Continued Eluate Problems

[Brenda K Hutson]

I brought this up before, but we continue to have problems that I cannot figure out.  We currently test Eluates using GEL (something I had not done until coming here; but I know others do it).  In the past several months, we have been having problems.  It seems to be intermittent; yet too frequent.  What we are seeing is:

1. All cells on Antibody Screen and  Panel appearing to be Positive
2. Often a mixed field appearance
3. Often see hemolysis in liquid in top of well
4. Repeat with Tube Eluate Testing is Negative

With the mixed field, I thought perhaps we were not performing a hard/long enough last spin of the eluate; so I changed that.  But it continues to happen; and the hemolysis is baffling.  It makes me think it has to do with the Reagents (maybe the Deionized Water used for the Wash Solution)??  But we have tried to make new Wash Solution.....to open a different Eluate Kit...etc.  Plus we are at 2 different Hospitals and it is occurring at both.  I don't think it is technique because my 2 Leads have the problems also; plus they have watched staff prepare eluates (for Annual Direct Observations) and know they are doing it correctly (and I have observed my Leads). 

Would love any ideas/thoughts/hypothesis any of you have to offer.  I would just say that we will switch to Tube Eluate Testing but currently, we only stock 2 Ortho GEL Panels so would have to concentrate the cells to perform Tube Testing.

 

Thanks,

 

Brenda Hutson, CLS(ASCP)SBB

[Brenda K Hutson]

Sorry, changed Deionized Water to Distilled Water....

 I brought this up before, but we continue to have problems that I cannot figure out. We currently test Eluates using GEL (something I had not done until coming here; but I know others do it). In the past several months, we have been having problems. It seems to be intermittent; yet too frequent. What we are seeing is:

1.All cells on Antibody Screen and Panel appearing to be Positive

2.Often a mixed field appearance

3.Often see hemolysis in liquid in top of well

4.Repeat with Tube Eluate Testing is Negative

With the mixed field, I thought perhaps we were not performing a hard/long enough last spin of the eluate; so I changed that. But it continues to happen; and the hemolysis is baffling. It makes me think it has to do with the Reagents (maybe the Distilled Water used for the Wash Solution)?? But we have tried to make new Wash Solution.....to open a different Eluate Kit...etc. Plus we are at 2 different Hospitals and it is occurring at both. I don't think it is technique because my 2 Leads have the problems also; plus they have watched staff prepare eluates (for Annual Direct Observations) and know they are doing it correctly (and I have observed my Leads).

Would love any ideas/thoughts/hypothesis any of you have to offer. I would just say that we will switch to Tube Eluate Testing but currently, we only stock 2 Ortho GEL Panels so would have to concentrate the cells to perform Tube Testing.

Thanks,

Brenda Hutson, CLS(ASCP)SBB

[goodchild]

1.All cells on Antibody Screen and Panel appearing to be Positive

2.Often a mixed field appearance

3.Often see hemolysis in liquid in top of well

4.Repeat with Tube Eluate Testing is Negative

 

When I see the issue with mixed field/hemolysis, my initial thought is a problem with the pH of the test system.

Do you use the gamma Elu-Kit II ?

[BrianD]

the hemolysis at the top of the well makes me wonder if your pipet tips have been coated with a pre-wetting agent....these often have a detergent activity and can lyse red cells....but you'd be seing this in all testing, i'd suspect.

[Brenda K Hutson]

When I see the issue with mixed field/hemolysis, my initial thought is a problem with the pH of the test system.

Do you use the gamma Elu-Kit II ?

Yes, we do; and once it starts to turn blue, we check the pH with pH paper after the addition of each subsequent drop of Buffering Solution.

Brenda

[Gnapplec]

We have a client that uses Gel for Eluates.  We always end up re-testing their samples using tube per the Elu-Kit's insert instructions because I have yet to see clear cut results from them.  I don't see our company ever approving a procedure for Gel Eluates due to the terrible inconsistencies.   Maybe look into keeping a 2 or 3 cell tube screen to start with?  That's where we start with our Eluates and then move onto a selected panel to minimize the number of cells that need concentrated.

