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Dear My Colleagues,

I am confused about which sample should be used in detection cold antibody, either allo or auto?

Shall I use serum or plasma? Or both are suitable…

My confusion arise from statement that, IgM depend on complement in its action and plasma has no complement….!!!!!

Thanks.

 

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We use EDTA, unless, of course, we are doing a Donath-Landsteiner test.

 

Thanks Malcolm, but could you please explain me, if IgM depend on complement in its action, what is the benefit of plasma in detection of cold agglutinin or allocold antibody?

Thanks

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Well, there are two reasons.

 

The first, and main reason, is that we don't want to detect cold antibodies in the first place, because, unless they react at 30oC or higher, they are most unlikely to be clinically significant.  Therefore, detecting them, and then having to investigate them for specificity is a complete waste of time and reagents - and staff time is the single most expensive thing in the laboratory.

 

Secondly, it is highly unusual that you would only detect an IgM antibody by the presence of activated complement (not impossible, but highly unusual) and, therefore, the fact that EDTA chelates the Ca++, Mn++ and Mg++ ions required for the complement cascade to initiate, is irrelevant.  The IgM molecules will still be present in the plasma, and are normally easily detectable.

 

As I said above though, obviously, if we are trying to detect the biphasic anti-P that causes PCH, we would need complement present, and we would then use serum - BUT, very often in these cases, the patient's own complement is already exhausted, and so we would use the indirect two-stage DL test, where fresh complement from another source is added.

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Well, there are two reasons.

 

The first, and main reason, is that we don't want to detect cold antibodies in the first place, because, unless they react at 30oC or higher, they are most unlikely to be clinically significant.  Therefore, detecting them, and then having to investigate them for specificity is a complete waste of time and reagents - and staff time is the single most expensive thing in the laboratory.

 

Secondly, it is highly unusual that you would only detect an IgM antibody by the presence of activated complement (not impossible, but highly unusual) and, therefore, the fact that EDTA chelates the Ca++, Mn++ and Mg++ ions required for the complement cascade to initiate, is irrelevant.  The IgM molecules will still be present in the plasma, and are normally easily detectable.

 

As I said above though, obviously, if we are trying to detect the biphasic anti-P that causes PCH, we would need complement present, and we would then use serum - BUT, very often in these cases, the patient's own complement is already exhausted, and so we would use the indirect two-stage DL test, where fresh complement from another source is added.

 

Thanks Malcolm, usually I learned much from this forum especially from you Malcolm.

Regarding to first point I aware and for autoanti P I know, only confusion about the second point.

Thanks for your clarification

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  • 3 weeks later...

Not sure if you are still confused about IgM and one's ability to detect it.....

 

Paraphrasing Malcolm; complement is not required to be present for an IgM antibody to bind to it's cooresponding antigen.  With or without complement, the IgM antibody will bind to the antigen which most likely will result in visable agglutination.

 

If detecting hemolysis is your end goal, then yes you would need a source of complement (serum or antibody free "fresh serum") to accomplish that goal.

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I'll have a go (but don't hold me to this being entirely correct)!

 

A cold auto-antibody is an auto-antibody that reacts against the patient's own red cells, but is not active at 30oC or above.  So, I could have, as a group A1 individual, and probably do have, an auto-anti-H or auto-anti-Hi, but it is clinically benign.  In other words, the cold auto-antibody is present in my plasma, but, unless tests are performed at temperatures below 30oC, I would never know, because the presence of this auto-antibody is not clinically significant.

 

If, on the other hand, I had CAS, my auto-anti-H or auto-anti-HI (in the example I have given) would be reactive at 30oC or greater (30oC is estimated to be the temperature to which the extremities - such as the toes, fingers, ears and nose - amongst other things - will drop in cold weather or other cold conditions), under which conditions the auto-antibody would be clinically significant, as I would start to destroy my own red cells.

 

The higher the thermal amplitude of the antibody, the more clinically significant the antibody becomes (irrespective of the specificity of the antibody, or the titre of the antibody, DESPITE what a lot of doctors would have you believe).

 

In addition, however, it also depends upon the turnover of your complement.  If you consume your complement quicker than you can produce complement, then your red cells will not be destroyed as quickly as you may think, particularly as they will be "covered" in C3dg, which protects the autologous red cells.  BUT, if the Hb drops to a dangerously low level ( and I do mean a DANGEROUSLY low level - not just what is traditionally thought of as a "low" level), then the patient may require a transfusion.  This transfusion may not be as successful as would be assumed, however, as the "virgin" allogeneic red cells will not be protected by the coating of C3dg, and so will be vunerable to complement coating to C3d level and beyond, and so be removed from the circulation quite quickly.  This may result in the requirement for a splenectomy, possibly leading to the chance of Evan's syndrome setting in.

 

I hope that explanation is of some use, but others may have other (valid) explanations, may disagree with what I have written, or may well disagree entirely with what I have written.

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  • 2 months later...

A VERY good question DCeDCe!

 

The answer is that transfused red cells are destroyed quicker than the autologous red cells, but the transfused red cells will, quite quickly, also be coated in C3dg.  Although a proportion of them will be destroyed quite quickly, a proportion of them will also last as long as autologous red cells.  That having been said, because a proportion of the transfused red cells are destroyed quite quickly, it may be necessary to either transfused more blood than normal at one go, or to transfuse with a smaller gap between transfusion episodes - both of which expose the recipient to an increased number of foreign antigens.

 

Certainly, a transfusion is justified if the patient is in extremis, but I have my doubts as to the true efficacy of transfusion as a "top up".

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