Working up cold aggl.

[Steve72]

For gel antibody screen, you get 1+ in all three cells and the panel is inconclusive with only 2 negative cells (autocontrol is weakly positive). Some of our techs feel that we should repeat the screen using prewarm technique and if it comes out negative we can report cold auto without any further work-up. What steps should be followed before coming to this conclusion (i.e. cold auto)? Thanks.

[TVC15]

For gel antibody screen, you get 1+ in all three cells and the panel is inconclusive with only 2 negative cells (autocontrol is weakly positive). Some of our techs feel that we should repeat the screen using prewarm technique and if it comes out negative we can report cold auto without any further work-up. What steps should be followed before coming to this conclusion (i.e. cold auto)? Thanks.

Hi Steve,

I would scroll down to the #6 post by Dr. Garratty and head to his advice...he is a very seasoned Blood Trasnfusion PhD pathologist!

http://www.cbbsweb.org/enf/2009/prewarm_missab.php

[Malcolm Needs ☆]

For gel antibody screen, you get 1+ in all three cells and the panel is inconclusive with only 2 negative cells (autocontrol is weakly positive). Some of our techs feel that we should repeat the screen using prewarm technique and if it comes out negative we can report cold auto without any further work-up. What steps should be followed before coming to this conclusion (i.e. cold auto)? Thanks.

We do the same as you as far as the serology is concerned, but we would not automatically call it a cold-auto antibody. We wouls err on the side of caution and call the reactions "non-specific" (whilst acknowledging that there is no such thing as a "non-specific" antibody).

[Joanne P. Scannell]

With gel, the first thing we consider: 'Is there mixed cell reactivity?' Mixed cell reaction is highly suggestive of cold agglutinin reactivity (given the screening cells are single donor) or strong rouleaux.

If yes: To confirm that, test pooled O cells vs the patient's plasma at Room Temp. If they react, you likely have a cold agglutinin or strong rouleaux (or both) which you can actually visualize under the microscope if you'd like.

Final Interpretation: Cold Antibody of Undetermined Specificity or Cold Auto-Antibody of Undetermined Specificity (depending on if you perform testing to make the differentiation)

If no: Run a panel. You stated your panel was inconclusive which is strongly suggestive of being either HLA or HTLA antibody(ies). Gel is very good at picking up these antibodies so the old 'HLA is weak reacting' doesn't apply to gel. To 'confirm' this, we run an antibody screen using an enhancement medium that is not so sensitive to HLA/HTLA, e.g. OES (Ortho Clinical). More often than not, we have found that this test is negative, i.e. 'no clinically significant antibodies detected'.

Final Interpretation: = 'Probably HLA/HTLA Antibodies'

[Mabel Adams]

Someone once killed a patient with anti-Vel using a pre-warmed technique without doing any additional workup. Some 10-15 years back there was a big debate and an article or two in Transfusion on appropriate use of the pre-warmed technique. The one thing everyone could agree on is that you should not use the pre-warmed technique to just make reactivity go away. You need to have at least proven that you have a cold antibody. More cautious experts like John Judd called it the "pre-fried" technique and would not allow it in their lab. His lab did research and found that a fair number of significant antibodies were missed using the pre-warmed technique--partly because it relies on a less sensitive (saline) technique. Often, the need for it can be avoided with other approaches except in the case of a very strong cold antibody.

[Malcolm Needs ☆]

partly because it relies on a less sensitive (saline) technique.

I agree with you Mabel that the pre-warmed IAT should be used with caution, and also I would say that competency and experience in using the technique should be proven before anyone is let loose using the technique on real patients, but it does not rely on using saline. We regularly use it in my Reference Laboratory using Low Ionic Strength Solution (LISS), but very rarely use it using isotonic buffered saline.

[Joanne P. Scannell]

Hear, hear!

And yes, I believe the 'missed clinically significant antibodies with pre-warm' is more of a function of it being a less sensitive method (it used to be 'no enhancement, 1hr inc, do not read at 37oC) rather than the pre-warming. (Observe, all antibody screens are 'prewarmed' in the ProVue because the cards are set in a 37C incubation rack as they wait to be loaded ... haven't heard any stories of missing antibodies.)

