either due to dilution or radical change in pH causing loss of ab reactivity. 

 

What elution kit are you using?  I use Immucor's though I did try HemoBioscience's and found it satisfactory.

What elution kit are you using and what type of cards.  I would only ever test eluates on IgG cards.

We do our elutioin exactly the same way as David, and get results exactly as David.

 

We also quite often get positive throughout - even if, on occasions, the DAT is negative, but the diagnosis/symptoms suggest an AIHA of some sort.

 

As David says, if your last wash is negative, I really wouldn't worry about it Brenda.

Malcolm - I remember years ago you discussed DAT neg positive eluates.  At that time we had a patient who was having fevers but no one could diagnose anything - as I had a student we ran an elution and got a decently strong panaggl in the eluate.  I brought the article you had mentioned and the results to my Medical Director who discussed it with the pt's MD.  "what do I know?"  Anyway, they put her on steroids because? . . . and her eluate became negative.  Good experience for the student (and me).

We also test eluates in gel.

We don't test pH but rely on technologist evaluation of 'blue.'

We see the same problem occasionally (saw it within the last few weeks, training a brand new technologist).

After adding the buffered solution, we give it a hard spin and transfer it to a clean tube. Then we do it again. And again. Essentially you keep respinning until no stroma/discoloration can be seen on the inside wall of the tube. That's worked out the best for us.

Edited September 9, 20159 yr by goodchild

Actually, yes, I agree with goodchild, and I forgot to mention that we give the eluate a good hard spin to get rid of any crud/stroma that is in the liquid phase, and you do get much, much cleaner reactions.

Ok, so it sounds like we are getting the same results as you guys....but then my question is, when you get all 3 tubes positive (with a negative Last Wash), are you then repeating it in Tube and going with your tube results?  I guess I am just questioning the fact that it seems to happen more often than I would like and just haven't known whether to attribute it to "junk" in the eluate (granted the Last Wash is Negative, but you have continued manipulate the cells after that and add solutions; that is why I questioned technique), or, whether they are Warm Autos that GEL is just more sensitive to than Tube??  I guess the question I keep asking myself....is if we are getting that many that we have to repeat in Tube, should we just perform a Tube Screen with the Eluate in the first place?  But sounds like you guys are saying no.....you just "deal with it?"  

We use the Gamma ELU-KIT.

Thanks for all of your input!!  

Brenda

For me the questions are

• Are these panagluttinins unexpected based on the patient's history?
• How recent were direct observation competency assessments performed on the technologists you're seeing this from most? [i mean no offense by this. Prior to 2011 we weren't following CLIA stringently and were rotating which analytes/processes were competency assessed. When we started doing every analyte/process, every year, by each element of competency assessment, we definitely found some issues.]
• What is your algorithm/policy to perform an eluate? e.g. elution performed with every positive DAT, certain DAT strengths, within a certain timeframe of transfusion, physician order.

I don't repeat my eluates in tubes.

I don't repeat my eluates in tubes.

So are you reporting them out as Warm Autos or Panagglutinins (assuming the Panel looks like the Screen....or maybe you go straight to a Panel)???

Thanks

Brenda

 

For me the questions are

• Are these panagluttinins unexpected based on the patient's history?  Don't always know for sure....sometimes I feel they can easily be a Warm Auto for that patient....
• How recent were direct observation competency assessments performed on the technologists you're seeing this from most? [i mean no offense by this. Prior to 2011 we weren't following CLIA stringently and were rotating which analytes/processes were competency assessed. When we started doing every analyte/process, every year, by each element of competency assessment, we definitely found some issues.] We do a Direct Observation on ALL of our functions, every year; including Eluates.  And these Positive results are obtained with my Leads, as well as the less experienced (and we did change to a double hard spin at the end....some time ago).
• What is your algorithm/policy to perform an eluate? e.g. elution performed with every positive DAT, certain DAT strengths, within a certain timeframe of transfusion, physician order. If they have been transfused, we perform an elution with same frequency as antibody ID (I know a lot of people do the elutions less frequently than serum ID).  If no transfusion in past 3 mos., if it is the first time we have seen them, we do an elution (I just like to have that baseline); if we have previously performed an elution and no transfusion in past 3 mos., we do not perform an elution.

 

I don't repeat my eluates in tubes.

I guess I also just like to see the word "Negative" over "Positive"   (i.e.if  positive in GEL but negative in TUBE); but that was partly because I have not been sure if some of them were false positives (i.e. technique vs. warm auto or panagglutinin).

Brenda

Brenda, sounds a lot like what we do/see.

 

I would recommend instead of a routine double spin, re-spinning it until the tube appears clean, which cleared up a lot of the 'unexpected' reactions for us. Depending on the cells/acid ratio and the length of time it takes to take off the supernatant, it can take a wide range of spins to get it clear.

 

We only occasionally switch to tube eluates, if we're grasping at straws hoping to find an underlying antibody.

So are you reporting them out as Warm Autos or Panagglutinins (assuming the Panel looks like the Screen....or maybe you go straight to a Panel)???

Thanks

Brenda

I usually just run a panel not the screening set.  I report a panagglutinin - it is not for me to say the patient has a WAIHA.

I usually just run a panel not the screening set.  I report a panagglutinin - it is not for me to say the patient has a WAIHA.

 

Absolutely correct David.  There are just so many reasons for a positive DAT and an auto-antibody/panagglutinin that a "diagnosis" of WAIHA, without checking all symptoms, is a) impossible and dangerous.