Antibody Titration

[Malcolm Needs ☆]

I'm curious, can anyone site a current/recent reference study indicating the titer significance when "enhancement" techniques are used for titration studies of antibodies during pregnancy?

Point #1: The original and only antibody studied for HDN is Anti-D.

Point #2: The technique utilized was saline and 60 minutes incubation.

Point #3: This is the study most OB docs are made aware of during training and they were trained to those numbers. Any numbers provided to them using alternate techniques will be confusing to them and possibly dangerous to the fetus.

A little historic food for thought.

Oh John, I do apologise, but I'm going to have to disagree again!

Anti-D was most certainly the first antibody studied for HDN, but it is by no means the only antibody to have been studied.

Anti-c has also been studied extensively.

Over here, for these two antibodies we do not perform titrations, but rather we perform quantification.

All below assumes that the baby's red cells express the antigen (otherwise, of course, it would be nonsense).

For anti-D, if the level is below 4IU/mL, there is little chance (not no chance, but little chance) of an affected baby.

Between 4 and about 15IU/mL there is a moderate chance of an affected baby.

Between 15 and 20IU/mL, there is a high chance of an affected baby.

Over 20IU/mL, there is an almost certain chance of hydrops, unless there is intervention.

For anti-c, up to 7.5IU/mL, there is little chance (once again, not no chance, but little chance) of an affected baby.

Between 7.5IU/mL and 20IU/mL, there is a moderate chance of an affected baby.

Over 20IU/mL, there is an almost certain chance of hydrops, unless there is intervention.

We titrate all other antibodies, and are not really worried, unlesws the titre reaches well over 32, but in the case of anti-K and anti-k, we know that the antibody titre does not have to reach these stellar titres before severe HDNF can occur, because the Kell antigens are expressed extremely early on the red cell precursors in the bone marrow (much earlier than the Rh antigens).

There are other antibodies that have been studied though (not least what used to be called anti-Tja, which caused recurrent spontanious abortions in the first trimester).

Sorry John.

:redface::redface:

[Susan Patti]

We use 60 minutes, no enhancement and we have not adopted the uniform procedure.

[John C. Staley]

Nothing to be sorry about. I certainly misspoke by using the words "only" and "studied" in the same sentence. What I was trying to get across was that in my (limited) experience the only antibody most OB docs are even aware of is anti-D and their data comes from very old studies and providing them with data based on different techniques could be dangerous.

Obviously I've been out of the loop so enlighten me. What techniques/technology is utilized to determine the quantification of the antibodies you referred to and can you provide references to the information you have provided? I'm not sure how wide spread the quantificaiton of antibodies is on our side of the "pond". I know it was not readily available in my past practice and we still relied on titration as an initial indicator.

[Malcolm Needs ☆]

Hi John,

Thanks for that.

I'll probably have to do this early next week, as I'm on-call tonight (Friday) and Sunday.

Had a good start too. A group O patient with anti-V, anti-VS, anti-N, anti-S, anti-Lua, anti-Jsa, anti-Fya and anti-Fy3, (who also requires HbS- blood as she is a sickler) with an Hb of 4g/dL, and another MDS patient with mixed warm and cold auto-antibodies who requires a cross-match.

I wonder why these people can ONLY require a cross-match on a Friday night!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

And to cap it all, it so hot here that the milk for my coffee has curdled.

Edited July 10, 2009 by Malcolm Needs

[AMcCord]

Extra fun patients always show up at my hospital on holidays so we have to figure out some way to get the sample to the reference lab in the first place before we can even think about bugging the folks on call.

Good luck and my sympathies!

[Malcolm Needs ☆]

Extra fun patients always show up at my hospital on holidays so we have to figure out some way to get the sample to the reference lab in the first place before we can even think about bugging the folks on call.

Good luck and my sympathies!

Thanks for that. Well, I survived! I had another DAT positive sample in after I wrote that post and was actually in the lab for 10 hours from 17.00 to 03.03. Boy, was I craving for the coffee by the end!

