Can change the order in gel card test? Why?

[sunan20090128]

Can change the order in gel card test? Why?

When using gel card (anti-human globulin) for cross-matching , can be added by first serum , then, added red blood cells ?

Edited November 6, 2009 by sunan20090128

[David Saikin]

You need to add the cells first . . . you don't want the plasma traveling down the column with the cells layered on top of the plasma.. Yes, the cells do on occasion, but the directions are cells first. With tubes it does not matter, with gel I think it is more than appropriate to add the cells first.

[TimOz]

I have always added serum first. The reason is that the earliest DiaMed and OCD BioVue instructions were written this way and I find it better as it is more reliable at obtaining the air bubble. I believe this has to do with the serum being more viscous than 0.8% red cells in LISS suspension reagent. We also validated the CAT methods by this method.

After all, the mix in the incubation chamber is the same no matter what the order. I think the importance of the addition order comes form the tube LISS Additive method where the LISS Additive must not be added directly to serum as this can cause precipitation of immunoiglobulins. I have heard this and taught it but never seen it proven with modern LISS Additive reagents.

And David. I agree in that the manufacturers instructons should always be followed or any deviation characterised and validated.

Any column that does not have a clear and visible bubble cannot be used and must be repeated, no what the order of addition.

And an anecdote - I was in an Asian lab last week where a lab worker I was training said she went to a great deal of trouble tapping the card to get rid of that pesky bubble! READ THE MANUFACTURER INSTRUCTIONS.

[galvania]

I have always added serum first. The reason is that the earliest DiaMed and OCD BioVue instructions were written this way and I find it better as it is more reliable at obtaining the air bubble. I believe this has to do with the serum being more viscous than 0.8% red cells in LISS suspension reagent. .

Tim - I am curious about this. I have been using the DiaMed cards since 1990 and have never seen anything that says that the serum/plasma should be added before the red cells, but always the contrary.

[Steven Jeff]

I am puzzled too. In the Ortho BioVue User manual which sits on our shelf, every procedure shows the sera/plasma as being added last. The exception being DAT's where only the cells are used

Steve :)

[TimOz]

Well I NEVER advise doing anything except the manufacturer's instructions...but there is always an exception (golden rule No.3).

I think the science of an IAT CAT is simply an evolved tube method. After all, it is just a LISS method with the AHG wash and agglutination display modified. If you go back in time, the order of reagent addition into tube is usually serum, cells, LISS Additive or the other way around. What you cannot do is add serum and LISS additive together as (in theory anyway) the lowered ioinic strength can cause precipitation of proteins including immunoglobulins. I have never seen this proven but it comes from the Low and Messeters work.

Maybe it was a specifically Australian thing, but many CAT users (specifically BioVue users) chose to do their antibody screening using 3% cells with a tube LISS additive. A few workers found that the LISS suspension reagents were insensitive (compared to their LISS Additive tube method) and the stability of the cells over time was not great. In this case, it is easiest to add the serum first, 10uL of 3% cells and then 40 or 50uL of LISS Additive. You can't easily add 10uL of anything and keep the bubble. This protocol is still available on the OCD AutoVue/Innova instrument and is still used.

So, my experience is that the serum viscosity actually makes it far easier to get the column bubble, even in DiaMed ID-MTS. While DiaMed advise to add the cells first, in theory there is no difference in adding the 25uL of serum first or last as there are only 2 reagent addition steps into a clean incubation well. Whatever works best to keep the bubble seems the way to go. I even did a few antibody screens yesterday during a training exercise. I was trying to show how to mess it up and I had to try pretty hard to not get the bubble when adding the 25uL of serum first. It was much easier to "burst the bubble" when adding the 50uL of cells first and I feel this is due to the low viscosity.

Anna, what do you think.

[galvania]

Hello TimOz

Well - actually, the gel technique is not really just a tube technique, especially when it comes to indirect antiglobulin tests - mainly because you have the Coombs reagent underneath - in tubes it goes on top. So it REALLY is important that the plasma doesn't go directly onto the gel. This is why it's always been said to pipette the cells first. If the cells should fall directly on to the gel it CAN (but doesn't always have to) weaken the reaction or even give a 'double population', because a proportion of the red cells won't have been in contact with the plasma at all. Obviously it's best to maintain the air gap. Usually, if you pipette the red cells at an angle of about 45°, it's really not difficult to do this. In any case, the cells should form a barrier between the gel and the plasma. If, when you pipette, the cells fall down the side of the reaction chamber, leaving the 'mouth' of the column free, then the card should be GENTLY tapped, so the cells cover the mouth. This is really important, because if the plasma falls directly on to the gel then there is a real danger that it won't come into contact with the red cells - or at least not all of it. This could seriously weaken a result, or even give you a false negative. You may also get a certain degree of neutralisation of the Coombs reagent. If red cells fall directly onto the gel, you can see them -- but if the plasma falls directly on to the gel, it could easily be missed - potentially disasterous. Also you pipette 50ul of cells and only 25ul of plasma, so the chance of 25ul NOT covering the mouth of the tube is much higher than for the cells.

There is another reason why the cells should always be added firs, and it's to do with mixing. Ideally the cells are pipetted into the reaction chamber, at an angle, and finish up covering the mouth of the tube evenly. Most people I know achieve this almost 100% of the time with a bit of practise. Then the plasma is pipetted vertically on to the red cells. They either mix directly, or filter through the red cells, allowing the antibodies to meet their antigens during the incubation phase. If you pipette the plasma firs, then even with an air gap, I'm not sure that the mixing effect would be quite as efficient.

The volumes I am quoting are of course for the DiaMed gel technique; I can't talk for Ortho.

Edited November 26, 2009 by galvania
Added last 2 sentences for clarification.