Differentiating room temperature anti-M from AHG using gel

[Sue Miller]

When using gel method anti-M sometimes shows as a mixed field which indicates to us it is probably and IgM cold reacting antibody. It does not seem clear cut to me though if it is not showing mixed field does that mean it is an AHG reacting antibody? According to CAP J-B educational challenge it states that only antigen negative units should be given to a patient who has an AHG or 37 degree reacting anti-M. What does everyone do with anti-M who uses gel? Do you give crossmatch compatible or antigen negative?

[Seveets]

If the anti-M shows up in gel in the antibody screen and is identified in the gel antibody panel then we give M- fully compatible units. I have not seen a mixed field in gel.

My last one was wishy washy in gel and would not go away with prewarming anything. So I had to screen the units. I proved it with a cold antibody id panel with the gel cards.

[Mabel Adams]

I have seen many anti-M come up in gel. Cold antibodies do have a characteristic look to them--not in my mind quite like the mixed field reactions Ortho put in the Interpretation Guide but with small amounts of cells spread throughout the column and a bigger button in the bottom than one would expect for the amount of reaction in the column.

The only way I know to prove that anti-M is "cold only" is to do a tube test (I do LISS) at the AHG phase only. If it does not react at AHG, I conclude it is "cold only." I think Issitt said that even if anti-M does react at AHG it is only necessary to give xm compatible units, not screened with commercial antiserum. (My experience with anti-M in gel is that there are seldom any weak ones.)

I often find the fastest way to find compatible units with a strong anti-M is by doing an IS XM and then doing a gel XM on those that don't react at IS. There is even a CPT code for screening for compatible units using the patient's serum/plasma which I use in these cases.

[Geriann]

When anti-M is suspected in Gel-AHG we use the tube method through the AGH testing phase. If anti-M is identified we run M- panel cells to rule out additional clinically significant alloantibodies. We perform a prewarm technique with screening cells (make sure to include at least 1 M+N- cell sample) to determine if the Anti-M reacts in the AHG testing phase under these conditions. If the prewarmed results are negative in the AHG testing phase we use prewarm technique through the AHG testing phase for crossmatching. We only screen units for M if the anti-M reacts in the AHG phase using prewarm technique. Rule outs for additional clinically significant alloantibodies are performed using M- cell samples in Gel-AHG. I have found many anti-Ms in Gel that do not react in the tube AHG testing phase using prewarm technique. I use Saline-AHG tube method for prewarm technique.

[janet]

I think Issitt said that even if anti-M does react at AHG it is only necessary to give xm compatible units, not screened with commercial antiserum.

We follow what ISSITT said and give crossmatch compatible.

I have seen more than a few strange looking M's that go along the side of the gel (almost like the card wasn't pushed down in the spinner all the way)

[OPUS104]

We call the Gel cold reactions "waterfall" reactions in our reference lab. We also see quite a few "mixed field" (yes we know mixed field is impossible with reagent cells) appearing Kells. These have a really cool banded appearance. (We think these are IgM/IgG mixes) If we detect an M or other usually cold antibody in Gel, we give negative units. Most people I have talked to consider Gel "Coombs phase". If it reacts in Gel, we are not going to test in tubes to see if it will go away. We have several uncommon antibodies that are going through coombs right now, including a P1 that is 4+ in Gel. We screen for anything that reacts in Gel since most of our hospitals will croosmatch the units in Gel. You can do a "pre-warmed" in Gel, but the centrfuge is of course not going to be 37 degrees.

[Eagle Eye]

We give gel-compatible units for all anti-M reacting in Gel. We also perform tube method screening and prewarm it out to make sure they are cold.

I belive some cold antibody do react in gel eventhough they are IgM.

[Julie]

It is important to remember if identifying anti-M in a patient undergoing open heart surgery to perform thermal amplitude studies to determine at what temperature IgM anti-M reacts. This is due to the patient being "cooled down" for the surgery and prevents problems with performing the surgery. With prior knowledge of the thermal amplitude, the surgeon can either alter his/her approach, or not cool down the patient as much.

[Cathy]

What do you all do when you find an M on your OB patients? We have found several that react in gel and are negative in tubes and the OB physician insists on having a titer.

[Mabel Adams]

I got good advice once from John Judd on an OB with an anti-M reacting in tube with anti-IgG (so it apparently had at least some IgG portion). I couldn't really titrate just the IgG part which is all that really matters in an OB. So he suggested we titrate the "whole" antibody and, as long as the total did not have a titer above 16, we knew the IgG portion alone couldn't. Since many of these anti-M's in pregnancy may not be really significant, this worked great. The titers never rose. We dealt with at least one of these women through more than one pregnancy.

We still do our titers in tube because we don't really have a way to correlate the results of gel titers with patient outcomes without collecting data for 30 years (and I don't have the energy right now to figure out how to just correlate the titer results between methods). When we find an antibody in gel, I have been known to run it neat by the titer procedure and if it won't react, I have turned it out as "too weak to titrate." Otherwise we end up turning out titer results of "< 1".