[Malcolm Needs ☆]

We have NEVER experienced problems with testing eluates in gel.

[goodchild]

I don't think gel testing eluates has been a problem for us either, although in the past there have been isolated incidents where the results were questionable that we have attributed to technique and have cleared up with retraining/competency observations.

 

You know step 9, where it says to mix well and centrifuge to remove any precipitate or cellular debris, then transfer to a clean labeled tube? We do that step several times. Since we've been doing that there are very few problems with weird reactions.

[rravkin@aol.com]

Wrong question

Edited July 10, 2013 by rravkin@aol.com

[rravkin@aol.com]

We have NEVER experienced problems with testing eluates in gel.

Hi Malcolm, can you give detail of your testing method; or is it the same as described here??

[rravkin@aol.com]

 

Brenda, also are gel reactions occurring primarily at the top of the column or are they dispersed throughout the gel?  One other item; have you considered trying a reagent grade water to make your 10% Wash solution? I am not sure that it would make any difference but it's something else to try.

Edited July 10, 2013 by rravkin@aol.com

[David Saikin]

We have NEVER experienced problems with testing eluates in gel.

Ditto - occasionally the wash buffer needs to be remade because of hemolysis, but the elutions have always reacted (or not) in a reasonable fashion.

[Malcolm Needs ☆]

I will certainly post our method, but I am in the middle of moving back to our repaired home, and my own laptop has fallen over big time, so it may take a couple of days.

[AMcCord]

I will certainly post our method, but I am in the middle of moving back to our repaired home, and my own laptop has fallen over big time, so it may take a couple of days.

Happy homecoming!

[macarton]

We use DI water when making up the wash solution.  We also wash cells  2 or 3 times in saline before starting the wash.  We use gel and usually don't have issues unless the patient is having active hemolysis. 

[David Saikin]

I also wash a few times with saline prior to using the wash solution.  Depends on the presence/strength of any plasma abs.

[Byfaith]

I seem to recall seeing a kit made by Hemobioscience at a recent meeting - claimed to be validated for use in Gel.  We have yet to try it but may in the near future.

[Mabel Adams]

We had no problems with eluates in gel until our last Elu-Kit and then problems with the CAP survey.  We will see how the new kit works on the latest survey.  We had some problems with tube testing on the last one too.  Anyone using the Hemobioscience kit and want to report their experience?

[Malcolm Needs ☆]

Hi Malcolm, can you give detail of your testing method; or is it the same as described here??

Hi Ronald, 

 

I am back on much more of an even keel, although we are still living out of cardboard boxes to a certain extent!

 

We usually wash the red cells x 1 in phosphate buffered saline, kept at room temperature once.

 

Then we wash the red cells at least 4 times in the Gamma RLU-KIT II wash solution, which has been kept at 4oC (I usually wash them 8 times myself, just to make sure, but that's just me using a belt and braces).  The last wash is kept as a negative control.

 

THe elution solution is then put on to the packed washed red cells, well mixed and then centrifuged on a high setting for 1 minute.

 

The supernatent is then taken off and the buffer solution added until we get a blue colour.

 

We then centrifuge both the last wash and the eluate on a high setting for at least 1 minute to get rid of an contaminating cells or cell debris, and transfer each to a clean tube.

 

We then test in gel as normal.

[rravkin@aol.com]

Hi Ronald, 

 

I am back on much more of an even keel, although we are still living out of cardboard boxes to a certain extent!

 

We usually wash the red cells x 1 in phosphate buffered saline, kept at room temperature once.

 

Then we wash the red cells at least 4 times in the Gamma RLU-KIT II wash solution, which has been kept at 4oC (I usually wash them 8 times myself, just to make sure, but that's just me using a belt and braces).  The last wash is kept as a negative control.

 

THe elution solution is then put on to the packed washed red cells, well mixed and then centrifuged on a high setting for 1 minute.

 

The supernatent is then taken off and the buffer solution added until we get a blue colour.

