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A positive with ? anti-A1


frenchie

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Hi everyone!

I have an OB patient (first semester of pregnancy) who has a history of A positive, 3 previous pregnancies, who is puzzling me and I would appreciate your input. My results are:

In gel,

Anti-A 4+

Anti-B 0

Anti-D 4+

Ctl 0

A1 cells IS (no RT inc.) w+, 15 min RT inc. 3+

A2 cells 0

B cells 4+

In tube, I have a weak A1 microscopic (and no rouleaux). A1 lectin is negative (weak pos micro, which the package insert says that a weak pos should be considered negative). 

Antibody screen negative at 37 C and RT. I ruled out P1 and M with gel.

Is the patient really A with anti-A1 IGM? I am just concerned because she is pregnant, I want to make sure that there is not something I am forgetting...

Thanks in advance,

Helen

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Sounds remarkably like an anti-A1 to me, which means that there is absolutely nothing to worry about as far as the pregnancy is concerned.

Anti-A1 is almost always IgM (which means, of course, that it will not be transported through the placenta) rarely reacts at 37oC (as in this case), and, in any case, the expression of the A antigen on feotal red cells is weak, due to the fact that the N- acetyl-D-galactosyltransferase (the actual gene product of the A gene) is not fully functional at birth.  In addition, ABO antigens are histoantigens and, should the maternal anti-A1 get stronger during the pregnancy (a remote possibility at the best of times), or should it become IgG (usually IgG2 anyway, which rarely gets transported through the placenta), will attach to other foetal tissues in preference to the foetal red cells.

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Another thing to consider...  Although it looks like Anti-A1, it could be some other room temp reactive IgM antibody.  Regarding the A2 cell reaction that was negative, was that also incubated at room temp like the A1 cells were?  In this scenario we would set up A1 cells, A2 cells and group O screening cells as well as a patient auto control and incubate all at Room temp.  Occasionally, an antibody other than anti-A1 (e.g. cold auto antibody, anti-P1 etc.) will be reacting with reverse grouping cells.  Some of those cold reactive antibodies can give variable reactions,some cells reacting immediate spin others requiring room temp incubation.

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  • 2 weeks later...
On ‎1‎/‎8‎/‎2016 at 6:40 PM, frenchie said:

Hi everyone!

I have an OB patient (first semester of pregnancy) who has a history of A positive, 3 previous pregnancies, who is puzzling me and I would appreciate your input. My results are:

In gel,

Anti-A 4+

Anti-B 0

Anti-D 4+

Ctl 0

A1 cells IS (no RT inc.) w+, 15 min RT inc. 3+

A2 cells 0

B cells 4+

In tube, I have a weak A1 microscopic (and no rouleaux). A1 lectin is negative (weak pos micro, which the package insert says that a weak pos should be considered negative). 

Antibody screen negative at 37 C and RT. I ruled out P1 and M with gel.

Is the patient really A with anti-A1 IGM? I am just concerned because she is pregnant, I want to make sure that there is not something I am forgetting...

Thanks in advance,

Helen

Helen,

At my current employer we are required to perform a tube panel and Auto Control, read at all phases, with the typing results presented. It's a rare occurrence to see the Anti A reaction so strong with a potential A variant.

If all antibodies are ruled out with your tube panel and your Auto control is negative at all phases then it would add much validity to the A1 lectin result. Without the auto control or a Poly DAT the possibility of a substance coating the red cells and preventing the A1 lectin reaction can not be ruled out; especially when your forward type Anti A reaction is so strong. Also, I would not rule out an anti M with gel in this scenario although your screening results lend some validity we do not know the number of cells used for the screen nor the distribution of M and other antigens; therefore a full tube panel at all phases is more sound. I hope this helps.

 

Edited by rravkin@aol.com
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I fundamentally disagree with this post frenchie.

I can assure you that we, as a Reference Laboratory, see large numbers of samples from group A patients who have an A subgroup, and with the monoclonal reagents used these days, it is extremely common to see a very strong reaction with anti-A, when the red cells have an A subgroup, such as A2, Aint or ABantu.  Granted, this is not so for A3, Ax or Am samples, but these are exceedingly rare, and your results suggest that your patient is anything but an A3, Ax or Am.

As Dolichos biflorus is a lectin, and not an antibody, I know of no substance that could be coating the red cells that would stop this lectin reacting with the red cells.  Indeed, the fact that there is a microscopic reaction suggests just the opposite, as Dol. biflorus is not, actually, an anti-A1, but will react with the A antigen (and the Cad antigen, come to that) unless it is diluted so that the lectin only reacts with A1 red cells (which is why the insert tells you to ignore microscopic reactions).

Lastly, gel technique is probably the VERY BEST and MOST SENSITIVE method for detecting an anti-M!  The reasons for this are that 1) the reactants are introduced to each other in the well at room temperature, and a "cold reacting" anti-M (or any other specificity that reacts at room temperature, come to that) will sensitise the red cells very, very quickly (certainly before the cassette is put at 37oC to incubate), 2) The incubation time is too short a time for total dissociation of the anti-M from the red cells, 3) you then take the cassette out of the incubator, back to room temperature (see point 1), 4) you then centrifuge the cassette, which brings the red cells into closer proximity (one way of enhancing antibody/antigen reactions - which is why, for example PEG works) and then 5) the cassette columns containing the AHG are at a slightly acidic pH, and, as you will see from most textbooks, the reaction between anti-M and the M antigen is greatly ENHANCED by a low pH (as low as pH4 has been recorded).

