Pregnant patient with an Anti-M reacting through all phases(immediate spin, RT, 37oC and IgG). 4+ IS, 4+ RT, 3+ 37oC, 2+IgG with homozygous M (heterozygous is slightly weaker). 37oC and IgG was with Lo Ion enhancement. .01 M DTT treated serum and PBS control reacted same strength indicating incomplete denaturation of IgM class. We had some suspicion that the DTT was not working however we had used the same DTT on other patients and it worked. Could it be the strong reactivity of the Anti-M? We performed some titration studies using neat plasma/serum, the DTT treated serum and PBS control with results as follows:

 

Neat: IS 32  30 min RT- 128   37 Lo Ion-32 and IgG- 16

 

60 minute 37 incubation IgG phase no enhancement: <1 

 

DTT treated no enhancement IgG: <1

 

PBS control no enhancement IgG: 32

 

Any thoughts are appreciated!!!!!!

 

Did you try the prewarm testing technique to see if the Anti-M reacts at AHG?

Did you try the prewarm testing technique to see if the Anti-M reacts at AHG?

Am I right in thinking you are doing this in tubes? And that you are taking the same tubes through the different techniques? If so, I would pre-warm as jill above suggests and ONLY do an indirect antiglobulin test (without LISS) at 37°C, using warm saline to wash with.

We performed a prewarm Lo Ion panel and Peg panel.The results were: 1 out of 3 M homozygous cells reacting and 3 heterozygous negative. Yes we are testing in tube. The titer for monitoring maternal specimen was performed using no enhancement and was negative at 1:1. At this point the anti-M appears to be IgM but the DTT does not seem to concur.

Are you trying to determine the class of the antibody or the clinical significance for transfusion?  Even though often natually occurring and cold reacting, many/most anti-M's contain an IgG component.  Your DTT treatment results appear to have confirmed that the antibody is or contains an IgG component.

Many anti-M's have a cold-reacting IgG element to them (as SMW infers).

 

As long as the anti-M does not react strictly at 37oC, it should not be clinically significant, whether it be IgM, IgG or a mixture of the two.

I wonder if there's an anti-M1 in there too?

From a strictly practical approach, I don't understand the quandry.  Since it's reacting at 37 degrees, just crossmatch using M ag negative units and move on.  Why was all the additional testing necessary?

Actually, it seems that the OB folks in our area read about a baby developing HDN from potent anti M.. We have been asked to distinguish between an IgG and IgM anti-M to decide how to proceed with the serological evaluation of the pregnancy.

 

In our case DTT treatment has not eliminated the reactivity at IgG.  Thus the concern for a mixed IgG/IgM antibody.

 

I was thinking that our anti-IgG reagent was made to the determinant on the Fc region of the IgG and should not react with IgM monomers formed after DTT treatment that might be hanging on to the RBC membrane after washing and the IgG reagent is cross- or non-specifically reacting. Strange things seem to happen when the antibodies don't read the textbooks. 

 

Another possibility is that the IgM was not completely disassociated by the DTT and there is residual pentamer on the RBC causing the residual reactivity.  I have rarely seen a 4+IS reaction with an anti-M.  When DTT treated, 1-2+ reactivity disappears in our lab. So we are going to cook the antibody a little longer in DTT.

 

Thanks in advance for any other suggestions.

 

Update:  Cooking longer (2 hr and 3 hr) did not result in negative reactivity. It is 1-2 +.  It seems to me this is a mixed IgG/IgM anti-M antibody so I guess I will recommend serial titers to OB.

Edited October 9, 201311 yr by Kip Kuttner

Do you have a reference for the article about HDN from potent anti-M?

There was an article published in the mid-80s about an M induced HDN.  We did not think it was very scientific as I recall.

Geoff Daniels quotes no less than 7 papers in the 3rd edition of his book Human Blood Groups. They are:

Yasuda H, Nollet K, Ohto H. A review of hemolytic disease of the fetus and newborn due to MN incompatibility in Japan. Vox Sang 2009; 97 (Suppl. 1): 125 (Abstract).

Stone B, Marsh WL. Haemolytic disease of the newborn caused by anti-M. Br J Haematol 1959; 5: 144-147.

Macpherson CR, Zartman ER. Anti-M antibody as a cause of intrauterine death. A follow-up. Am J Clin Path 1965; 43: 544-547.

Yoshida Y, Yoshida H, Tatsumi K, et al. Successful antibody elimination in severe M incompatible pregnancy. New Eng J Med 1981; 305: 460-461.

Duguid JKM, Bromilow IM, Entwistle CD, Wilkinson R. Haemolytic disease of the newborn due to anti-M. Vox Sang 1995; 68: 195-196.

Furukawa K, Nakajima, T, Kogure T, et al. Example of a woman with multiple intrauterine deaths due to anti-M who delivered a live child after plasmapheresis. Exp Clin Immunogenet 1993; 10: 161-167.

Kantra T, Yuce K, Ozcebe OI. Hydrops fetalis and intrauterine deaths due to anti-M. Acta Obstet Gynecol Scand 1996; 75: 415-417.

Hinchliffe RF, Nolan B, Vora AJ, Stamps R. Neonatal pure red cell aplasia due to anti-M. Arch Dis Child fetal Neonatal Ed 2006; 91: F467-F468.

Wikman A, Edner A, Gryfelt G, Jonsson B, Henter J-I. Fatal hemolytic anemia and intrauterine death caused by anti-M immunization. Transfusion 2007; 47: 911-917.

Geoff speculates that anti-M could have a similar aetiology to anti-K, in that it attacks very early erythroblasts, leading to a sort of suppression of erythropoiesis, hence the neonatal pure red cell aplasia.

Tagging along on this one - interesting...

By coincidence, I found out this morning that the following manuscript has just been accepted for publication in Transfusion Medicine Reviews.

 

Hiroyasu Yasuda MT, PhD, Hitoshi Ohto MD, PhD, Kenneth E. Nollet MD, PhD, Kinuyo Kawabata MT, Shunnichi Saito MT, Yoshihito Yagi MT, Yutaka Negishi MD, Atsushi Ishida MD.  Hemolytic disease of the fetus and newborn with late-onset anemia due to anti-M: a case report and review of the Japanese literature.  PII:  S0887-7963(13)00065-5.  DOI:  doi: 10.1016/j.tmrv.2013.10.002.  Reference:  YTMRV 50382.

 

Although I have printed off a copy, this is so hot off the press, as it were, that I have not yet had time to read it myself.

Another point of concern, for me is, when antibodies are made due to a first time antigen exposure IgM class antibodies appear first.  When working up a prenatal, how do you know that the anti-M IgM just found is not due to a first time exposure and the antibody that follows in 8 or so weeks will not be a clinically significant IgG?

@Kip, good question I'd like to know what everyone else thinks about that.  We just had one and determined the anti-M was IgM but the doctor insists he wants to keep performing a titer in case it becomes IgG.  So what was the point of having us determine the Ig classification?!

Well, the thing is with anti-M that, quite often, it is a mixture of IgM and IgG, BUT, the IgG is "cold reacting", and so will still not be clinically significant in pregnancy. It is more useful to look at the thermal range of the antibody, once it is determined that it is IgG.