Why to add first serum when grouping and crossmatching

[fiza]

Hi Anyone,

I wonder why serum is first added when grouping & crossmatching is performed?

I would be happy if anyone could explain me the reason behind this.

Thanks in advance.

fiza

[Malcolm Needs ☆]

I am assuming you are talking about adding the serum first when performing tube techniques?

As far as I know, it is so you can check that the serum has been added to all tubes prior to the addition of red cells.

Once the red cells have been added, it is very difficult to tell whether or not the serum has been added, as the volumes used are usually so small that it may be impossible to be certain just by looking at them.

Of course, if the serum has not been added, you may get a false negative (false compatibility) result.

:):)

[fiza]

Thanks for the help Malcolm Needs. It was really helpful & I appreciate your answer.

Regards

fiza

[fiza]

Isn't it because albumin first will block antibody sites or red cell will adhere to glass or tube surface if serum is added first? Could you please explain the answer scientifically?

Thanks in advance

fiza

[fiza]

Sorry! in my previous reply before this, I've said "if serum is first added"

The correct way is "if serum isn't first added" will the albumin block antibody sites and or will red cell adhere to glass or tube surface?

Thanks in advance

fiza

[kate murphy]

As Malcolm has stated, it really is just good lab practice. If you are interrupted during pipetting, you would not be able to tell if serum had been added if cells went in the tube first. False negative results. It has no bearing on the reaction, as the potentiators, heat and centrifugation are what really get the cells close enough to agglutinate.

[fiza]

Can I conclude your answer as that adding washed red cells to a glass tube initial to adding serum has no bearing on the reaction.

The only thing we would know is that adding serum first to the tube would help us to prevent from false negative results.

And if washed red cells is added to the tube initially, we would not be able to tell if serum had been added to the tube, if we are interrupted during pipetting.

If my conclusion is wrong please assist me further; with your answer.

Regards

fiza

[Malcolm Needs ☆]

Can I conclude your answer as that adding washed red cells to a glass tube initial to adding serum has no bearing on the reaction.

The only thing we would know is that adding serum first to the tube would help us to prevent from false negative results.

And if washed red cells is added to the tube initially, we would not be able to tell if serum had been added to the tube, if we are interrupted during pipetting.

If my conclusion is wrong please assist me further; with your answer.

Regards

fiza

Hi fiza,

Yes, that is exactly what I meant.

The actual order that the reactants are added should make no difference whatsoever to the results obtained. The serum is added first purely because it is easier to see it in the tubes before the red cells are added, rather than vice versa.

Best wishes,

Malcolm

:)

[fiza]

Thanks guys for your great help on the subject. I do really appreciate all your replies & it helped me a lot.

[trisram]

error

Edited March 13, 2010 by trisram
it was too mean

[fiza]

Hi trisram,

If you don't have an appropriate answer to my question, I guess you have the right to remain silent.

I hope nobody has given you the authority to make decisions on me.

Moreover,my advice to you is; please mind your own business.

And if you want to reply to any member's question in the future, please reply to the subject. Don't try to make decisions on them.

[trisram]

Hi trisram,

If you don't have an appropriate answer to my question, I guess you have the right to remain silent.

I hope nobody has given you the authority to make decisions on me.

Moreover,my advice to you is; please mind your own business.

And if you want to reply to any member's question in the future, please reply to the subject. Don't try to make decisions on them.

I wasn't trying to make decisions on you. I apologize if it seemed that way. I am just tired of incompetent people working in the medical industry. Enough patients have died already.

Nothing against you personally because I don't know anything about you. But, anyways, I apologize, so sorry.

Edited March 14, 2010 by trisram

[fiza]

It's Ok. I don't mind that.

But in the other hand if you are that much worried. Have you tried any methods to avoid this?

Since this is a forum, people will post different kind of questions.

It's not that they aren't confident for their work or they don't know anything.

It's to give messages to the coming generations so that all types of errors could be avoided.

I hope you would in the future think twice before you post issues in this forum.

Anyways keep smiling & enjoy life.

[heathervaught]

Fiza - I'm not doubting the answers given by the previous posters (I agree with them), and I am not suggesting that your question was at all inappropriate for this forum (because I think it is a valid query), but your question of "does the addition of serum or cells impact the validity of test results" could easily be tested. Set up two identical testing systems, but add the serum first in one system and the cells first in the second. You can try it with various antibody strengths and specificities. At the end, you can come up with a summary for your management, and maybe even a piece of work that would lend itself well to being published as an AABB abstract (you would want to check the literature first and make sure that your findings were unique or otherwise not well documented).

We are, after all, scientists. It is within our nature to have a question and seek out the answer. Don't always be satisfied with taking someone else's word for it. Trying something hands-on can provide a valuable opportunity to learn more about the work that you do every day.

