Jump to content
dwalters

Gel card with 1+ reactions showing negative after spinning a second time

Recommended Posts

An ER trauma patient's plasma showed 1+ reactivity in Gel with Screening cell 1 of Immucor's Trio screen. The other two cells were negative. (We buy 3% Immucor screening cells, then dilute them to 0.8% for gel testing.) Using an Ortho 0.8% panel for antibody identification, we found 7 of the 11 cells reacting 1+ with a negative auto control. The reactions did not fit any pattern what-so-ever. Our workup was stopped at that point because the patient was transferred to a larger facility. On follow-up the next day, we found that the other facility had gotten a negative antibody screen, also using Gel cards. Within three hours of performing the antibody screen on the ER trauma patient, the remaining three wells of the gel card were used to perform an antibody screen on another patient. So, the card with the positive Screening cell 1 was spun for another 10 minutes. After the 2nd spin, the reaction had changed to negative. Later, we spun the two cards containing the antibody id with the seven 1+ positive reactions. After the 2nd spin (for a total of 20 minutes) most of the reactions were now negative with the others only questionable, definately not 1+ as after the orginal spin.

I've checked the rpms and timer on the centrifuge, and they are okay. Has anyone else seen positive reactions change to negative after spinning a second time?

Share this post


Link to post
Share on other sites


That's very interesting. I have never seen that before but generally, we used all the wells in our gel cards so we never really had the opportunity to respin them. I do remember getting some weak to 1+ reactions, that when repeated, were negative on the repeat. I wonder if real small fibrin pieces could be responsible. I'm not using gel at my facility currently but we are discussing it's use so I will have to watch for this when we do get it.

Share this post


Link to post
Share on other sites

:sleepy:

Yes...we've seen this phenomenon at our facility as well. Am not certain if it's a problem with centrifugation or if it could be remedied by pre-spinning the gel cards prior to use. I vaguely recall some facilities having pre-spun the cards on receipt but never got an explanation of this practice. Upon getting a positive reaction on the 3-cell screen, I always set up a new card and repeat prior to running a panel to avoid this problem. Have not seen anything from Ortho to suggest that others have this problem and/or explanations to this.

Also...I like the Immucor cells myself, and do often reduce them to 0.8% and run them in gel as you do ... does Immucor sell 0.8% panels? If not, they should...have found their cells to have a stronger antigen expression than the Ortho cells.

Share this post


Link to post
Share on other sites

No, Immucor doesn't offer 0.8% cells. If they did, we would use them for our panel cells also. Ortho states in their package insert that E and K might not be detected rarely, however, I've seen this also with Jka.

Share this post


Link to post
Share on other sites

Yes, we've seen this phenomenon for once at our facility in Taiwan. Instead of (ID-MTS) Gel Test commonly used in US, we use ORTHO BIOVUE system column agglutination technology. The principles of these two systems are similar.

We had a blood sample with a negative DAT, yet a positive autocontrol. Upon second centrifugation, the positive autocontrol turned to be negative. We then performed the autocontrol using tube method and found obvious rouleaux formation after immediate spin. We thus concluded that rouleaux formation of red blood cells might have caused a false positive reaction in CAT.

Share this post


Link to post
Share on other sites

You know, I hate to be the bearer of bad news but the manufacturer's instructions for gel cards do NOT support recentrifugation of the cards as a valid test method. There may be other reasons that results are not reproducible (e.g. fibrin, weak antibodies, variations in samples collected), but centrifuging the card again is not the way to resolve the problem! I hope no one is using this method to interpret patient test results!

From the insert:

1. False positive or false negative test results can occur from bacterial or chemical contamination of test materials, inadequate incubation time or temperature, improper centrifugation, improper storage of materials, or omission of test samples.

2. Proper centrifuge calibration is particularly important to the performance of the MTS Anti-IgG Card. TheMTS Centrifuge has been exclusively designed to provide the correct time, speed and angle.

and

For best results, it is recommended that following centrifugation, results should be read immediately. If tests are not read immediately, results may be affected by the drying out of the gel; hemolysis of the red cell and slanting of the reaction patterns due to storage in a non upright position.

Share this post


Link to post
Share on other sites

Yes, I've heard techs discussing this at my facility and I don't think anyone at my place of work agrees with that tact either.

Just last night, ran a gel card and one cell was "iffy". Set up a new card and the reaction was stronger, but the ortho 0.8% cells from the panels were negative...resorted to reducing the Immucor 3% cells to 0.8% and running them in gel. It was an anti-E reacting with homozygous cells only (but non-reactive with ORTHO homozygous cells). This same tired scenario happens 1-2 times weekly.

