anti D titration

[Desoki]

Dear colleague;

I have 3 questions regard to anti D titration: -

1. We already use saline manual tube method for anti D titration without any enhancement media (albumin or Liss), my question is if I add enhancement media to the test like albumin or Liss it will affect the result?
   

1. Also shall I use coombs liss gel card in anti D titration or this may affect my result?
   

1. I always selected cell R₁R₁ for this titration but due to long interval between titration and next one, we use also cell R₁R₁ but from different panel, is this will affect my result?
   

[Malcolm Needs ☆]

Dear colleague;

I have 3 questions regard to anti D titration: -

1. We already use saline manual tube method for anti D titration without any enhancement media (albumin or Liss), my question is if I add enhancement media to the test like albumin or Liss it will affect the result?
   

1. Also shall I use coombs liss gel card in anti D titration or this may affect my result?
   

1. I always selected cell R₁R₁ for this titration but due to long interval between titration and next one, we use also cell R₁R₁ but from different panel, is this will affect my result?
   

The answer to your first question is "yes"; that is why it is, afterall, called an enhancement medium. Anti-D will sensitise the red cells in less time, but there will also be an increase in the amount of antibody senstising the red cells, because you are pushing the reaction equation (governed by the Law of Mass Action) to the right.

In answer to your second question, we went over to using gel cards for titration several years ago now, and found that, when we did parallel testing with the tube technique in the Chagne Control, in reality, there was very little difference (maybe on dilution stronger in gel).

In answer to your last question, although anti-D does not classically show dosage (as is the case with certain other antibody specificities, such as anti-M), the number of D antigen sites per red cell varies from phenotype to phenotype (R2R2 having, in general, the highest number of the common Rh types), they also vary from individual to individual of the same Rh phenotype. This is why we always used a pool of, for example, R1R1 donors when we are doing titrations, to try to minimise this variation.

[Desoki]

The answer to your first question is "yes"; that is why it is, afterall, called an enhancement medium. Anti-D will sensitise the red cells in less time, but there will also be an increase in the amount of antibody senstising the red cells, because you are pushing the reaction equation (governed by the Law of Mass Action) to the right.

In answer to your second question, we went over to using gel cards for titration several years ago now, and found that, when we did parallel testing with the tube technique in the Chagne Control, in reality, there was very little difference (maybe on dilution stronger in gel).

In answer to your last question, although anti-D does not classically show dosage (as is the case with certain other antibody specificities, such as anti-M), the number of D antigen sites per red cell varies from phenotype to phenotype (R2R2 having, in general, the highest number of the common Rh types), they also vary from individual to individual of the same Rh phenotype. This is why we always used a pool of, for example, R1R1 donors when we are doing titrations, to try to minimise this variation.

Thanks malcolm but which best cell to be used to anti D titeration (R1R1 which represent the the phenotype of 75% of asian like Saudi arbia where I work, or R2R2 which represent more antigen epitopes)

[Malcolm Needs ☆]

Well, neither actually!

If you think about it, the foetus/baby must inherit a "d" gene (I know that this doesn't exist - maybe I should call it a "D Negative" gene) from the mother, and so, at most, they can only be heterozygous for the RHD gene (Or, if the dad is also D-, they would be D- too). To mimic the "D-ness" of the foetus/baby, therefore, I would use a pool of R1r red cells (and there is a difference between the number of D antigen sites per red cell between R1R1 and R1r - albeit this is so slight that it is very difficult to detect serologically), but, if this phenotype is rare, as I believe it is in certain parts of Asia, I would certainly use a pool of R1R1 red cells in preference to a pool of R2R2 red cells for titration.

By the way, sorry to be pedantic, but the number of epitopes is the same for R1R1 and R2R2, but the number of antigen sites per red cell differs.

Edited May 6, 2012 by Malcolm Needs
Forgot a bit.

[Mabel Adams]

Nowdays the mother may not be the genetic mother so the baby could be homozygous in cases of in vitro fertilization with a donor egg. Not common but worth remembering if you get seemingly impossible results on a baby's type.

We titrate with R2R2 cells but can't necessarily use the same ones every month. Nor do we have the resources to pool R2R2 cells readily (although that seems like a good idea). Whatever you choose for phenotypes, it is important to stay as consistent as possible with sequential titrations. Also, the proficiency testing results the ASCP publishes look like gel titers run at least one dilution or maybe two higher. I think you should do tube to gel correlations to see how much higher gel titers run in your hands. Doctors would need to be informed of the change in expected results so that they use an appropriate titer as a cut-off for follow-up testing--especially if it is invasive. The original cut-off titers were determined before LISS existed so using a more sensitive method (either LISS or gel) may increase the titer and give the doctor the idea that the problem is worse than it is because his textbooks refer to a saline titer.

[galvania]

dear msdesoki

If you freeze down an aliquot of the plasma that you used to do the first titration and then use it in parallel with your new plasma when you do your second titration you can see if any difference in titre that you observe is due to the cell or the amount of antibody in the plasma. For example if on day 1 your titre is 64 and 1 month later it is 128 - if your original plasma is now also 128 then it has not really risen - the difference is due to the cells or technique. the biggest advantage here of using cards instead of tubes is that you can keep pictures of the results and compare them. It's also a lot easier to read them. If you don't have access to a reader you can photocopy them

[Halima]

what if we use R0r for gel method do think it will affect the results?

we use R2R2 for tube method and R0r for the gel and when compare the results the they almost show the same dilution

[Malcolm Needs ☆]

what if we use R0r for gel method do think it will affect the results?

we use R2R2 for tube method and R0r for the gel and when compare the results the they almost show the same dilution

Halima, I'm sorry, but I've got to ask.

If you have only performed serological testing of these red cells, as opposed to molecular techniques, how do you know whether they are RoRo or Ror? One is homozygous for the RHD gene, and the other is heterozygous.

[Halima]

we get them commercially from Diamed kit

[Malcolm Needs ☆]

If they are Fy(a-b-), then there is an extremely good chance that they are RoRo.

[Halima]

do u think the use of R0r is a good idea for the gel titration? I will confirm the duffy group by tomorrow

[Malcolm Needs ☆]

Can't see any problem - as long as you are consistent with what you use.

[Mabel Adams]

But, Malcolm, isn't part of the question the fact that R0r cells from this month's panel might be R0r but those labeled R0r on next month's panel might really be R0R0? Then you would titrate this month with a heterozygous D cell and on her next specimen you might use a homozygous D cell. If you can freeze cells and use them month after month that would be different. Of course we also repeat the previous specimen with each new titer but we would not want to introduce more variables than necessary.

[Malcolm Needs ☆]

I agree, Mabel. That was the point I was, rather clumsily, trying to make; that with R0 red cells you can only really "guess" whether they are RoRo or Ror.

The problem is that, with R1R1 and R2R2, you are also "guessing", because they could be R1r' or R2r" in reality; but these are much, much less common than are Ror red cells.

This is why I would much rather use R1r (or R2r) red cells. These mimic the "obligate" Rh type (at least, as far as D is concerned) of the foetus/baby and will give a titre "closer to the reality" of the conditions the foetus/baby is experiencing.

That having been said, of course, R1r (or R2r) red cells could be Ror' (or Ror") in reality - but that doesn't matter so much, as the D will only be expressed in the heterozygous state (that is, if you ignore the number of D antigen sites that are different between the R1, R2 and Ro haplotypes).

[Mabel Adams]

Really they need to start producing reagent cells from those stem cell clones so we can always have a known cell from the same line to titrate against without freezing them.

[Malcolm Needs ☆]

Now that, Mabel, is an excellent idea!