[Dr. Pepper]

I agree with Malcom. And for the same reason (that it's easier to see that you added it), had it been up to me I'd have made anti-D green instead of AHG, which you know you've added if your AHG-control cells work.

[fiza]

thanks guys for the replies. I do really appreciate them

[clmergen]

I wanted the Anti-A,B sera to be green because everyone knows yellow and blue make green.

[GilTphoto]

On the other hand, if you get distracted while adding panel cells to a 12-16 cell panel that already have the plasma, lets say to hand out a unit of blood, the tubes will have different incubation times at room temp. This could cause antibody ID to be more difficult. Prolonged incubation at room temp may cause insignificant cold antibodies to raise their head and interfere.

[GilTphoto]

I wanted the Anti-A,B sera to be green because everyone knows yellow and blue make green.

The color green is already spoken for by AHG antisera!

[Malcolm Needs ☆]

On the other hand, if you get distracted while adding panel cells to a 12-16 cell panel that already have the plasma, lets say to hand out a unit of blood, the tubes will have different incubation times at room temp. This could cause antibody ID to be more difficult. Prolonged incubation at room temp may cause insignificant cold antibodies to raise their head and interfere.

True, but then the same would apply if you were disturbed adding serum/plasma last; those first few tubes would have the serum/plasma sensitising the red cells much longer.

It is also easier to see where there are no red things in the tubes, rather than where there is no yellow stuff in the tube that has already got red things in it.

:confused::confused::confused:

[havatselet]

Hi folks. I hate to say this but I nearly always drop my cells into the tube before adding serum or plasma! My reasoning is that if my cell drop is too heavy (too large a volume or too heavy a suspension) I can only correct this before I've added serum/plasma. The patient sample is generally more precious and hard-to-come-by than the commercial reagent. Also, this is the way I was taught to do it 30 years ago! As for false negatives - I really think you'd realize you haven't added serum, if not before than at least after spinning your tubes. My question is - how do you know you've added plasma to gel cards, which require that you add cells first, and don't even have the benefit of check cells?

[Dr. Pepper]

You could put green dye in it.

[Malcolm Needs ☆]

. My question is - how do you know you've added plasma to gel cards, which require that you add cells first, and don't even have the benefit of check cells?

You are supposed to layer it, rather than mix it, so a quick visal check should be made.

:)

[vilma_mt]

Hi folks. I hate to say this but I nearly always drop my cells into the tube before adding serum or plasma! My reasoning is that if my cell drop is too heavy (too large a volume or too heavy a suspension) I can only correct this before I've added serum/plasma. The patient sample is generally more precious and hard-to-come-by than the commercial reagent. Also, this is the way I was taught to do it 30 years ago! As for false negatives - I really think you'd realize you haven't added serum, if not before than at least after spinning your tubes. My question is - how do you know you've added plasma to gel cards, which require that you add cells first, and don't even have the benefit of check cells?

I have done this "exercise" for Proficiency/Competency testing....Techs would visually judge spun Ortho gel card, tubes have varying amounts of plasma and cells. They were asked to recognize which tube has plasma, <0.8% or >0.8% suspension of cells. And yes with this exercise one can tell if the right amounts of plasma and cells were added.

[rravkin@aol.com]

Sorry! in my previous reply before this, I've said "if serum is first added"

The correct way is "if serum isn't first added" will the albumin block antibody sites and or will red cell adhere to glass or tube surface?

Thanks in advance

fiza

Hey Fiza,

I think that you answered your own question here. I have not responded to this thread because I could not remember the exact reason why we add non-cellular reagents first. I'm remembering! It has to do with the way we detect agglutination using the tube method. The only way we detect agglutination is through the dispercement of the cell button. If one takes a test tube, holds it horizontally, places a drop of cellular reagent in it, and spins the tube immediately at the most minimal time in the serofuge one would see a partial cell button at the bottom and a streak of cells running along the side of the tube. We would not be able to detect agglutination in the streak of cells therefore they would be excluded from any reaction interpretation. The procedure I state here is an extreme case but brings home a valid point. We add our non-cellular reagents first, not only to know that they were added, but also to coat the side of the tube so that, as you said, the cells will not stick to the side of the tube. Also remember that when we make our cell suspentions there can be some varience in concentration. Idealy you would want to try to place the cellular reagent drop similarly as the noncellular reagent in the tube. This will assure that all the cells make it to the cell button so that they can be a part of the reaction, if any. Given the fact that we can see weak reactions macroscopically; in these cases every cell counts. Most importantly, however, is that in the Blood Bank the very worst interpretation that can occur is the false negative and in practice we do everything we can to avoid this, even something as simple as adding noncellular reagents first. I hope this helps.

rravkin :)

Edited March 30, 2010 by rravkin@aol.com