Re-incubating a gel card??

[RR1]

I thought your Lab was working well today!!!!!!!!!!!

:devilish::devilish::devilish:

Bad Malcolm!!!

:giggle:

[Steven Jeff]

I know of Hospital Laboratories that do this jcdayaz, but it is not as effective as washing tubes manually with pre-warmed saline.

The reason I can say this with some certainty is because they will very often say that they have tried pre-warming the cards and got nowhere, before they send us the samples, but we find nothing by manual tube technique with warm-washing.

Sorry!

;)

I can confirm Malcolm's statement here, I recently had a patient with an anti-M, discussed on another thread. I pre-warmed the cassette, the BLISS reagent (ortho), the cells, and the serum and still got a 3+++ positive reaction at 37°C. Malcolm's laboratory confirmed the same sample as a cold reacting Anti-M, unreactive at 37°C. Cassette techniques do not appear to work, no matter how much pre-warming you do. The Ortho spin time is only 5 minutes.

Steve

[jcdayaz]

We were not successful using the prewamed technique in a gel card. I think it is because of the 10 minute spin at room temp. Wish it did work.

Mary,

I must confess to being confused by your post. In our prewarm procedure there is no room temp

&/or 37 spin/read. We spin/read only at the AHG phase.

[PaulSunV]

I believe that Mary's comment related to the spinning of the gel card after incubation at 37 degrees so unless you incubate your centrifuge the spin will be at room temp. for 10 minutes.

Edited December 24, 2009 by PaulSunV
Added

[jcdayaz]

I believe that Mary's comment related to the spinning of the gel card after incubation at 37 degrees so unless you incubate your centrifuge the spin will be at room temp. for 10 minutes.

Sorry to be dense! Why would one spin a gel card other than one time at the end of incubation time! That is the AHG phase.

[Malcolm Needs ☆]

Sorry to be contrary, but I don't think that the spin phase has much to do with it. I am much more convinced that it is the time when the reactants are mixed.

Consider this.

You have warmed both your reactants (presumably!) and your card; but have you warmed your pipette tips?

The reactants are very small in volume (50uL and 25uL). When you draw these up into your tips, the surface area to volume ratio is very much in favour of the surface area. Therefore, the reactants are going to lose temperature extremely quickly.

"Cold-reacting" antibodies tend to sensitise red cells extremely quickly (which is why setting up the tests at room temperature and then incubating them at 37oC causes so many problems); the k1 of the Law of Mass Action. But, dissociation between a "cold-reacting" antibody and its antigen happens relatively slowly, and probably does not happen within the incubation time; the k2 of the Law of Mass Action.

Therefore, you get unwanted reactions.

Using hand-held pipettes for the manual tube technique on the other hand, where larger volumes of both reactants are used, and larger volumes tend to be drawn up into a pipette that has a thicker bore, means that the surface area to volume ratio is nearer 50:50, and so there is a slower drop in temperature, and that may make all the difference.

I may be talking complete rubbish, but it is something to think about.

:confused::confused:

[jayinsat]

Sorry to be contrary, but I don't think that the spin phase has much to do with it. I am much more convinced that it is the time when the reactants are mixed.

Consider this................

I may be talking complete rubbish, but it is something to think about.

:confused::confused:

Another brilliant post from Malcolm:winner:. You really got me thinking here. My next cold, I'm going to try warming the pippette tips. I wish I could put the entire MTS centrifuge in an incubator! I have often wondered why prewarm is ineffective on gel and what you said makes a lot of sense. I'm sure my manager will think, for sure now, that I have completely gone off the deep end!:crazy::crazy:

[Malcolm Needs ☆]

Another brilliant post from Malcolm:winner:. You really got me thinking here. My next cold, I'm going to try warming the pippette tips. I wish I could put the entire MTS centrifuge in an incubator! I have often wondered why prewarm is ineffective on gel and what you said makes a lot of sense. I'm sure my manager will think, for sure now, that I have completely gone off the deep end!:crazy::crazy:

Well, if your manager does think that (and, especially if it doesn't work) blame me!!!!!!!!!!!!!

:D:D:D:D

[Steven Jeff]

I have to confess Malcolm I didn't warm the pipette tips in my experiment with the cold Anti-M, but what you say makes an awful lot of sense to me. Even after a few beers and wine on Christmas night

Steve

[Malcolm Needs ☆]

The thing is though, with the surface area of the tips, compared to their volume, and the fact that they are made of plastic, which also loses temperature quickly, I'm not convinced that warming them is the answer; but it may help.

:confused:

[Steven Jeff]

I am sure you are right, it is more likely the volume of pre-warmed reagents used in the manual tube technique decreasing the chance of the antibody antigen reaction.

Steve

[jcdayaz]

We currently put a pipette in each of the tubes while they are prewarming. Yes, Malcolm, they are the larger-bore pipettes----do you think it is feasible to also put the smaller pipette tip in the incubator (gel procedure vs. tube) while the reactants are prewarming??

I think I am fixated on this topic....Maybe I will will develop a successful method of Gel Prewarms and name it the "Jean Method"!!!!

[jcdayaz]

I can confirm Malcolm's statement here, I recently had a patient with an anti-M, discussed on another thread. I pre-warmed the cassette, the BLISS reagent (ortho), the cells, and the serum and still got a 3+++ positive reaction at 37°C. Malcolm's laboratory confirmed the same sample as a cold reacting Anti-M, unreactive at 37°C. Cassette techniques do not appear to work, no matter how much pre-warming you do. The Ortho spin time is only 5 minutes.

