Is anyone else aware of the possible reaction variability caused by different pipetting techniques when using gel?

There was an article in Vol 24 no 3 (summer 2011) of Clinical Laboratory Science that seems to say that it is a really bad idea to NOT have an air bubble over the gel during incubation. Weaker positives (up to 1+) are often missed if the incubation mixture of plasma and cells is touching the gel.

Currently, I do not think our procedure (Ortho) says much about it one way or the other.

I remember during training by the Ortho rep many years ago as we instituted gel a discussion to this effect. The rep did not however say that a lack of an air bubble was a reason to throw the test card out and repeat. Not sure of the official stance Ortho has at the moment.

Our rep said while it is not ideal, there is no reason to discard a card/tube because of this. What happens is a small amount of cells do not get to react with the plasma. Is it clinically significant? I can't really say. There are days when my cards are filled to perfection and then there are other days . . .

We played around with the bubble or no bubble with some samples that were showing weak antibody reactivity and saw no significant difference.

The study I cited above seems pretty definitive. If a bubble is not present, you are going to miss weaker positives.

Because the gel technique is a standardised technique, any departure from the norm MAY cause false pos or neg reactions. For example, if your cell suspension is too weak, you MAY miss a weak pos DAT, or you may mis-identify a very weak weak D as D neg. The bubble is controversial. What the lack of the bubble will often do, when you are doing a reverse group, is give you a double population instead of a clear positive. In terms of indirect antiglobulin tests I have seen rare cases of VERY weak antibodies coming out negative when there was no air bubble and the cards were pipetted manually (this is not an issue in the automats). Having said that, I would not automatically repeat if I didn't have an air bubble. I would take it case by case - and, more importantly, if you're regularly not getting an air bubble when you pipette, then check your pipetting technique. Best is to pipette the red cells in at a 45° angle and then pipette the plasma vertically on top.

Edited November 8, 201113 yr by galvania
missed out bracket

When I was trained I was told that the reactions happen better if kept in the well - and was taught to pipette at an angle down the side of the well (but not touching). The reason they say this is that if the cells end up down the column part you don't get a complete vortex when you you add the plasma and thus don't get proper mixing. It *can*, in very low titre sample, result in the prozone effect. Correct pipetting technique completely eliminates this problem

Perhaps I am missing something here, but my understanding is that a bubble ensures that there is an incubation period prior to the cell/Liss mixture meeting the gel, beads(in the case of ortho BioVue) which contain the AHG/reactants etc. In the case of AHG reactions I would think it important to have a bubble, reverse groups which are immediate spin and no reagent in the gel less so.

Steve

:)

Perhaps I am missing something here, but my understanding is that a bubble ensures that there is an incubation period prior to the cell/Liss mixture meeting the gel, beads(in the case of ortho BioVue) which contain the AHG/reactants etc. In the case of AHG reactions I would think it important to have a bubble, reverse groups which are immediate spin and no reagent in the gel less so.

Steve

:)

That too but less so - the contact surface is actually quite small... Lack of vortex mixing and prozone has more of an effect.

Thanks all for your comments. The link to the article I referenced is:

http://findarticles.com/p/articles/mi_qa3890/is_201107/ai_n58121039/?tag=content;col1

From the posts given it sounds like the bubble will ensure that all of the reaction volume rbc's remain within the reaction volume and not prematurely migrate into the gell thus ensuring that all of the reaction volume rbc's have an opportunity to react. I was just wondering how is this bubble accomplished? Is it part of the gel-card manufacturing process?

. I was just wondering how is this bubble accomplished? Is it part of the gel-card manufacturing process?

The well narrows to a column towards the bottom. If the cells/reagent/plasma are pipetted at an angle towards the side of the well, it shouldn't go towards the column.

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Edit - I still feel that the weak reactions are more to do with improper mixing and prozone than contact with the gel itself (the actual surface are in contact is small).

If the red cells are pipetted and go into the column, when the plasma is added there is no way for it to mix. During incubation the red cells are circulation and fall through the plasma. If the cells are below the plasma already, this can't happen. Think of it as a combination on convection currents, brownian motion and the ESR . If nether the twain shall meet there won't be a reaction...

Edited November 9, 201113 yr by Auntie-D

Ortho has always been pretty ambivalent about the need for an air bubble between the reactants added and the gel already in the card. They say they have validated the US MTS system without a bubble so it is acceptable but you can miss very weak reactions if you don't have the bubble. I have known others that validated the difference and found it significant so always require a bubble. We required it at my prior workplace (partly because it seemed to reduce some hazy reactions we were getting) but not at my current workplace. It is always up for debate whether we must detect really weak antibodies as no method will get them all without picking up nuisance reactions as well. The bubble is one change that I think adds sensitivity without reducing specificity so I favor it.

In reference to Mabel's last couple of sentences: The article added that they found that non-specific "nusance" reactions seem to be reduced when a bubble is left during incubation. As some others pointed out here. So the bubble does indeed seem to increase both sensitivity and specificity.

I used the Ortho BioVue system (in the UK) which you will know from posts on another thread is quite different to that used in the USA. The design of the test well and the incubator with the Ortho BioVue demand that the contact point of the heat block with reactant area is in the upper area of the well, away from the beads i.e. there must be an air bubble. The whole cassette card is warmed with the Diamed system unlike the Ortho BioVue card

Steve

:)

Edited November 13, 201113 yr by Steven Jeff

Adding the link to this

 

http://europepmc.org/abstract/MED/21905581