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Clinically Insignificant Panagglutinin


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I have a pt with Mantle Cell Lymphoma.  Just started transfusions in October.  He is also being transfused at our tertiary care referral center.  In mid-November his gel absc looked like he was developing a cold agglutinin.  On Monday he returned with all screening cells 2+mf in gel.  He had been transfused at the referral hosp last week - they use Capture technology and decided he had the "clinically insignificant panagglutinin".  In our gel he reacted with 8 of 11 cells and his auto: 2+mf.  His DAT is IgG negative.  2 step Bromelase pretreatment and everything is negative.  Peg/IgG crossmatches are totally compatible and my Medical Director would like him worked up with the PeG for the time being.

 

I am just curious so if there are any ideas I'd like to hear them.  I anticipate this pt being tranfused on a weekly basis either here or at the referral.  Thanks in advance.

Edited by David Saikin
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Hi David,

Unless they have performed thermal amplitude tests, which, of course, they may have done for all I know, I can't see that they can say it is a clinically insignificant panagglutinin. If the antibody is detectable at 30oC or above, then it is clinically significant, whatever the specificity.

In this particular case, however, I am a little worried about the specificity - not from the transfusion point-of-view - but from the patient outcome point-of-view. As the antibody/antigen reaction is destroyed by the action of proteolytic enzymes, it is unlikely to be either an auto-anti-I or an auto-anti-HI, but sounds more like an auto-anti-Pr, which, as far as I am aware, foretells a worse prognosis for the patient than does an auto-anti-I or auto-anti-HI.

Of course, from this distance, I could be talking complete rubbish!

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I have a pt with Mantle Cell Lymphoma.  Just started transfusions in October.  He is also being transfused at our tertiary care referral center.  In mid-November his gel absc looked like he was developing a cold agglutinin.  On Monday he returned with all screening cells 2+mf in gel.  He had been transfused at the referral hosp last week - they use Capture technology and decided he had the "clinically insignificant panagglutinin".  In our gel he reacted with 8 of 11 cells and his auto: 2+mf.  His DAT is IgG negative.  2 step Bromelase pretreatment and everything is negative.  Peg/IgG crossmatches are totally compatible and my Medical Director would like him worked up with the PeG for the time being.

 

I am just curious so if there are any ideas I'd like to hear them.  I anticipate this pt being tranfused on a weekly basis either here or at the referral.  Thanks in advance.

 

There is an article in Transfusion, "How We evaluate Panagglutinating Sera" on page 1540, volume 49, August 2009.  The authors present an algorithm for panagglutinating sera.  Are you familiar with the information in this article and how does it relate to this patient?

 

I am curious about this situation and it completely baffles me, and hope you don't mind answering these questions...

 

Am I correct in thinking that if, in your workup, the patient's serum reacted with 8 of 11 cells, then this is not really a panagglutinin?  The definition of a panagglutinin, as presented in the aforementioned article, is an agglutinin that reacts with all red cells tested. 

 

Also, you mention that in a later workup the sera was nonreactive at 4C and RT, so doesn't this mean that this is not a cold agglutinin?  You mentioned that in an earlier workup it looked as if a cold agglutinin might be developing.

 

What is the explanation for the nonreactivity with enzyme-treated cells?  Is this an antibody to a HFA or a HTLA that is denatured by enzymes?  In the aforementioned article, in the flowchart presented on page 1541, there is a step where the autocontrol is negative or weaker than the reaction with panel cells, there is persistence of panagglutination after auto- and alloadsorption, there is no reactivity after papain treatement, and the flow leads one to think maybe there is an anti-HFA or anti-CH/RG.....

 

Why are the PEG/IgG crossmatches all compatible, and are they incompatible without the PEG?

 

I would like to understand this situation better, and hope you don't mind my questions...

 

Thank you!

