Gel vs Tube methods in IRLs

[Malcolm Needs ☆]

I have an example of an anti-M that is being monitored in a maternal patient for antibody titration. Since there is a collective wealth of knowledge here I figured there would be somebody with an answer. The father is an M-positive individual and the patient has had three titers performed now, the first in October was anti-M reactive at AHG - titer of 1:8, the second titer was 1:1 - AHG, the titer performed this month was again 1:8 at AHG but had an IgM component reactive at room temperature at a 1:2 titer. My question pertains to the significance of the titer results, is the mother considered a high risk pregnancy now that both antibody types are present in her sample? Also, what woud be considered a significant rise in titer in this patient, In my limited knowledge I have the understanding that the titer should have doubled to be considered significant, is this correct in relation to anti-M?

Many thanks

You have to be even more careful with anti-M titres than most other antibody specificities, a there are so many low frequency antigens associated with the M and N (but particularly the M antigen), which may mean that the red cells you are using may not be straightforward M+N+.

All that having been said, severe haemolytic disease of the newborn associated with anti-M is exceedingly rare and, as far as I know, there has only ever been one case of foetal death associated with anti-M (and, to be honest, that particular case leaves many unanswered questions as to the actual cause of the death, as many other possible causes were not ruled out).

In the UK, we do not usually get worried about maternal antibodies until they get to a titre of at least 32.

:comfort::comfort:

[Joanne P. Scannell]

Woah.

First, if you are detecting Anti-M in gel, it may be simply because gel is acidic (and if you remember from years ago, acidifying the serum was a way of enhancing Anti-M). To test if this is the case, test the patients plasma vs M-pos cells using one of the traditional tube testing procedures. We see this a lot ... perhaps we would have all those years before gel if we used acidified serum for routine testing.

Second, Anti-M is not considered clinically significant for HDN, therfore, we don't run titers on this antibody. (Not all IgG antibodies cause HDN for various reasons ... that's a whole 'nother conversation!)

Even if we did, there is no answer to your question about what titer is to be considered significant for Anti-M because there is no strong literature to support it. Furthermore, it's not the particular value of the titer, it's the significant movement of the titer (i.e. greater than one tube change) during the pregnancy (common practice is to test every month) that provides one of the clues that there may be a problem brewing that requires a closer look (e.g. amniotic tap). If the titer does not move, it is likely the child is negative for the corresponding antigen. Yes, we all know there are exceptions, etc. I'm just stating general practice here.

nb. Whenever we identify an antibody that is clinically significant for HDN (we are using gel), before preparing the titer dilutions, we test the patient's plasma vs antigen positive cells using tube technique which is essentially the 'neat' (or 0) tube of the standard titer (= tube testing, dilute with saline, no enhancement, see AABB Technical Manual, literature is based on this method). If it is negative, there is no titer and the result is reported with 'Below Titration' (or one could say 'less than 2').

Why are you titering IgM activity? IgM does not pass through the placenta.

Different for transfusion: If we find Anti-M, we test again prewarmed. If prewarmed test is negative, the result is 'Cold Agglutinin: Anti-M' and we transfuse just as any other cold agglutinin, i.e. use a blood warmer, period.

btw: Patients who are producing Anti-M but type 'M-positive' may actually be 'Mg-positive'. Mg is a common antigen that reacts with some reagent 'Anti-M'. Hence, it is not fruitful to type these patients for M when you know you are likely to get a positive result ... so don't bother. We don't even purchase Anti-M anymore.

That's not to say patients cannot develop an Auto-Anti-M ... I have a great case I call 'The Purple Lady'.

Careful ... Reference Labs test for and report EVERYTHING, significant or not. Just about every report I have from our reference lab states 'Cold Antibody at 4oC'. Yes, we are forced to pay them to test for and say that even though we don't care what happens at 4oC and most human beings have some Anti-I activity at that temperature anyway. It is up to the Medical Director of the Transfusion Service to determine what is clinically significant for any given case. e.g. our 'Purple Lady' and a clinically significant COLD IgM Auto-Anti-M. Cool case ... no pun intended!

[Malcolm Needs ☆]

Careful ... Reference Labs test for and report EVERYTHING, significant or not.

Whoa! Excuse me, but SOME Reference Labs test for and reort EVERYTHING, significant or not. Some are a bit more responsible.

:judge::judge::judge::judge::judge:

[Joanne P. Scannell]

Good news! Let me clarify that statement, 'reference labs that I have had experiences with'.

Thank you!

Whoa! Excuse me, but SOME Reference Labs test for and reort EVERYTHING, significant or not. Some are a bit more responsible.

:judge::judge::judge::judge::judge:

[L106]

Want to tell us more about the "Purple Lady?"

[hunb]

Woah.

Second, Anti-M is not considered clinically significant for HDN, therfore, we don't run titers on this antibody. (Not all IgG antibodies cause HDN for various reasons ... that's a whole 'nother conversation!)

Even if we did, there is no answer to your question about what titer is to be considered significant for Anti-M because there is no strong literature to support it. Furthermore, it's not the particular value of the titer, it's the significant movement of the titer (i.e. greater than one tube change) during the pregnancy (common practice is to test every month) that provides one of the clues that there may be a problem brewing that requires a closer look (e.g. amniotic tap). If the titer does not move, it is likely the child is negative for the corresponding antigen. Yes, we all know there are exceptions, etc. I'm just stating general practice here.

nb. Whenever we identify an antibody that is clinically significant for HDN (we are using gel), before preparing the titer dilutions, we test the patient's plasma vs antigen positive cells using tube technique which is essentially the 'neat' (or 0) tube of the standard titer (= tube testing, dilute with saline, no enhancement, see AABB Technical Manual, literature is based on this method). If it is negative, there is no titer and the result is reported with 'Below Titration' (or one could say 'less than 2').

Why are you titering IgM activity? IgM does not pass through the placenta.

Different for transfusion: If we find Anti-M, we test again prewarmed. If prewarmed test is negative, the result is 'Cold Agglutinin: Anti-M' and we transfuse just as any other cold agglutinin, i.e. use a blood warmer, period.

btw: Patients who are producing Anti-M but type 'M-positive' may actually be 'Mg-positive'. Mg is a common antigen that reacts with some reagent 'Anti-M'. Hence, it is not fruitful to type these patients for M when you know you are likely to get a positive result ... so don't bother. We don't even purchase Anti-M anymore.

That's not to say patients cannot develop an Auto-Anti-M ... I have a great case I call 'The Purple Lady'.

Careful ... Reference Labs test for and report EVERYTHING, significant or not. Just about every report I have from our reference lab states 'Cold Antibody at 4oC'. Yes, we are forced to pay them to test for and say that even though we don't care what happens at 4oC and most human beings have some Anti-I activity at that temperature anyway. It is up to the Medical Director of the Transfusion Service to determine what is clinically significant for any given case. e.g. our 'Purple Lady' and a clinically significant COLD IgM Auto-Anti-M. Cool case ... no pun intended!

I agree with you regarding the reference labs, all this did was confuse the ordering physician by having 2 different titer results on the most recent sample. For the anti-M there has not been a tube change in the titer it remains 1:8 from the initial sample. The

reference lab is still doing tube method for most testing and is using Quotient BD for their reagent supplier. In the future I will only report the anti-M IgG titer as this is the relevant result for the physician.

I questioned the initial order from the physician as there has been little in my career to indicate the anti-M would be problematic in regard to HDN. Now an anti-Kell or anti-E by all means I would pursue the titer to assess the risk for HDN. Thanks for the wonderful answers

TX Blood Banker