Fyb Antisera Problems???

[Brenda K Hutson]

I am going to call Immucor torrow (today is Sunday), but just curious; has anyone else had problems with the current Lot numbers of anti-Fyb Antisera from Immucor? We have tried 2 different lot numbers; and 2 different heterozygous panel cells. The Positive Control is only macroscopically W+ at best (can actually appear to be negative).

Thanks,

Brenda Hutson, CLS(ASCP)SBB

[marvy1]

I am going to call Immucor torrow (today is Sunday), but just curious; has anyone else had problems with the current Lot numbers of anti-Fyb Antisera from Immucor? We have tried 2 different lot numbers; and 2 different heterozygous panel cells. The Positive Control is only macroscopically W+ at best (can actually appear to be negative).

Thanks,

Brenda Hutson, CLS(ASCP)SBB

We have been experiencing weak reactions with Fyb antisera for quite some time. Tried Ortho, Immucor and Biotest (Biorad) and all are weak.

[lacs]

We have been experiencing weak reactions with Fyb antisera for quite some time. Tried Ortho, Immucor and Biotest (Biorad) and all are weak.

We've had the same issue on and off too.

[Brenda K Hutson]

Oops...sorry, my mistake! These Lot numbers are from Ortho; not Immucor!

FYB073C and FYB073A.

Brenda Hutson

[Brenda K Hutson]

Ok, so here I am replying to my own Thread again! But thought you guys might be interested in what I found out.

I called Ortho and they asked the pH of the saline we use (in that the Fyb antisera is a coombs reactive reagent). I told them it was Blood Bank Buffered Saline but I did not have the exact pH in front of me. They suggested we try washing it by hand after incubation (before adding AHG) and said they would call back (so waiting for that). Much to my surprise (since we use the same saline cubes for the cellwashers as we do in the saline bottles), the positive control was now 2+!! It will be interesting to hear the explanation.

Any "thoughts/guesses" out there?

The only thing I can think of is some difference in the dryness of the button between washing with a cellwasher, and washing manually; but have no other thoughts at this point.

Stay tuned.....

Brenda Hutson

[Brenda K Hutson]

So, Ortho believes this indicates a problem with our cellwashers. While I cannot explain the discrepancy, I do not believe we have any cellwasher problems. We regularly perform PM and change tubing. We use coombs control cells for AHG testing with no problems noted. We perform Daily Fill Level Checks and Weekly Volume Checks.

Anyone else have any thoughts on this? :tongue: Would be interested in hearing some theories.

Thanks,

Brenda

Ok, so here I am replying to my own Thread again! But thought you guys might be interested in what I found out.

I called Ortho and they asked the pH of the saline we use (in that the Fyb antisera is a coombs reactive reagent). I told them it was Blood Bank Buffered Saline but I did not have the exact pH in front of me. They suggested we try washing it by hand after incubation (before adding AHG) and said they would call back (so waiting for that). Much to my surprise (since we use the same saline cubes for the cellwashers as we do in the saline bottles), the positive control was now 2+!! It will be interesting to hear the explanation.

Any "thoughts/guesses" out there?

The only thing I can think of is some difference in the dryness of the button between washing with a cellwasher, and washing manually; but have no other thoughts at this point.

Stay tuned.....

Brenda Hutson

[GilTphoto]

Two things:

1.Have you tried incubating longer?

2. How long do you spin for?

The results of our Serological Centrifuge Calibration always indicates that 20 seconds is optimal....... but....

There seem to be several reagents that don't work well at that time.

I believe the best time is 15 seconds (the default for the Immufuge), regardless of the silly Sero Cent Cal procedure we have to do yearly.

My night shift tech always reports 1+ for K+ control spun at 20 seconds.

I get 3-4+ when spun at 15 seconds. I also find Anti-A is hard to shake off at 20 seconds, amoung others.

So what to do? Somehow make 15 seconds work, the next time the Serological Centrifuge Calibration procedure is done?