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To put in my twopennyworth:

In my experience, the most common reasons for mixed field agglutination in an antibody screen in gel, in the absence of a mixed cell population, of course are the following:

1.  IgM antibodies

2.  Fibrin mimicking agglutination

3.  White cells physically blocking the negative cells from falling through the gel

 

Anna

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ok - I got the results of 8/11 cells reacting.  The tertiary care place called in a panagglutinin (they use solid phase).

It looks like a cold ab is r/o.  I mentioned a possible cold aggl with a previous encounter where the gel looked like it does when a cold is present - small red line on top/mf:  could also be due to fibrin or wbcs.  don't think it was fibrin as I spun my specimen prior to use.  I like that the enzyme destroyed it - my feelings are some HTLA/Ch/Rg that is enzyme sensitive.  Even the auto ct was reactive in gel.  The PeG xm are comatible since Peg is a less sensitive enhancement than gel.  The gel xms (ahg) are all incompatible.  We could not autoabsorb as the patient has rec'd 6u rbcs in the past month.

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The patient may have a cold auto and an HTLA/Ch/Rg antibody of course, but the fact that it is positive in gel makes me think that there is certainly a cold auto of some sort there. Have you done a thermal range David? As the auto is positive by gel, and the antibody is enzyme sensitive, I am still thinking along the lines of an anti-Pr - BUT, if the patient has had 6 units in the past month, the auto could be due to the transfused cells being sensitised. Is there any mixed-field in the DAT?

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I recently had a patient with reactions almost like Davids. Just curious what others think of it. I sent it out to an IRL. But the patient had a negative ABSC with a previous sample was transfused 2 units and now is positive. Looks like mixed field in gel (the top layer on the gel seemed a little to thick to be fibrin), gel panel looked the same but the auto control was 1+ (no top layer), 2 of the 11 cell panel looked negative (with maybe fibrin on the top), and 1 of 11 looked like a good 4+ (really no cells on the bottom). So I was thinking an IgM/cold antibody. DAT- IgG negative, C3d  positive (Pre transfusion DAT was negative). LISS tube method was negative at all phases. Cold Screen only the I negative cell reacted 4+ at IS, RT,15C and 4C, the AC was 3+ at 15C and 4C and Screening cells were 1+wk at 15C adn 4C. 

 

Any thoughts?

Edited by Justina
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I recently had a patient with reactions almost like Davids. Just curious what others think of it. I sent it out to an IRL. But the patient had a negative ABSC with a previous sample was transfused 2 units and now is positive. Looks like mixed field in gel (the top layer on the gel seemed a little to thick to be fibrin), gel panel looked the same but the auto control was 1+ (no top layer), 2 of the 11 cell panel looked negative (with maybe fibrin on the top), and 1 of 11 looked like a good 4+ (really no cells on the bottom). So I was thinking an IgM/cold antibody. DAT- IgG negative, C3d  positive (Pre transfusion DAT was negative). LISS tube method was negative at all phases. Cold Screen only the I negative cell reacted 4+ at IS, RT,15C and 4C, the AC was 3+ at 15C and 4C and Screening cells were 1+wk at 15C adn 4C. 

 

Any thoughts?

Justina's case has pos cold screen result, but David's case is neg cold screen.

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David , you say Bromelase pretreatment the result is neg, I dont know after Bromease treat you run the panel on gel or  other method, if you do gel ,then we can say the antibodies are enzyme sensitive, but if not on gel, I think we can't get the conclusion like this. 

This antibody only react at gel, maybe because gel is more sensitive than other method, or there is something in the patient's plasma react with the additive in gel.

We fear there is some clinical significant antibdies under the autoantibody, and this patient has been transfused last weak , so I prefer alladsorption ( use R1R1,R2R2,rr) .

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I did run some tube studies at immed spin, r.t. and at 4C.  They were all negative. 

Sorry for my poor English, I misunderstand it as thermal testing, and you mentiened before this patient's DAT is IgG neg, not mention C3, I misunderstand it is pos or not do it.

I will study harder to improve my language ability.

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Maybe I'm taking the simplistic approach but when we see mixed cell reactivity, we consider the 3 reasons given by the manufacturer for causing such results ...

