I was once told (or read) that when using the IgG gel cards, it is best to leave an air bubble between the serum and the gel layer. Supposedly this enhances the incubation of the reagent and the patient serum to better pick up weak antibodies. Has anyone else been told to do this or are they leaving an air pocket when testing antibody screens in gel? Sometimes it is very difficult to leave that air pocket when pipetting, just wondering what everyone else was doing.

I am not aware of such thing. I don't think you can really do that. You may develop a bubble during pipette or you may not.

As far as I know gel is suspended in low ionic strength solution. If you look at the card there is some liquid above gel layer. If you think logically it is better not to have bubble!!!!!!!!!!! I may be wrong but just a thought:) .

How could you hope to control where the plasma and cells mingle.....as long as they are together, I would think sensitization should happen. I'll have to check what happens in the Provue!

It's been so long I don't remember why, but I leave an air bubble between the reaction part of the microtube and the gel. I think it had something to do with the lack of mixing when you add plasma.

We were taught that it is best to leave an air bubble between the original contents of the gel tube and the cells. I know of another lab that also requires it and they have seen reduced sensitivity when the bubble is not present. Our Ortho trainer did not say it was essential but that it was best (in 2003) to have a bubble. At first, I did not require it, but I found that many techs didn't make much effort to get the bubble and we had more hazy reactions that were hard to interpret. Then I talked to this other lab so now we require it. As I understand it, the logic is that you don't want to mix anti-IgG with your plasma/serum before it has a chance to react with your cells or it could bind up some of the antibody.

How do the try to get the bubble?

I will try to watch ProVue on monday.

I finally found a way to describe the technique for getting a bubble. We use the tipmaster pipets. I am not good at it with other pipets. When pipeting the cells, hold the pipet at a 45 deg angle, stabilize it with your other hand, and place the tip as close to the far side of the gel tube opening as you can without touching it. The pressure on the pipet is gentle--just strong enough to complete the stroke. With a little practice it works pretty slick. Hope that helps.

We were taught that leaving the air space in the microtube was the goal, but not a necessity and reactions would not be affected. We don't require it.

We were taught that leaving the air space in the microtube was the goal, but not a necessity and reactions would not be affected. We don't require it.

We were told the same... that when the bubble is gone it is merely a case of "aggresive pippetting" and the tech should try to back off a bit.

Susan

We try our best to leave the bubble but do not require it. I do train to hold the pipet at a 45 degree angle, stabilize the pipet, dispense as close to the far edge as possible and "slow down". This works quite well.

Initially when we first started using Gel (2003) we were taught to hold the pipetor at a 30-45 degree angle to dispense the 0.8% cells and plasma into each well of the Anti-IgG Cards. Since then we have had several questions arise about Gel procedures. The technical support staff at Ortho told me on two separate occasions that there is no actual documented procedure as to how to hold the pipetor when dispensing. I was also told that the you should try to keep the bubble intact, but as long as the cell suspension has not descended into the gel before centrifugation it will be ok, but it may decrease if the bubble gets dislodged and separates the sample so disproportionate amounts of cells and plasma get trapped on either side.(I've found that it does sometimes affect the strength of reactivity that you see when weaker antibodies are present.) but it is definitely not optimal, and I've trained my staff that if the top of the gel is not touching the cells that they just added, then it is fine. If it is touching the top of the gel then they are to repeat it in another well of the cards and not to use the reactions in those wells that weren't pipetted correctly.

I looked at what the Provue does.....it seems to consistantly leave a space between the plasma/cells and gel !!

I was once told (or read) that when using the IgG gel cards, it is best to leave an air bubble between the serum and the gel layer. Supposedly this enhances the incubation of the reagent and the patient serum to better pick up weak antibodies. Has anyone else been told to do this or are they leaving an air pocket when testing antibody screens in gel? Sometimes it is very difficult to leave that air pocket when pipetting, just wondering what everyone else was doing.

Air bullbles in reaction volumes, such as that of the gel card, are not a good practice because they can cause an unequal distribution of the reactants and heat throughout the volume, such that, the reactions, if any, can be significantly altered and/or missed (ie false negative).

Labman,

I think that what you are eluding to in your statement about the air bubble would apply if the cells were dispenced into the gel and let standing at room temp for a significant length of time before the plasma was added. The plasma should be added immediately after the cells and the card placed into the incbator right away. Therefore the cells do not have the opportunity to disipate into the gel prior to mixing with, and potentially reacting with, the free IgG in the plasma, if any.