 

We then centrifuge both the last wash and the eluate on a high setting for at least 1 minute to get rid of an contaminating cells or cell debris, and transfer each to a clean tube.

 

We then test in gel as normal.

Hi Malcolm, it's good to have you back. I sent you an email prior to reading this so there may be some redundancy.

[rravkin@aol.com]

Hi Brenda, it has been a while since we have heard from you. Were you able to resolve this problem? If so, how?  Thank you in advance.

[Brenda K Hutson]

I have tried making new Wash Solution; and have also changed the centrifuge used for the final centrifugation step to one that spins harder and longer (but only do it once; maybe we will spin X2 as goodchild suggested). Can also try a different water source for Wash Solution. Rravkin...reactions often have a mixed-field appearance but sometimes have some cells dispersed throughout cell; and sometimes hemolysis at top). We only wash X1 with Saline (as per Manufacturer's Instructions; though can't see how it would hurt to wash more).

What is odd is the inconsistency. Sometimes we get perfect results; other times we have this problem. And I have been doing Direct Observations on staff preparing and testing the eluate and see no differences.

So our current approach is to first have them Test the Eluate with just a GEL Antibody Screen; then if they get this type of reactivity, I have them repeat it in Tube.

It is just a mystery to me!

Thanks for all of your suggestions and replies!

Brenda

[Brenda K Hutson]

And again, my apologies for not spending more time on this website (would really love to).  Just the challenges of getting ready to move into a new Hospital, plus some family illness has kept me busy.  But I am very grateful for all of your input.  I know the day will come when I can spend more time on the site....  

 

Brenda Hutson

[Mabel Adams]

We just finished the latest CAP survey and had some problems again.  The first sample was tested a couple of weeks ago and the tech made up fresh wash solution.  The other tech assigned a sample was on vacation so she did hers last weekend. The second tech found reactivity in all cells in gel so had to do the eluate with the remainder of the sample using a new lot of ELU-Kit II and freshly made wash solution from the new kit.  The new eluate worked fine so she decided to test the two kits further by mixing anti-D with some O pos cells and then eluting it off.  She had to make up fresh wash solution for the old kit but used the wash solution from the new kit that had been in the fridge a couple of hours in the usual squirt bottle that we have used for the wash solution for years.  She found that both eluates worked fine!  The one from the new kit was a little junkier looking than the one from the old kit but readable.  We are doing some head-scratching.  It was almost as if the wash solution stored in the fridge was causing the junkiness.  Could the old wash solution bottle somehow be causing a change in pH or something?  Have they changed the ELU-Kit?  I can't see why cold wash solution would give false positives.  It is recommended for antibodies that wash off too easily as I recall.  I hope these clues help you, Brenda, or someone can help me resolve our issue.

[rravkin@aol.com]

I have tried making new Wash Solution; and have also changed the centrifuge used for the final centrifugation step to one that spins harder and longer (but only do it once; maybe we will spin X2 as goodchild suggested). Can also try a different water source for Wash Solution. Rravkin...reactions often have a mixed-field appearance but sometimes have some cells dispersed throughout cell; and sometimes hemolysis at top). We only wash X1 with Saline (as per Manufacturer's Instructions; though can't see how it would hurt to wash more).

What is odd is the inconsistency. Sometimes we get perfect results; other times we have this problem. And I have been doing Direct Observations on staff preparing and testing the eluate and see no differences.

So our current approach is to first have them Test the Eluate with just a GEL Antibody Screen; then if they get this type of reactivity, I have them repeat it in Tube.

It is just a mystery to me!

Thanks for all of your suggestions and replies!

Brenda

Brenda, since these problems are sporadic have you checked your Gel Card Centrifuge. At maximum speed the cards are suppose to orient to a horizontal position; this ensues even migration of the cells through the gel of each column in the card. If these cards do not orient horizontally then the cells do not migrate evenly and can give the appearance of reactivity. This can happen in any of the gel card seats on the wheel lending to the sporadic nature of the problem you are experiencing.

Just a suggestion and let us know what you find if and when you can.