Cold agglutinin: Prewarming patient specimen???

[Generalist Jan]

Hi and best wishes for a happy new year!

This is my first post, but I am a little troubled by something that occurred in our hospital transfusion service, so I thought I should check with others.

Patient background: Hx of a strong cold agglutinin that causes an ABO discrepancy. The patient forward types as B+ and reverses as Group O.

According to the reference lab report, the ABO discrepancy could not be resolved by any of the techniques that were used. The transfusion recommendation is

to crossmatch for compatibility with group O positive RBCs.

The patient is frequently transfused on an outpatient basis. The antibody screen is consistently negative with Ortho gel, but a comment in the patient notes says to use prewarm technique. I'm not sure why the note is there, but I've used both methods.)

Recently I found a crossmatch requistion for this patient in the in-basket at the start of my shift. The patient specimen was nowhere to be found.

A few hours later, my BB partner wondered aloud if the specimen was in the incubator. "What incubator?" I asked.

As it turned out, the sample had been sitting in a 37 deg. C incubator in a different lab section for over 3 hours -- and its stay was actually longer, because my partner didn't remove the specimen from the incubator for another 3 hours.

My thought was that the specimen had been compromised by this incubation and should not be used for antibody testing or crossmatch purposes. Such incubation is not part of our prewarm procedure or part of any other procedure that we have, so I left a note for the day shift to have the patient sample redrawn.

I later learned that my request was overruled, the specimen was tested the following day, and units were crossmatched. The transfusion was later canceled for unrelated reasons.

If prewarm technique carries some risk of missing allo-antibodies, wouldn't prewarming the entire specimen for antibody screening/compatibility testing carry as much or even more risk? I have never heard of doing this, although an SBB friend tells me that there are special techniques where that is done.

[Generalist Jan]

Ack! The word "patient" in the subject thread is forever misspelled! Sorry ...

[Yanxia]

I don't think prewarm the specimen at 37 degree C will cause some risk.

[Dansket]

To properly comment, it would be useful to know if compatible crossmatches are demonstrated without prewarming to thoroughly characterize the patient's antibody reactivity.

Given a negative antibody screen and that the purpose of prewarming is to minimize false-positive agglutination, I don't see the point in prewarming crossmatches. The comment in the computer may have been appropriate at the time, but the transient nature of patient antibody requires that information be updated as situations change.

I would discard a specimen that had been incubated at 37C for over 6 hours. Our SOP requires that specimens be stored at 2C-8C when not being tested.

Based on your narrative, it seems that the processes in your laboratory foster problems rather than solve them, e.g., incubator not located in blood bank, no visual indicator posted in blood bank that a specimen is being prewarmed, computer comments out of date, prewarming an entire blood sample rather than an aliquot.

[Malcolm Needs ☆]

To properly comment, it would be useful to know if compatible crossmatches are demonstrated without prewarming to thoroughly characterize the patient's antibody reactivity.

Given a negative antibody screen and that the purpose of prewarming is to minimize false-positive agglutination, I don't see the point in prewarming crossmatches. The comment in the computer may have been appropriate at the time, but the transient nature of patient antibody requires that information be updated as situations change.

I would discard a specimen that had been incubated at 37C for over 6 hours. Our SOP requires that specimens be stored at 2C-8C when not being tested.

Based on your narrative, it seems that the processes in your laboratory foster problems rather than solve them, e.g., incubator not located in blood bank, no visual indicator posted in blood bank that a specimen is being prewarmed, computer comments out of date, prewarming an entire blood sample rather than an aliquot.

I agree with everything posted by Dansket, but would add one or two things myself.

If you have a patient with a known "cold" auto-antibody, it is important (obviously) to detect any clinically significant alloantibodies that may be masked by the auto-antibody. Such alloantibodies are usually only masked if the "cold" auto-antibody is of wide thermal amplitude (detected at 30oC or above). You want, therefore, to "get rid" of as much of the "cold" auto-antibody as possible. In such a case, the first thing we would do is to incubate the sample, not at 37oC, but at 4oC. In this way, you are reducing the temperature of the sample to the optimal temperature for the reaction to take place between the antigens on the patient's red cells and the auto-antibody. In other words, a large percentage of the free auto-antibody will be adsorbed onto the surface of the autologous red cells. The bad news is that, when a "cold" auto-antibody has a wide thermal amplitude, it also usually, although not always, has a very high titre, so you may wish to perform this auto-adsorption step several times with fresh autologous red cells.