Labman,

Also, the 30 to 45 degree angle of holding the pipette is a convention of practice formed to enable all practioners; laboratory techs; to dispence the same drop. As you know the angle that the pippette is held can alter the size of the drop such that if you have fourty employees practicing the same proceedure but all of which hold the pipette at different angles the procedure will potentially give different results per employee.

Hey Anyone,

I am noticing that the posting I am veiwing are from 2006. Are there any current postings????

Hey Anyone,

I am noticing that the posting I am veiwing are from 2006. Are there any current postings????

Hello rravkin@aol.com,

These posts are from 2006, because that is the post you selected. The forum has over 20,000 posts, many of them recent and many from today.

Go to the forum main page and select a section to view.

We were told that as long as the plasma and the cells were in contact, it did not matter if there was or was not contact between the plasma/cells and the gel.

We were told that as long as the plasma and the cells were in contact, it did not matter if there was or was not contact between the plasma/cells and the gel.

I think you will find that an air gap between the gel and the reactants is essential because the gel sits below the heat transfer portion of the incubator.

Regards

Steve

:)

On 10/5/2006 at 1:20 PM, lef5501 said:

I was once told (or read) that when using the IgG gel cards, it is best to leave an air bubble between the serum and the gel layer. Supposedly this enhances the incubation of the reagent and the patient serum to better pick up weak antibodies. Has anyone else been told to do this or are they leaving an air pocket when testing antibody screens in gel? Sometimes it is very difficult to leave that air pocket when pipetting, just wondering what everyone else was doing.

I know this thread is really old. I was wondering if anyone else does this technique and what is your principle behind it. 

This thread is pretty old but as it comes up again.... this "air gap" is required to avoid having the Anti-Human Globulin (AHG) present in the gel matrix getting partly "neutralized" by the excess of human immunoglobulin from the plasma. Keep in mind that plasma is full of various human Immunogloblins which will be recognized by the rabbit AHG. This "partial" neutralization may weaken the reaction indeed. 

Same as in tube but there is no washing step requires as the air gap is there to prevent this neutralization. Once cells are sensitized after the 37°C incubation step, the card is spun and the AHG will catch the Ig bound to the RBCs leading to positive reaction.    

Edited March 17, 20223 yr by Arno

We were told by the Ortho rep that trained our staff that you need to have an air gap between the patient plasma/reagent rbcs and the liquid on top of the gel in the column for the same reason described in Arno's post.  Years ago we had a tech that always got weaker reactions with daily QC and we finally figured out that after she pipetted the rbcs and patient plasma into the gel columns, she would tap the card until the plasma/rbcs mixture touched the liquid layer in the column.  Her reactions were always 1-2 grades weaker than everyone else.  Once she stopped doing that, she got much stronger reactions.  We use a Biohit pipettor and hold the pipettor at a slight angle and pipette towards the side of the well.  This works well.  Should the rbc/plasma mixture touch the liquid layer in the well, we discard that well and repipette using a new well.

On 3/17/2022 at 5:32 AM, knelson said:

We were told by the Ortho rep that trained our staff that you need to have an air gap between the patient plasma/reagent rbcs and the liquid on top of the gel in the column for the same reason described in Arno's post.  Years ago we had a tech that always got weaker reactions with daily QC and we finally figured out that after she pipetted the rbcs and patient plasma into the gel columns, she would tap the card until the plasma/rbcs mixture touched the liquid layer in the column.  Her reactions were always 1-2 grades weaker than everyone else.  Once she stopped doing that, she got much stronger reactions.  We use a Biohit pipettor and hold the pipettor at a slight angle and pipette towards the side of the well.  This works well.  Should the rbc/plasma mixture touch the liquid layer in the well, we discard that well and repipette using a new well.

Since a representative from Ortho says we need the “air gap”, does Ortho have actual printed material with instructions on how to do this air gap method? 

We were trained that you pipette the 0.8 RBC at about 45’ down the side (not hard) this ensures the RBC sit on top. The plasma gets pipetted straight  down. Inspect and incubate. I was just told that the air space ensures a space for the plasma and RBC to mix. No air space and many RBC go down the tube reducing the number that incubate with the plasma.

I find it interesting that various users have been told/advised to use a pipette in a manner that may compromise the accuracy of the volume delivered. I'm sure the pipette instructions indicate to use vertically.

Thankfully, the serological assays that are used by the Transfusion Medicine field have a wide range of tolerance. The "1-drop to 1-drop" concept is horrifying to many other pathology disciplines.