Respinning the patient specimen, and repeating with a new card is best...I'm not happy with the Ortho cells either and their lack of potency. This is a big problem and I don't think it's restricted to just E and K. Am sure the provue has better "optics" than the human eye but am not sure instrumentation would correct this problem.

Share this post


Link to post
Share on other sites

We totally agree that the instructions on the package insert should be strictly followed and the result of second spin should not be considered valid.

However, a negative DAT and a positive auto control created a discrepancy needed to be solved. We took the negative result of second spin only as a reference.

Actually we resorted to tube method. The results of tube method was rouleaux formation after immediate spin and 37 C incubation, but negative in indirect antiglobulin phase.

Share this post


Link to post
Share on other sites

We have also seen positive reactions become negative when we've reused the card for another patient. When we first started with gel (1998) the Ortho trainers had said a respin was acceptable. Later they changed their tune so I'm guessing real antibodies had spun away. We didn't specifically track what we found because I believe it's been a mixed bag - both antibodies and rouleaux.

Share this post


Link to post
Share on other sites

We have also seen positive reactions spinning down to negative when respinning the gel card while finishing a second screen on a new patient on the same card. We have found that anything truly positive does not spin away. The positive reactions we had that spun away were either rouleaux or a non specific cold agglutinin. We have also found that any agglutination, no matter how little, is an antibody.

Share this post


Link to post
Share on other sites

Have you considered antibodies that aren't listed on the panel. We recently had a patient (prenatal) with a postive antibody screen (1+ in one cell). When we did the panel, we had 3 positive cells but were able to rule out all the clinically significant antibodies. If this had been anything but a prenatal we would have probably quit there with negative crossmatches through AHG. But with the prenatal we sent it off to the ARC ref lab. It turns out to be an Sd. So you could always have something on the cells that is not listed on the panel sheet.

Also, I would never go by results gotten after respinning the cards. This is a lot like respinning and rereading tube reactions- never as good as the first time. I would repeat the screen and make sure pos and neg controls with all 3 screen cells were reacting correctly just to make sure nothing was contaminated. Probably not the case here since you got positive panel reactions as well.

just my .02

Share this post


Link to post
Share on other sites

We have also seen the respin phenomenon. But remembering chemistry training involving SDS Gel columns (ie Ortho Gel Cards) ,the packing problems with this molecular seive, and the package insert, we opt to repeat rather than respin. Could this be a DILUENT ADDITIVE PROBLEM? When LISS became a new attenuating medium for antibody identification, LISS antibodies drove us all crazy. Everyone has always said IF IT IS A LISS PROBLEM ALL REACTIONS SHOULD BE THE SAME. Our experience has not been all reactions the same, however, PEG Antibody Id's have shown it to be an additive phenomenon this phenomenon . This led us to use PEG ABID as a back-up to our Ortho Gel Card system for Antibody Identification when Gel results do not make sense.

Share this post


Link to post
Share on other sites

We have seen this same thing when re-spinning a card with another patient later on-it goes to negative. It is usually rouleaux or some other clinically insignificant problem. We have never had a true antibody go away when re-spun - that we know of - it is not something we would be actively looking for! We also have had the pre-diluted Ortho cells miss a Jkb that I knew was there from previous results. That was many years ago and the reason that we started diluting our own cells. The only problem we have had with using Immucor cells with the Gel system is that they are not screened for Gel use and may cause false positive reactions.

Share this post


Link to post
Share on other sites

We see this quite often at our donor center. Often it is a cold antibody of some sort. (often characterized by a hazy waterfall appearance) Rouleaux is only a problem with auto's. When we inspect our donor interview cards, we have noticed a large amount of these donors are taking some sort of "natural" supplements, but this is just a "Mad Scientist" theory of ours. If it is a pan-agglut., we often find these are reactions to some component in the reagent cells. And yes we detect MANY antibodies by Peg or other methods that Gel misses........but also detect many in Gel that Peg or other misses.......as usual there is no "be all" "know all" method as we all know. If we get a neg. screen in Gel on a sample sent to us from one of our hospitals, we also do various tube testing to detect any of these ghosts.

Share this post


Link to post
Share on other sites

We too have seen the weak reactions with gel...we back up our gel testing with LISS and/or PEG. Some of the weak reactions cannot be repeated by either method and some give nice patterns (E, K, Jka) in PEG. Each methodology has pros/cons. We also pick up Anti-M and non-specific cold reacting antibodies....then go back to the tube methods.

How many gel users make your own screening cell solutions using MTS diluent as opposed to the manufacture ortho gel cells (1,2,3)?