1. Mixed population - this is not the case for screening/panel cells so that out.

2. Cold Agglutinin - you investigated that and got negative results so no cold agglutinin there.

3. Rouleaux - I'll assume you checked that out as well and found none otherwise you would have said differently. n.b. Logic tells us that rouleaux should be seen with all cells, but experience tells us that it doesn't always happen that way.

 

In addition, the premise for reactivity in gel is essentially that the cells that are 'not smooth' don't make it to the bottom.  Anything that causes the surface to be 'rough' causes a 'positive result'.  Irregular shape (e.g. sickle, acanthrocytes), abnormal immunoglobulin coating, etc. etc. so those things need to be added to that list.  (Keeping in mind that reagent cells are aged.)

 

Also, gel is acidic, so we see more Anti-M than we did with tube testing.  I'll assume you checked that out.

 

Anti-Pr ... aren't all human cells Pr positive?  However, if I remember correctly, isn't Anti-Pr also enhanced by acidity?  Again, your patient doesn't have a cold agglutinin.

 

Gel is also great at picking up HLA/HTLA antibodies.

 

Currently, with no cold agglutinin demonstrated, no rouleaux and a negative DAT, and 'some pos with some neg results', we'd run the antibody screen with OES (Ortho's version of LISS/Albumin) and if that is negative, call it 'HLA/HTLA Antibodies' and move on with our lives.

 

P.S. We do have two active patients right now who's plasma reacts with everything in gel ... negative DAT, no cold agglutinin, no rouleaux and totally negative with tube testing ... calling it 'interferring substance with the method' as we'll never know what is causing this .. meds? abnormal proteins?

 

 

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We do see antibodies, as Malcolm mentioned, that are directed against some component of the Gel test system.  We suspect that they are directed at some antibiotic in the reagent cell suspending medium.  They often give that characteristic “mixed field-ish” looking agglutination typical of IgM antibodies.  Some are quite strong (even 4+).  The Auto Control in Gel is typically negative because the patient cells are in MTS diluent and not exposed to the same antibiotics that are in the reagent cells.  It seems like the 0.8% cells manufactured by Ortho for Gel testing have the offending ingredient and the 3% cells do not.  When we see this pattern of all screening and panel cells positive, autocontrol negative we will prepared our own 0.8% dilutions of 3% screening cells to test in Gel and get negative Gel reactions.  Of course all of this does not help with solid phase but the mechanism may be similar.

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We do see antibodies, as Malcolm mentioned, that are directed against some component of the Gel test system.  We suspect that they are directed at some antibiotic in the reagent cell suspending medium.  They often give that characteristic “mixed field-ish” looking agglutination typical of IgM antibodies.  Some are quite strong (even 4+).  The Auto Control in Gel is typically negative because the patient cells are in MTS diluent and not exposed to the same antibiotics that are in the reagent cells.  It seems like the 0.8% cells manufactured by Ortho for Gel testing have the offending ingredient and the 3% cells do not.  When we see this pattern of all screening and panel cells positive, autocontrol negative we will prepared our own 0.8% dilutions of 3% screening cells to test in Gel and get negative Gel reactions.  Of course all of this does not help with solid phase but the mechanism may be similar.

 

So true ... the reagent antibiotic antibody issue has been around for years, too.  This is covered by my 'interferring substance' interpretation. 

 

I guess I'm taking the attitude 'I'm looking for clinically significant antibodies.'  All the rest is academically interesting but doesn't change how we choose the units from the refrigerator.

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Hi David,

 

I have in the past seen lymphomas throw out atypically presenting "antibodies or antibod-like" reactions. What I had noted over the many years (pre-gel or enhanced IDC) that the more undifferentiated the lymphoma, the more reactions you are likely to encounter (often reacting at 4 deg C).

 

Away from the questions posed re auto or not - are you considering phenotyping for common antigens (as far as it is possible to still supply phenotypically identical blood) to supply matched blood if available, if the patient is becoming transfusion dependant?

Cheers

Eoin

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