Once the auto-antibody has been adsorbed to an extent that it no longer interferes with the detection of alloantibodies, there may be no need to pre-warm the reactants (and, of course, any clinically significant alloantibodies will remain in the plasma, as they will not be adsorbed onto antigens on the patient's red cells, and will not have been denatured by prolonged exposure to high temperature incubation).

By incubating the patient's sample at 37oC, there is a chance that the "cold" auto-antibody that is already complexed with the patient's own red cell antigens will dissociate from these antigens, making the concentration of the auto-antibody in the plasma that much higher, and so compounding the problem.

If auto-adsorption at 4oC does not remove the auto-antibody suffficiently to make a "normal" screen practicable, then there is little choice than to pre-warm the reactants, BUT, all that is required is for the plasma to be brought to 37oC and the screening cells/antibody panel cells/donor cells to be brought to 37oC before mixing, and then for the test to be performed. There is no need (or excuse) for prolonged incubation at 37oC (although washing the IAT at the end of incubation with saline brought to 37oC, and using monospecific anti-IgG reagent may be of adavntage).

BUT, as Dansket and you both observe, in this case the screen is negative anyway, so why not perform the cross-match by routine techniques, without pre-warming?

I should, perhaps, add this caveat. If I were performing a test for CHAD, I most certainly would NOT advocate pre-incubation at 4oC. I would ask for the sample to be taken and kept warm, as, for such tests, as much of the auto-antibody as possible should be kept free in the patient's plasma for thermal amplitude testing (although testing for antibody specificity and/or antibody titre are tests that are about as useful as a chocolate tea pot).

Edited December 30, 2012 by Malcolm Needs
Adding a caveat.

[vilma_mt]

"Patient background: Hx of a strong cold agglutinin that causes an ABO discrepancy. The patient forward types as B+ and reverses as Group O.

According to the reference lab report, the ABO discrepancy could not be resolved by any of the techniques that were used. The transfusion recommendation is to crossmatch for compatibility with group O positive RBCs.

The patient is frequently transfused on an outpatient basis. The antibody screen is consistently negative with Ortho gel, but a comment in the patient notes says to use prewarm technique. I'm not sure why the note is there, but I've used both methods.)"

In your case, I see no reason performing pre-warming techniques for AB Screens when you consistently have negative Gel AB Screens. I would look more at the cause of ABO discrepancy and try to resolve it, is it weak or missing reactivity in the forward typing or extra reactivity in reverse typing.

Performing prewarm techniques "to get rid of weak antibodies" is not an acceptable practice, clinically significant antibodies can easily be "prewarmed away". Prewarm techniques can be a powerful tool but never assume what you're detecting is merely cold reacting clinically insignificant antibody without first trying to determine if a clinically significant antibody is present. If a technologist suspects a cold antibody, he/she must prove a cold is present, not necessarily identify it. The technique should be used with caution especially on patients with recent transfusion and not used to eliminate unidentified reactivity.

[BloodBank101]

Due to shortage we trained generalists to work in blood bank, scary some techs use pre-warm or saline technique when the reactions are weak on gel cards and report results as negative. Sometimes able to catch the error have repeated and have found significant antibodies. Unfortunately our supervisor refuse to recognize there is a problem.

[tbostock]

Prewarming, in my opinion, should be used very rarely, and VERY carefully. It takes a long time to break that culture of "let's just prewarm it to make it go away".

[Yanxia]

I don' think the specimen keeps at 37 degree C fr 6 hours wil cause the miss identify of clinical signficant antibodies, beause our bdy temp is the same,if it will cause miss, how can we explain the in vivo reaction.

As to the rewarm technique, because it not only prewarm the plasma but also the reagent to be used to do the test, the prewarm may lower the sensetivity of the reagent , so it will cause antibodies miss.

I agree from Malcolm, to keep the specimen at 37 degree will dispense the cold auto from the RBC, it is not a good way to avoid cold auto's disurb. To do auto adsorb at 4 degree is better.