Now, all we have to do is wait and see if the patients survived my cross-matching!

:rolleyes:

[carolyn swickard]

There has been a lot of discussion on this topic in the CAP Antibody Titer Survey. They have presented and recommended tha "standardized" method and we too had a reaction one tube lower when we incubated for 30 mins instead of our SOP of 60 mins. The standardized method also called for unbuffered saline, but since we don't have that anymore, all of our testing was in pHix buffered saline (we use Capture solid phase for routine testing). I don't know if that would make a difference too.

The results of the titer surveys have consistently shown a higher titer achieved in gel studies - I assume this is because gel acually has LISS enhancement(?) involved in the method. There are 2 distinct curves for tube testing vs gel testing over and above the curves involved when enhancement medias are used in tubes.

CAP has been trying to get some standardization in this test for some time. The CAP AB Titer survey final critiques have several references in them - might be useful. The best article I ever found was a Transfusion article - Transfusion Vol 41, Nov 2001 - titled "Practice Guidelines for Prenatal and Perinatal Immunohematology - revisited". We designed our SOP around that article and have been on the CAP survey mean for tubes since. I really hope they have another conference and put out another set of guidelines in the next decade too (seems to be about how often they do that).

Still one controversy even in that article - What kind of cell to use? heterozygous or homozygous for the antigen the mother has an antibody against. We still have trouble with picking a consistent cell depending on the antibody and the cells available to us. The CAP survey just gives you the cell now because they have tried to eliminate that part of the variability in the test results too.

[Malcolm Needs ☆]

Surely, as the foetus can only inherit the gene encoding for the red cell antigen in a heterozygous state, the antigens on the red cells used for titration should be expressed in a "heterozygous" state?

[Malcolm Needs ☆]

Nothing to be sorry about. I certainly misspoke by using the words "only" and "studied" in the same sentence. What I was trying to get across was that in my (limited) experience the only antibody most OB docs are even aware of is anti-D and their data comes from very old studies and providing them with data based on different techniques could be dangerous.

Obviously I've been out of the loop so enlighten me. What techniques/technology is utilized to determine the quantification of the antibodies you referred to and can you provide references to the information you have provided? I'm not sure how wide spread the quantificaiton of antibodies is on our side of the "pond". I know it was not readily available in my past practice and we still relied on titration as an initial indicator.

Hi John,

I've tried to attach a PowerPoint, with explanations under the slides, concerning the technology/technique. Unfortunately, even when I compressed it, it was too large to attach. If you would like to send me a private message with your email address on it to malcolm.needs@nbs.nhs.uk, or malcolm.needs@blueyonder.co.uk andI will try to send it that way.

As far as the clinical interpretation of the results is concerned, the most cited paper is:

Nicolaides KH, Rodeck C. Maternal serum anti-D antibody concentration and assessment of rhesus immunization. British Journal of Haematology 1992; 304: 1155-1156.

I hope that this is of help. If not, please feel free to get back to me.

[BBK710]

There has been a lot of discussion on this topic in the CAP Antibody Titer Survey. They have presented and recommended tha "standardized" method and we too had a reaction one tube lower when we incubated for 30 mins instead of our SOP of 60 mins. The standardized method also called for unbuffered saline, but since we don't have that anymore, all of our testing was in pHix buffered saline (we use Capture solid phase for routine testing). I don't know if that would make a difference too.

The results of the titer surveys have consistently shown a higher titer achieved in gel studies - I assume this is because gel acually has LISS enhancement(?) involved in the method. There are 2 distinct curves for tube testing vs gel testing over and above the curves involved when enhancement medias are used in tubes.

CAP has been trying to get some standardization in this test for some time. The CAP AB Titer survey final critiques have several references in them - might be useful. The best article I ever found was a Transfusion article - Transfusion Vol 41, Nov 2001 - titled "Practice Guidelines for Prenatal and Perinatal Immunohematology - revisited". We designed our SOP around that article and have been on the CAP survey mean for tubes since. I really hope they have another conference and put out another set of guidelines in the next decade too (seems to be about how often they do that).

