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Confirming Weak A or B by adsorption elution

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Could someone share their procedure for confirmation of weak A or B antigen and antibody by adsorption elution. Specifically controls, how to make sure that the findings are weak A or B and not something else.

Thank you a lot

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This is me going back to the early 1970s, when I was first working at the International Blood Group Reference Laboratory (IBGRL) when it was still in London, and so my memory might not be 100% reliable, but I'll have a go.

Obviously, in those days we used to use polyclonal anti-A or anti-B that was derived from human donors, rather than monoclonal antibodies (essentially, there were no such things in those days).  We did know enough, however, not to use anti-A,B from group O donors, because of the chance of cross-reaction.

We incubated the red cells against the antibodies at 4oC for about an hour, and then washed them at least six times in normal saline (we didn't even use buffered saline in those days).  The last wash was kept as a negative control.

Next comes the clever bit.  We used heat elution at 56oC, as did many people, but the really clever bit was the way we kept the supernatant at 56oC during the centrifugation stage, so that the antibodies would not go back onto the antigens as the tests cooled down.  The Director of the IBGRL at the time was the late, GREAT Dr Kenneth Goldsmith (I use the term "great" advisedly).  He built a wooden box that contained the centrifuge, but also had a common light bulb in it that heated the entire contraption when turned on (we had to turn it on a good half an hour before we used it, to allow it all to come to temperature), but the whole contained a thermostat, so that the temperature as close to 56oC, so that a higher temperature did not denature the antibody, and a lower temperature did not allow the antibody to go back on to the antigens (he really was an amazing person - a doctor who was also an expert scientist).

The eluate and the last wash were then tested against A1, B and O red cells at room temperature (23oC incubator), which, of course, all acted as internal controls.

Later, we realised that the Lui elution technique (using melting ice) was a more efficient technique for eluting ABO antibodies, but everything else (barring the heated centrifuge) was the same.

Nowadays, we hardly ever bother.  It is very time consuming for virtually no return.  If the subject is a patient, we would transfused group O red cells, and if the subject is a donor, it is cheaper and simpler to exclude them, as long as they are counselled, to ensure that they do not feel "stigmatised".

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On 5/29/2018 at 1:33 AM, Malcolm Needs said:

This is me going back to the early 1970s, when I was first working at the International Blood Group Reference Laboratory (IBGRL) when it was still in London, and so my memory might not be 100% reliable, but I'll have a go.

Obviously, in those days we used to use polyclonal anti-A or anti-B that was derived from human donors, rather than monoclonal antibodies (essentially, there were no such things in those days).  We did know enough, however, not to use anti-A,B from group O donors, because of the chance of cross-reaction.

We incubated the red cells against the antibodies at 4oC for about an hour, and then washed them at least six times in normal saline (we didn't even use buffered saline in those days).  The last wash was kept as a negative control.

Next comes the clever bit.  We used heat elution at 56oC, as did many people, but the really clever bit was the way we kept the supernatant at 56oC during the centrifugation stage, so that the antibodies would not go back onto the antigens as the tests cooled down.  The Director of the IBGRL at the time was the late, GREAT Dr Kenneth Goldsmith (I use the term "great" advisedly).  He built a wooden box that contained the centrifuge, but also had a common light bulb in it that heated the entire contraption when turned on (we had to turn it on a good half an hour before we used it, to allow it all to come to temperature), but the whole contained a thermostat, so that the temperature as close to 56oC, so that a higher temperature did not denature the antibody, and a lower temperature did not allow the antibody to go back on to the antigens (he really was an amazing person - a doctor who was also an expert scientist).

The eluate and the last wash were then tested against A1, B and O red cells at room temperature (23oC incubator), which, of course, all acted as internal controls.

Later, we realised that the Lui elution technique (using melting ice) was a more efficient technique for eluting ABO antibodies, but everything else (barring the heated centrifuge) was the same.

Nowadays, we hardly ever bother.  It is very time consuming for virtually no return.  If the subject is a patient, we would transfused group O red cells, and if the subject is a donor, it is cheaper and simpler to exclude them, as long as they are counselled, to ensure that they do not feel "stigmatised".

I recently used this procedure to differentiate anti-Pr from anti-En(a), which is the only time I have used it. Since our lab refer possible Asub for genotyping lab, we have no chance of trying this out to catch weak subgroup of A. 

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Thank you Malcom very helpful. We will do the same Incubate 4C, perform Lui elution. Incubate at room temperature and test against A1-, B-, and O -cells.

I was unable to log in the website for a while,  hence the long response time. I changed my personal data, which created the minor issue.

 

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😁 Something I remember!!  Back in the "Reference" lab days when we could innovate on our feet without reimbursement issues, and probably some safety issues too.  Malcolm, if I may address you informally, if not pardon me,. we did the same process as you described except, we found an old incubator we could adjust the temp and put our serofuge inside to centrifuge, running the plug wire up through the hole of the incubator that normally would have a black rubber stopper holding a glass thermometer!   

Good story, thanks for the memory.

🐀 mic

 

 

 

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11 hours ago, Malcolm Needs said:

PLEASE mrmic, feel free to address me informally; and the same goes for any other member of the group as far as addressing me informally.  I am nothing special!

Folks, please read this carefully, I suggest he is saying it is acceptable to call him Malcolm, as compared to Mr. Needs - hence informal.  He did not suggest he would find it acceptable to refer to him as a tosser, wanker, or some other (really cool sounding British) pejorative. 

I need to move to England, if only for a short time.  American English is so boring.

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On 7/6/2018 at 4:35 PM, Cliff said:

Folks, please read this carefully, I suggest he is saying it is acceptable to call him Malcolm, as compared to Mr. Needs - hence informal.  He did not suggest he would find it acceptable to refer to him as a tosser, wanker, or some other (really cool sounding British) pejorative. 

I need to move to England, if only for a short time.  American English is so boring.

I think the term "Chav" has recently become popular. Even more recently, calling someone "a right old Neymar" is not very flattering.:)

As the Irish writer George Bernard Shaw once said: "England and America are two countries divided by a common language."

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On 7/6/2018 at 1:35 PM, Cliff said:

Folks, please read this carefully, I suggest he is saying it is acceptable to call him Malcolm, as compared to Mr. Needs - hence informal.  He did not suggest he would find it acceptable to refer to him as a tosser, wanker, or some other (really cool sounding British) pejorative. 

I need to move to England, if only for a short time.  American English is so boring.

Whats boring is American quality control departments? Cannot do anything without running into red tapes. :~

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