[Cliff]

Ack! The word "patient" in the subject thread is forever misspelled! Sorry ...

Nothing lasts forever, fixed it.

[Malcolm Needs ☆]

I don' think the specimen keeps at 37 degree C fr 6 hours wil cause the miss identify of clinical signficant antibodies, beause our bdy temp is the same,if it will cause miss, how can we explain the in vivo reaction.

.

I see what you mean shily, but it is not exactly the same situation. With the in vivo situation, there is a constant break down of IgG molecules, but these are then replenished by active B lymphocytes. In the in vitro situation, there is still the constant breakdown of some of the IgG molecules, which is accelerated by the higher temperature, but these are not replenished by active B lymphocytes, and so there is a net loss of IgG molecules. This may not be a huge net loss, but it may be sufficient to make some clinically significant alloantibodies undetectable, particularly if, after this, the pre-warmed technique is used (and, particularly, if the pre-warmed technique is used in inexperienced hands).

[Yanxia]

.

I see what you mean shily, but it is not exactly the same situation. With the in vivo situation, there is a constant break down of IgG molecules, but these are then replenished by active B lymphocytes. In the in vitro situation, there is still the constant breakdown of some of the IgG molecules, which is accelerated by the higher temperature, but these are not replenished by active B lymphocytes, and so there is a net loss of IgG molecules. This may not be a huge net loss, but it may be sufficient to make some clinically significant alloantibodies undetectable, particularly if, after this, the pre-warmed technique is used (and, particularly, if the pre-warmed technique is used in inexperienced hands).

Thank you , Malcolm.

This is the reason to keep the specimen at-20 degree C to preserve the antibodies reaction, is it? Thank you

Edited December 31, 2012 by shily

[ElinF]

I agree that in this instance i would worry more about the ABO discrepency and do regular crossmatches if it negative, but if you are looking at a cold ab, could you just do your 4C incubation, determine it is a cold, and then do a tube screen with PEG to rule out clinically significants?

[Mabel Adams]

What about the effects of bacteria in a specimen that is stored a long time at 37C? If the sample had been opened and accessed, it might not be sterile and even a couple little bacteria would probably happily multiply and eat available protein, including immunoglobulins, wouldn't they?

[Mabel Adams]

Malcolm, your long post brings up a question I have long had. They usually say not to do cold auto-adsorptions in recently transfused patients. However, we don't consider cold antibodies significant (except maybe an IgM of a newly forming alloantibody). Many of us no longer separate cells from plasma and may store a "hold" specimen at 4C until tested later. In my experience, this will do a nice mild cold adsorption--which I sometimes think is just the ticket for cold autoantibodies. But do you think there is any real risk to this practice? How often would we adsorb out a significant IgM antibody this way? Also, if I were going to do this on purpose, I would separate out an aliquot of the patient's cells before I put the rest of the specimen in at 4C so I wouldn't have cells all coated with antibody for typing etc.

[Malcolm Needs ☆]

Mabel, many of my detractors would say "which of his posts are not long?", but I think I know the one to which you refer!

Even in a recently transfused patient, I would not be overly concerned abou what is, in effect, an alloadsorption, simply because antibodies reacting at 4oC are not going to be clinically significant. As I pointed out, even if an IgM antibody is going to be clinically significant, and there are some, apart from the ABO antibodies, they would not be adsorbed enough to become undetectable at 37oC. Personally, I would not lose any sleep over storing such samples unseparated at 4oC.

I have certainly never heard of such storage resulting in a missed clinically significant alloantibody that has caused a haemolytic transfusion reaction.

[Mabel Adams]

I have not lost sleep over our specimen storage protocol because my experience was the same as yours. It only makes me pause when reading the instructions for cold-autoadsorptions and they say not to perform it on recently transfused patients. With Malcolm's input, I would be comfortable in pulling off some patient cells, sticking the rest of the mixed, whole blood specimen in the fridge for an hour or so, spinning and separating it immediately after removal from fridge and using that plasma for testing in a patient with a not-too-strong cold antibody. I suppose one should write a procedure for this and validate it, but it is really just like our usual storage protocol so maybe that would not be necessary. This is sort of cold autoadsorption-light--easy and painless. What do you think?