If the centrifuge is the same, the rotor and tubes are the same, the RPM is the same, reagents the same, so how could the time vary?

The procedure is such a pain!

First dilute an ABO antibody to a magical 1+, with 6% Albumin (that you have to dilute from 22%)

and an Anti-D to the magical 1+

Years ago, we diluted Polyspecific antisera, but now with Monoclonal, the titer is just too high (512, >1000)

It's better to dilute a patient's plasma, so the titer isn't so high.

THe only thing is,...... the results don't resemble reality!

[Dr. Pepper]

And what does one use to get that magical 1+, the centrifuge that you're trying to calibrate????

[Brenda K Hutson]

Thanks for your thoughts!

As far as incubating longer; while that is often a tactic used in antibody identification, it would not then be strictly following the Manufacturer's Instructions for this antisera (I looked; it does not suggest a longer incubation in the event you obtain negative and/or weak reactions).

As you mentioned, our centrifuges and cellwashers are calibrated annually for the spin times. Also, the same spin time would have been used in this case, both for the tubes washed with the cellwasher, and the ones manually washed.

Brenda Hutson

Two things:

1.Have you tried incubating longer?

2. How long do you spin for?

The results of our Serological Centrifuge Calibration always indicates that 20 seconds is optimal....... but....

There seem to be several reagents that don't work well at that time.

I believe the best time is 15 seconds (the default for the Immufuge), regardless of the silly Sero Cent Cal procedure we have to do yearly.

My night shift tech always reports 1+ for K+ control spun at 20 seconds.

I get 3-4+ when spun at 15 seconds. I also find Anti-A is hard to shake off at 20 seconds, amoung others.

So what to do? Somehow make 15 seconds work, the next time the Serological Centrifuge Calibration procedure is done?

If the centrifuge is the same, the rotor and tubes are the same, the RPM is the same, reagents the same, so how could the time vary?

The procedure is such a pain!

First dilute an ABO antibody to a magical 1+, with 6% Albumin (that you have to dilute from 22%)

and an Anti-D to the magical 1+

Years ago, we diluted Polyspecific antisera, but now with Monoclonal, the titer is just too high (512, >1000)

It's better to dilute a patient's plasma, so the titer isn't so high.

THe only thing is,...... the results don't resemble reality!

[Eagle Eye]

Ortho sent out letter today regarding anti-S---again they are talking about use of specific saline.

[AMcCord]

Just a thought...how do you decontaminate your cell washer? Could this be affecting your reactions? We used to check the pH of the saline from the cell washer after we decontaminated (when we had a cell washer that is...a whole nother painful story there!).

[Brenda K Hutson]

Thanks.

It still does not make sense to me (or my staff) that our cellwashers are the problem (for reasons listed above). And we use the same saline for our cellwashers as we do the saline bottles.

Brenda

Ortho sent out letter today regarding anti-S---again they are talking about use of specific saline.

[Malcolm Needs ☆]

The only thing I can suggest is that you get a better mix of the cell button and the fresh slaine by hand, than the machine gives it. In other words, as we say over here, you can give the packed cells some real wellie, so you get a better wash and less inhibition of the AHG!!!!!!!!!

[Brenda K Hutson]

We use an outside vendor for our cellwasher preventive maintenance (they seem to be pretty good actually). I would have to ask; but my guess is that they do not check pH. That may be a good suggestion though in that Ortho also brought up the pH as a possible cause.

Thanks,

Brenda

Just a thought...how do you decontaminate your cell washer? Could this be affecting your reactions? We used to check the pH of the saline from the cell washer after we decontaminated (when we had a cell washer that is...a whole nother painful story there!).

[Brenda K Hutson]

Ok, so is "wellie" a British term? But yes, the cell button seems the most logical explaination to me also.

Brenda

The only thing I can suggest is that you get a better mix of the cell button and the fresh slaine by hand, than the machine gives it. In other words, as we say over here, you can give the packed cells some real wellie, so you get a better wash and less inhibition of the AHG!!!!!!!!!