Still one controversy even in that article - What kind of cell to use? heterozygous or homozygous for the antigen the mother has an antibody against. We still have trouble with picking a consistent cell depending on the antibody and the cells available to us. The CAP survey just gives you the cell now because they have tried to eliminate that part of the variability in the test results too.

Does anyone out there know why unbuffered saline and what difference it would make? Seems like alot to get unbuffered saline for a procedure that is done infrequently.

[Malcolm Needs ☆]

Seems to me that if they want standardisation, then buffered saline should be used, otherwise, potentially, everyone will be using saline at a slightly different pH (and, sometimes, at a wildly different pH), which affects the equilibrium constants of the Law of Mass Action (obeyed by antigen/antibody reactions) and there would be no standardisation whatsoever.

It appears to be contradictory.

[TimOz]

I hate to disagree with you Malcolm but I will.

I think it is NOT OK to use gel / CAT methods to do antibody titres - and I mean in average general and non-specialist labs. I can back this up with reports of terrible mistakes I have seen with this method and it is due to the issue you mention yourself. CAT systems can detect IgM and IgG and while they may look a little different in their reactions, they can be misinterpreted by inexperienced staff. I find that many immunohaematology staff I train are simply not aware of this attribute of gel / CAT systems. They use an AHG card and simply assume that the antibody they are seeing is IgG (and or C'). It never occurs to them that it could be IgM or both IgM and IgG. After all, the purpose of the antibody titre is almost always to determine the level of IgG that can cross the placenta and perhaps more importantly if that level is increasing.

I think gel / CAT is fine if the tests are also carried out in a saline card to rule out IgM or DTT is used to denature the IgM and allow only measurement if IgG. If not, it is too subject to misinterpretation by inexperienced staff. I would point out that the problems are NOT a problem with gel but a problem with misinterpretation of the class of antibody being observed.

I have seen cases where inexperienced staff reported high titre results that were due to IgM. Sadly, In this case it lead to foetal termination and it was likely the mother was in the process of seroconverting and would end up with a high level of IgG. Maybe the last chance at a normal pregnancy?

The other issue that has not been mentioned is the indicator cells used. We use and recommend a pool of at least 3 examples of R2R2 cells. The reason is that R2 is (supposed) to show less variability in antigen expression and therefore give better test precision. I have seen a case where a titre reactione were read like this:

4, 2, 1, wk, wk, wk, wk, wk, wk,........etc.

This was interpreted a a titre of > 10,000. While this is obviously wrong to an experienced Immunohaematologist, the lab reported this and the clinician unfortunatly acted on this result. The reactions were due to a HTLA and detected in gel and a single indicator cell was used.

I think the world needs an interbnational standardised method and it needs to be detailed as to diluent volumes, time, temp, indicator cells used, scoring system and interpretation. Australia has a method but is about 1/2 as complete as it needs to beand is still very undefined.

It may be that gel / CAT is actually better than tubes if DTT is used to pretreat the sample. After all, one of the major benefits of gel / CAT is that is it more precise and reproducable in inexperienced hands. I have yet to see this method studied, validated and published in a peer reviewed journal. Volunteers?

[Malcolm Needs ☆]

Actually Tim, I wouldn't disagree with what you have said at all, except that the people in my lab have been performing titrations on pregnant women day in, day out now for many years. As a result, they are extremely experienced in the methodology, and have shown the predictive correlation between titre by CAT and outcome of pregnancy.......BUT I agree entirely with you that performing these tests by CAT in inexperienced hands is simply asking for trouble.

[galvania]

.......BUT I agree entirely with you that performing these tests by CAT in inexperienced hands is simply asking for trouble.

I would add to that: Performing these tests by ANY methods in inexperienced hands is simply asking for trouble

[Malcolm Needs ☆]

Couldn't agree more Anna.