Share this post


Link to post
Share on other sites

Technically we are not supposed to respin gel cards after initial reading. We also back it up with conventional tube method. Anti-E, Anti-K are sometimes missed on gel cards and we now extend the incubation to 30 minutes as suggested by Ortho. The best method from our experience is to re spin the patient's sample using some beads and repeat the gel screen using a much clearer sample. It eliminates some of the fibrin that may be trap in the column. If it's still positive and antibody panel showed no specificity, I would check on patient's clinical condition and medications. Some reactions are related to patient's condition or drug interactions.

Share this post


Link to post
Share on other sites

Geriann,

We are actually in the process of getting rid of our 0.8% 3 cell screen and just converting our 3% daily. More cost efficient and stronger reactions. We prefer diluting our Immucor panel 16 to using the 0.8% panel A from Ortho.

Share this post


Link to post
Share on other sites

We would never respin the gel card and rely on that reaction. We will, however, respin the patient plasma and repeat the antibody screen on very weak initial reactions (Ortho suggested this).

Share this post


Link to post
Share on other sites

We also use gel cards and ProVue. If the patient has rouleaux, it almost always interferes with testing here. We perform saline replacement to confirm rouleaux. I have not had the problem with anti-E and anti-K. We use Ortho's 0.8% suspensions.

Share this post


Link to post
Share on other sites

I know it's probably a bit late, but I just came across this 'old' thread and wanted to comment on the phenomenon of weak reactions 'disappearing' after re-centrifuging. This is not unusual - in fact stronger reactions would become weaker too. The gel system is based on the fact that unagglutinated cells pass through the gaps between the gel beads, while agglutinated cells remain trapped. The distance travelled through the gel depends on a number of factors - the size of the agglutinates, the distance to be travelled, the speed of centrifugation and the centrifugation time. Altering any one of these factors will affect the distance travelled. The 'fixed' parameters - column length, centrifugation speed and time have been extremely carefully 'fine-tuned' so that negatives will fall to the bottom, but weak positives will still be visible. But if you spin twice, you are doubling the centrifugation time; therefore the cells will travel further down the column. Similarly, if you stop the centrifugation half-way, you will get false positives. Hope that helps.

Anna

Share this post


Link to post
Share on other sites

:sleepy:

Also...I like the Immucor cells myself, and do often reduce them to 0.8% and run them in gel as you do ... does Immucor sell 0.8% panels? If not, they should...have found their cells to have a stronger antigen expression than the Ortho cells.

I've done this on numerous occassions, also a colleague did this recently. We took a Panocell 3% and converted it to 0.8% and run them in gel. I personally would dilute them with the MTS regular diluent (not MTS Plus).

I had one patient where I ruled out Anti-S with the Panocell diluted to 0.8%, but I eventually decided to send it out to reference lab. Results came back positive for Anti-S.

My colleague said she couldn't rule out Anti-K with Panocell diluted to 0.8% -- it was reacting negative to those cell lines but the panel clearly shows multiple antibodies (1+ to 2+ reactions), and Anti-E was clearly present.

So as much as I thought theoretically it would work, diluting the Panocell from 3% to 0.8% may have decreased the number of antigen sites available on these cells. Maybe they weren't made to be diluted for Gel card use, I don't remember seeing that on the reagent insert though.

As for respinning the Gel cards -- I'm personally not comfortable with that practice. I've had cards where it didn't spin right (agglutination toward one side of the column), and I've had gel cards where it looks like it was dried up, very minimal liquid in the column present, and one Ortho rep had told us to pre-spin it before use.

Maybe our administration should just consider expanding the blood bank so that the cards are adequately stored in the right temperatures (although they claimed an Ortho rep did come in and said it was ok....I don't know about that), and maybe my BB supervisor should just get the MTS centrifuge fixed because respinning cards I think is just a bad way to practice Gel methodology.

But hey, my administration is another story ;)

Share this post


Link to post
Share on other sites

Spinning a gel card prior to use is OK and is necessary if the cards got shipped (or stored:mad:) upside down or on their sides.

Once a patient test is set up, it's one spin and one spin only. It is quite possible for a weaker reaction to appear negative if respun. If the card looks like it didn't get spun properly - stuck at an angle, for example - the test must be repeated, not respun. The product insert is very explicit about not respinning tests.

Share this post


Link to post
Share on other sites

Spinning a gel card prior to use is OK and is necessary if the cards got shipped (or stored:mad:) upside down or on their sides.

Once a patient test is set up, it's one spin and one spin only. It is quite possible for a weaker reaction to appear negative if respun. If the card looks like it didn't get spun properly - stuck at an angle, for example - the test must be repeated, not respun. The product insert is very explicit about not respinning tests.

I was told exactly this too!!!

Share this post


Link to post
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now

  • Advertisement

×

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.