If, however, you are still worried as to whether your antibody will cause problems in the pregnancy (IT WILL NOT) just treat the lady's plasma with 0.1M dithiothreitol, which will disrupt IgM antibodies, and then see if there is any IgG element.  If there is not, you have no worries.  If there is, then all you need to do is titre the antibody to see whether or not it is less than 32.

A full tube panel at all phases is TOTAL overkill, and will only serve to be an expensive exercise in reagents and technologists time (which is also expensive).

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3 hours ago, Malcolm Needs said:

I fundamentally disagree with this post frenchie.

I can assure you that we, as a Reference Laboratory, see large numbers of samples from group A patients who have an A subgroup, and with the monoclonal reagents used these days, it is extremely common to see a very strong reaction with anti-A, when the red cells have an A subgroup, such as A2, Aint or ABantu.  Granted, this is not so for A3, Ax or Am samples, but these are exceedingly rare, and your results suggest that your patient is anything but an A3, Ax or Am.

As Dolichos biflorus is a lectin, and not an antibody, I know of no substance that could be coating the red cells that would stop this lectin reacting with the red cells.  Indeed, the fact that there is a microscopic reaction suggests just the opposite, as Dol. biflorus is not, actually, an anti-A1, but will react with the A antigen (and the Cad antigen, come to that) unless it is diluted so that the lectin only reacts with A1 red cells (which is why the insert tells you to ignore microscopic reactions).

Lastly, gel technique is probably the VERY BEST and MOST SENSITIVE method for detecting an anti-M!  The reasons for this are that 1) the reactants are introduced to each other in the well at room temperature, and a "cold reacting" anti-M (or any other specificity that reacts at room temperature, come to that) will sensitise the red cells very, very quickly (certainly before the cassette is put at 37oC to incubate), 2) The incubation time is too short a time for total dissociation of the anti-M from the red cells, 3) you then take the cassette out of the incubator, back to room temperature (see point 1), 4) you then centrifuge the cassette, which brings the red cells into closer proximity (one way of enhancing antibody/antigen reactions - which is why, for example PEG works) and then 5) the cassette columns containing the AHG are at a slightly acidic pH, and, as you will see from most textbooks, the reaction between anti-M and the M antigen is greatly ENHANCED by a low pH (as low as pH4 has been recorded).

If, however, you are still worried as to whether your antibody will cause problems in the pregnancy (IT WILL NOT) just treat the lady's plasma with 0.1M dithiothreitol, which will disrupt IgM antibodies, and then see if there is any IgG element.  If there is not, you have no worries.  If there is, then all you need to do is titre the antibody to see whether or not it is less than 32.

A full tube panel at all phases is TOTAL overkill, and will only serve to be an expensive exercise in reagents and technologists time (which is also expensive).

Malcom, would what you say about the gel card testing hold true if testing was performed via an automated program? Also, would what you say about the Anti A reaction hold true with automated testing as well? I can say that I have never seen a 4+ Anti A reaction with a variant A; but I do not work in a reference lab either although we use monoclonal reagents. Without the A1 lectin testing the results given do look like an early forming Anti M; which would primarily be of the IgM class and which I have seen the gel card miss,  under automated and manual testing with no manipulation (ie extending inc time,etc). This is why I suggested the full tube panel with readings at all phases, along with an Auto control. But I would agree that the week microscopic A1 in tube is highly supportive of the Anti A1 conclusion.

 

Edited by rravkin@aol.com
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11 hours ago, rravkin@aol.com said:

Malcom, would what you say about the gel card testing hold true if testing was performed via an automated program? Also, would what you say about the Anti A reaction hold true with automated testing as well? I can say that I have never seen a 4+ Anti A reaction with a variant A; but I do not work in a reference lab either although we use monoclonal reagents. Without the A1 lectin testing the results given do look like an early forming Anti M; which would primarily be of the IgM class and which I have seen the gel card miss,  under automated and manual testing with no manipulation (ie extending inc time,etc). This is why I suggested the full tube panel with readings at all phases, along with an Auto control. But I would agree that the week microscopic A1 in tube is highly supportive of the Anti A1 conclusion.

 

However, I would also add that difference in rankings for Anti A reactions between the gel cards and tube is excessive; the gel card is more sensitive but not that much (ie 4+ to w+); I am leary about the microscopic positive for the A1 lectin and I think that a final ruling of Anti A1 would be more sound with the tube panel at all phases along with the auto control. I have seen the gel cards miss an IgM antibody; or give very week reactivity; and I never not seen dosage with the gel card either. If the antibody concentration is fully IgM the specificity will be appearent at IS and mostly at 37C. The degree of dosage can be seen as well with a tube panel read at all phases because reactions occur predominantly at 37C and IgG depending on the relative concentrations of IgM and IgG of the potential specificty. If all is negative then Anti A1 it is. As far as overkill, that really relative and dependent on how many cases like this your blood bank weekly, monthly, anually, etc...

To be continued:

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As I said previously - the simple thing is to put up the reverse group at 37°C.  I am sure the normal antibody screen was also negative. If you like you can also put up your antibody screen at room temperature. However - whether this is an anti-A1 or an anti-M, it is not clinically significant and there will be no problems for your pregnant mum.    And in my experience A2 samples (if you call A2 an 'A variant') almost ALWAYS gives a 4+ reaction

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