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We get lots of grouping problems due to weak reverse. When we cannot resolve it at RT we incubate at4Cx60'. This leads to nonspecific reactions due to cold agglutinins.

We set up a cold panel of 3 cell screen, A1 cells, A2 cells, B cells, and auto to find the specificity.

Q1. If everything comes up positive at 4C, how do you resolve this? We call it NTD.

Q2. Is there something else that we can do? We don't want to send samples for Geno?

Q3. If you have to do cold auto adsorption, how do you remove IGM coating the RBC? Same reagents as you use for ZZAP? DTT?

Q4. Most of the time these samples come to our lab for reference testing and we can not reproduce the initial results. The auto control becomes negative.  The nonspecific cold agglutinins disappear.

Initially, I thought it's due to cold auto adsorption but the cells are separated from the plasma. Any thoughts, what could be causing this? Antigen sites blocked?

There is a lag of one week between the initial testing and the reference lab testing. 

 

 

 

 

 

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3 hours ago, Learning from the experts said:

We get lots of grouping problems due to weak reverse. When we cannot resolve it at RT we incubate at4Cx60'. This leads to nonspecific reactions due to cold agglutinins.

We set up a cold panel of 3 cell screen, A1 cells, A2 cells, B cells, and auto to find the specificity.

Q1. If everything comes up positive at 4C, how do you resolve this? We call it NTD.

Q2. Is there something else that we can do? We don't want to send samples for Geno?

Q3. If you have to do cold auto adsorption, how do you remove IGM coating the RBC? Same reagents as you use for ZZAP? DTT?

Q4. Most of the time these samples come to our lab for reference testing and we can not reproduce the initial results. The auto control becomes negative.  The nonspecific cold agglutinins disappear.

Initially, I thought it's due to cold auto adsorption but the cells are separated from the plasma. Any thoughts, what could be causing this? Antigen sites blocked?

There is a lag of one week between the initial testing and the reference lab testing. 

With reference to the ABO reverse grouping problems that cannot be resolved at RT, instead of incubating the tests at 4oC for 60 minutes, have you tried incubating at 30oC, or, in extreme cases, 37oC, or using cord red cells.  Most of these "cold" antibodies have a specificity involving I or IH, and so, in the majority of cases these methods will resole the problems, without compromising the ABO antibodies, which, as we know from unfortunate transfusion reactions, work pretty well at 37oC!

Turning to your specific questions:

Q1.  Firstly, we wouldn't test the plasma at 4oC.  What is the point?  Apart from Zombies in Holywood films, how many humans do you know who survive with a body temperature of 4oC?  All we would do is test the antibody for its thermal amplitude, and if it does not react at 30oC, we ignore it (see Petz LD, Garratty G.  Immune Hemolytic Anemias.  2nd edition, 2004, Churchill-Livingstone).  If the antibody does react at 30oC AND is an alloantibody, we would be more proactive, identify the specificity, and give antigen negative blood.  If it was an autoantibody, we would ignore it for transfusion.

Q2.  Apart from the above, NO!

Q3.  Why would you want to remove the IgM from the red cells.  They will hardly ever be swamped by the autoantibody, but, if they are, we would use Rabbit Erythrocyte Stroma (RESt), which is available commercially.  Unfortunately, this also adsorbs anti-B, and so the plasma adsorbed like this should never be used for cross-matching, as a group B mis-match would never be detected.

Q4.  I am going to be VERY cheeky here, and I say this with no evidence whatsoever, and that I will probably be slated by most members on this site, possibly rightly so, but I think that, as you are a Reference Laboratory worker, your techniques for working with these samples are more "honed" than those of the average hospital laboratory, as you see them far more often than do the hospital laboratories.

I will now go and don body armour and a steel helmet!!!!!!!!

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2 hours ago, Learning from the experts said:

Thanks Malcom. We are trying to enhance the weak reverse, that's why we are testing at 4C. We do the test at RT first then at 4c x30', 4Cx60'.

Try 4 drops plasma and incubate for 30 min at RT, include an auto control. 

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11 hours ago, Learning from the experts said:

Thanks Malcom. We are trying to enhance the weak reverse, that's why we are testing at 4C. We do the test at RT first then at 4c x30', 4Cx60'.

If you want to enhance the weak reverse, have you tried pre-treating the red cells with a proteolytic enzyme, such as papain or ficin?  Obviously, this will require strict control.

Edited by Malcolm Needs
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