[Malcolm Needs ☆]

Ok, so is "wellie" a British term? But yes, the cell button seems the most logical explaination to me also.

Brenda

Basically, it means bash it to Kingdom Come (during the washing phase - NOT at the reading phase!!!!!!!!!!!!!!!!!!).

[Brenda K Hutson]

Ah.....

Brenda

Basically, it means bash it to Kingdom Come (during the washing phase - NOT at the reading phase!!!!!!!!!!!!!!!!!!).

[phalim]

Hey Brenda,

Our lab also experienced these problems with the Fyb antisera. We also investigated the age/condition of the panels and washing the cells before testing. We found that when tested with the Ortho panel the three sources of anti-Fyb we have gave us the reaction we wanted without having to wash the cells before use: score 9/ 3+. We have temporarily switched to using the Ortho panel only for our Fyb controls until the issue is resolved.

I think last year that Ortho reformulated the preservative their cells are stored in and their packaging is designed to protect the cells from exposure to light so it's possible the reactions are due to the affect of storage on the Fyb antigen on the Immucor panels.

[Brenda K Hutson]

Are you using the 0.8% Ortho cells? Because that would not be the correct concentration for antigen typing.

Also, I don't think the issue is with washing (or not), the cells being typed (at least not according to Ortho). What changed (dramatically) for us, was washing manually after the coombs incubation phase (vs the cellwasher).

But, it might be worth trying to wash our control cells (in that I know some antisera do state to do that).

Thanks,

Brenda

Hey Brenda,

Our lab also experienced these problems with the Fyb antisera. We also investigated the age/condition of the panels and washing the cells before testing. We found that when tested with the Ortho panel the three sources of anti-Fyb we have gave us the reaction we wanted without having to wash the cells before use: score 9/ 3+. We have temporarily switched to using the Ortho panel only for our Fyb controls until the issue is resolved.

I think last year that Ortho reformulated the preservative their cells are stored in and their packaging is designed to protect the cells from exposure to light so it's possible the reactions are due to the affect of storage on the Fyb antigen on the Immucor panels.

[Dr. Pepper]

There are published data saying that Duffy antigens elute off of saline-suspended cells, and this material can partially inactivate your reagent or patient sera resulting in weaker reactions. It is not supposed to happen with cells in reagent dilution media like Alseavor's, but it might be worth giving it a try.

[Dr. Pepper]

There are published data saying that Duffy antigens elute off of saline-suspended cells, and this material can partially inactivate your reagent or patient sera resulting in weaker reactions. It is not supposed to happen with cells in reagent dilution media like Alseavor's, but it might be worth giving it a try.

Oops, I meant to say you might give washing the cells before typing a try.

[marvy1]

Using the suggestions given in this thread for improving + control strength using Immucor Fyb antisera, I compared cell washer vs hand washing and Immucor panel cells vs Ortho panel cells.

Using Ortho panel cells and hand washing, I was able to achieve a 2+ grade for my positive control. Using cell washer and/or Immucor panel cell, the reaction strength was 1+ to weak macro.

I think we will change our Fy antisera procedure to use Ortho cell controls and hand washing.

[Mabel Adams]

Has the saline changed? Has the antisera changed? Have cell washers (or tubing) changed? Haven't we been using the same antisera and saline forever? Why problems now and not 10 years ago?

Hemo bioscience has anti-Fya and anti-S but I don't see Fyb on their website. Just looking for outside the box sources. How about Quotient/Alba? Do they behave differently?

[marvy1]

I've been noticing weaker-than-expected reactions using both Fy(a) and Fy( for a few years now. I have seen this in various labs both in Canada and US where I have worked, so I don't know if you could attribute this to problems with a cell washer. If someone is getting good strong reactions with their controls, I would love to hear what reagent they use. We have tried Ortho, Immucor and Biorad and they are all not so great.

Edited September 2, 2011 by marvy1

[Malcolm Needs ☆]

I agree.