Steve

Anti-M's can be "fun", can't they??

I am curious as to why you would try to prewarm it away. If we identify an Anti-M we simply give AHG crossmatch compatible blood.

[Steven Jeff]

The UK guidelines state that if the Anti-M is reactive at 37°C we have to give antigen negative blood. If the Anti-M is not reactive at 37°C then AHG crossmatch compatible blood is OK.

Regards

Steve

:)

[Malcolm Needs ☆]

We currently put a pipette in each of the tubes while they are prewarming. Yes, Malcolm, they are the larger-bore pipettes----do you think it is feasible to also put the smaller pipette tip in the incubator (gel procedure vs. tube) while the reactants are prewarming??

I think I am fixated on this topic....Maybe I will will develop a successful method of Gel Prewarms and name it the "Jean Method"!!!!

I see absolutely no reason why not, but, knowing that plastic takes longer than glass to heat up, and the air inside them would also need to heat, as there would be little circulation, how about keeping a supply of tips in the incubator at all times, so that they are already heated to the desired temperature.

:confused:

[Malcolm Needs ☆]

The UK guidelines state that if the Anti-M is reactive at 37°C we have to give antigen negative blood. If the Anti-M is not reactive at 37°C then AHG crossmatch compatible blood is OK.

Regards

Steve

:)

Yes, you can find it (and other really useful information) in;

Daniels G, Poole J, de Silva M, Callaghan T, MacLennan S, Smith N. The clinical significance of blood group antibodies. Transfusion Medicine 2002; 12: 287-295.

:D:D

[RR1]

We do not put more strips on the plate than we need for the current run. Immucor has told us that we can put whole plates on, so they don't seem to see the multiple incubations as a problem. There is a time limit to how long a plate can stay on the instrument, but no information about how the strips are affected by multiple incubations.

I have attached the warning that Immucor have sent about putting whole plates on and passing them repeatedly through the instrument until used.

Customers and Distributors information regarding Galileo Germany english.pdf

[cajcpsbb]

In using the ProVue system, the foil is not removed from the cards. The automated system pierces the foil of each microtube after the testing is ordered and initiated. I have reincubated gel cards again and again without incident but like you, I am not aware of documentation. Good topic ... I will do research on this topic.

Okay, this seems like a weird question, but my supervisor is pressing the issue: does it say anywhere (documentation) that you can incubate gel-card microtubes multiple times without removing the foil and have them be still good to use? I scoured the Instructions For Use and could not find anything with that kind of wording. It also doesn't state the contrary neither. I called Ortho tech-support and they stated something along the lines that "There have been no studies that have tested incubating multiple times before use."

Mind you, I've had rotations at other, larger labs that use the gel system. IMO, it seems obvious that you can incubate them a few times before use. Why else would they be designed as such?

Honestly, I don't think I'm going to find such documentation anywhere. This lab is going to be switching to the gel system for the first time and Joint Commission is right around the corner. Bottom line: I need a little help explaining to my sup that they're going to be okay and they're chasing red herrings. Tactfully of course! :D :D :D

Thanks!!

[cajcpsbb]

In using the ProVue system, the foil is not removed but simply pierced as testing is ordered and initiated. I have saved cards for weeks at RT and then reincubated them without incident. I am not aware of any time frame that the cards may be restricted to for reincubation. Good topic! I am going to do some further investigation.

[Steven Jeff]

Dear Carol

The following was written earlier in this thread:

This reply is from an Ortho technical specialist:

I got an email with your concerns for testing the gel card with multiple incubation times. This question came up in my very early years (1995!) and the answer was yes there many studies done incubating actually hotter than 37C(up to 40C). One microtube was opened and incubated followed by the second one etc. until all 6 were used. The main reason heating does not cause a problem to the gel is the gel does not come in contact with heat…….only the upper reaction chamber. Important to keep the foil intact and not open and try to incubate using that well later. Drying could occur which would not be good. Also the package insert states that storage should be between 2 – 25C (not testing environment).

So the bottom line is ……..multiple incubation times does not affect the gel card.

The design of the incubator is critical in that the reagent portion of the gel cards are not in direct contact with the warm bit!! Thats my understanding.

Regards and welcome to BBT, an unbelievably useful site

Steve

:)

[RR1]

The is Steve, the information needs to be a formal communication from the company to all users and should be included in the package insert.

[irshadaad]

we are re using left over tubes in the diamed cards and as i remember we did not see any trouble in re using the liss-coombs and nacl cards and normalyy we leave the cards @ room temp.im thinking now to have a survey of the finding...this is something nice been taken into account.....

[irshadaad]

Hey guys wish you all ....happy new year...

[Steven Jeff]

The is Steve, the information needs to be a formal communication from the company to all users and should be included in the package insert.

Hi Rashmi - now that I am back at work, there is nothing in the package insert about how frequently the cassettes can be re-incubated, which is not surprising. If anywhere the info should be provided with user manuals, not that I can find it there either.

Steve

:)

[Steven Jeff]

I am a little puzzled at how often you need to re-incubate a gel/bead cassette. We are only a small laboratory, processing approx 3000 group & antibody screens and doing 500 AHG cross matches a year plus 25 panels excluding NEQAS testing. The AHG cassette is the most likely one to be re-incubated a max of 3 times for cross matching (3x2 Unit cross matches), which I would have thought is fine.

So how often are you re-incubating your cassettes?

Steve