[OPUS104]

Mabel,

If you find some way to correlate titers in Gel, I believe you may receive the Nobel! If anyone knows anyway to compare, please share. I can titer out HTLAs for a year in Gel. Ortho (as most of you know) has no data and appears to not be pursuing any data on titers in Gel.

[Mabel Adams]

There certainly are labs that have validated gel titers and are using them routinely. I don't know how they did their correlations.

[Jane Pilz]

Mabel, we validated gel titers and found that there is an average of two dilution strengths between tube and gel for Rh antigens, gel being more sensitive. The difference is less for non-Rh antigens, averaging one dilution stronger for gel. MTS sent us a study done in the Netherlands that supports this. You will need to change your critical titer values for pre-natal testing if you change to gel.

The study from MTS showed that for higher titer antibodies, the difference between gel and tube are smaller.

[webersl]

Can someone explain the logic of why most of these blood bankers, even though they have proved that the anti-M is not reactive at 37C and therefore is not clinically significant, why they proceed with full AGH crossmatch, instead of just doing an immediate spin crossmatch? AABB Standards 5.15.1.1 are clear that you are only required to do and ABO crossmatch as long as CLINICALLY SIGNIFICANT antibodies are not found (and no history of such). I understand that by choosing to do ABO crossmatch only, you will get interference with the anti-M, so you will have to crossmatch many more that you actually need to see a clealy negative ABO crossmatch, but this saves a lot of time and reagents. What is the logic - why continue with the AHG crossmatch?

[Malcolm Needs ☆]

I agree with the vast majority of these posts (particularly Mabel Adams, Geri Ann, Janet, aakupaku and webersl).

Cathy, if the OB physician insists on a titre for the anti-M, and the anti-M does not react by pre-warmed, warm-washed tube IAT, why not cut your losses and report the the antibody as "too weak to titrate" (sorry about my UK English spelling), without titrating in gel. You are, after all, not lying. The antibody is too weak to titrate by a method that measures antibody titres that may affect the foetus/baby.

[Cathy]

We are reporting the titers as <1 if reacting in gel but not in tubes.

I think the reason we continue to perform gel crossmatching is just for consistency, positive antibody screen = coombs crossmatch.

[TimOz]

Just to wade in on the question of Gel antibody titres.

In Australia there has been (sometimes fierce) debate about antibody titre methods. Some say you must use a defined tube method while others move to gel. My own opinion is that if you use the gel method it is natural to want to titre antibodies in gel BUT I often find great misunderstanding about what gel can an cannot do and what you are actually detecting. I realise that this will be obvious to some here but I think not others. You can validate the gel method with a tube assay and correlate the results but if you do this using a number of IgG samples you will not understand the test performance with samples containing IgM. You must not forget that a very significant difference between gel and the tube method is that an AHG gel card can and will detect both IgM and IgG. The commonest use for an antibody titre is to try to determine a rising titre of IgG that may cause HDFN. For this purpose you do not want to detect IgM and want to report only the IgG titre. Gel cannot do this unless you understand whether you are seeing IgG only or if IgM is also present. You can do this by using a direct tube test, Saline (neutral) gel cards or DTT treatment to ensure no IgM is present.

I have been told (anecdotally) of 2 sad cases where preterm foetuses were terminated on the basis of a single high antibody titre result. One case was a IgG with a titre of 1 (neat) with high titre IgM (256 in tube if I recall) that was simply reported as a the 256 titre. I may be misinformed but I understand that it was the woman's first pregnancy

The second case was a low titre IgG but the patient had an anti-Bga that was high titre and the lab used unpooled cells that by chance were Bga pos. The titre was reported as >10,000. Note that these cases are NOT gel systems failing but cases of poor clinical decisions and cases of users not testing properly and misinterpreting the test.

Australians tend to use a standard tube based antibody titre method called the NICE (National Immunohaematology Continuing Education) method. This is a reasonable method but still does not define the indicator cells and I feel that is a failing and source of imprecision. More and more users are moving to gel and I hope that characterise the test well and even though the correlation with tube can be validated, whatever method is used, attention must be paid to antibody class. Australasian External Quality Assurance Programmes show a wide variety of antibody screening methods in common use and an unsurprising but frightening imprecision in this test.

For those that are interested in this topic, some work was done many years ago on chemiluminescant assays that appeared to provide very good prediction of an antibodies clinical effect. Unfortunatley this assay has very poor correlation with serological methods as it measures different attributes of an antibody and it never caught on.

[Malcolm Needs ☆]

Quite agree with what you say Tim.

As for the chemiluminescence test, the same can be said for most bioassay techniques tried over the years - poor corrolation.

To a certain extent, this was not surprising, as the white cells used were from random individuals, instead of from the mother. Even if the tests were to show what we wanted them to show (if you see what I mean), they would still not mimic the actual situation unless the mother's own immune system was involved in vitro.