Let me add to Mabel's notation ...

Recently, we sent a sample that was reacting 'same strength' with all cells in the panel to our local reference lab. nb We test for cold agglutinin = negative at Rt with pooled O cells. Their answer: Cold Agglutinin. Their reasoning: Reactions were seen at 4oC and reactivity diminished/disappeared after treatment with RESt. Ummm ... hmmm. Knowing RESt has been documented to absorb some clinically significant antibodies, well, luckily, the patient went home and didn't need a transfusion.

So, not only do we need to be mindful of the capabilities of pre-warming, we need to be fully aware of the capabilities of the 'specialities' we employ.

[Malcolm Needs ☆]

Knowing RESt has been documented to absorb some clinically significant antibodies.

So, we need to be fully aware of the capabilities of the 'specialities' we employ.

Agree entirely with this Joanne. RESt adsorbs out anti-B for one thing!!!!!!!!!

[KKidd]

We currently have a patient with Cold Agglutinin Disease. We sent a sample to the reference lab because he had been transfused in the past. In addition to that very strong cold agglutinin, he has an Anti-K. You never know what lurks in the plasma/serum of patients.

[Malcolm Needs ☆]

We currently have a patient with Cold Agglutinin Disease. We sent a sample to the reference lab because he had been transfused in the past. In addition to that very strong cold agglutinin, he has an Anti-K. You never know what lurks in the plasma/serum of patients.

Any Reference Laboratory WORTH THE NAME should be able to detect th anti-K.

Sadly, I know there are some around that are not worth the name!

[L106]

We currently have a patient with Cold Agglutinin Disease. We sent a sample to the reference lab because he had been transfused in the past. In addition to that very strong cold agglutinin, he has an Anti-K. You never know what lurks in the plasma/serum of patients.

So, do you know how the reference lab handled this particular work-up to identify the Anti-K? Did the Anti-K simply react obviously stronger than the Cold Auto Antibody, or did they use RESt, or did they do the PreWarm Technique, etc?

I am seeing an increase in the use of the PreWarm Technique among our staff (and I don't like it.)

I have often found the RESt Adsorption helpful when dealing with strong Cold Auto Antibodies. I realize that the RESt adsorbs out Anti-B and several clinically insignificant antibodies. I would like to hear from others if they have had the experience of the RESt adsorbing out clinically significant antibodies (other than Anti-B.)

Your input and comments about RESt are appreciated.

Donna

[KKidd]

I agree Malcolm. Thankfully, I have a great reference lab. We don't have the man power or reagents to perform some of the testing I would like and they are a fantastic resource.

Back to the subject, Cold Agglutinins are like having a cold(kind of a nuisance).

[KKidd]

So, do you know how the reference lab handled this particular work-up to identify the Anti-K? Did the Anti-K simply react obviously stronger than the Cold Auto Antibody, or did they use RESt, or did they do the PreWarm Technique, etc?

I am seeing an increase in the use of the PreWarm Technique among our staff (and I don't like it.)

I have often found the RESt Adsorption helpful when dealing with strong Cold Auto Antibodies. I realize that the RESt adsorbs out Anti-B and several clinically insignificant antibodies. I would like to hear from others if they have had the experience of the RESt adsorbing out clinically significant antibodies (other than Anti-B.)

Your input and comments about RESt are appreciated.

Donna[/quote

Sorry Donna,

That information is not on my report. The cold agglutinin was so strong that they could not resolve the discrepancy between the front and reverse group. I do remember that at least 2 absorptions were performed and the anti-K was detected uusing PEG@AGT.

Karen

[Joanne P. Scannell]

Transfusion, 2002 Sep;42(9):1180-3 Anti-Vel reactivity diminished by adsorption with rabbit RBC stroma

Transfusion, 2010 May:50(5):1139-43 Immunoglobulin M red blood cell alloantibodies are frequently adsorbed by rabbit erythrocyte stroma.

To cite a couple ... there are more out there.

[Malcolm Needs ☆]

I don't know if this will help, but I can tell you what we do in my lab when we receive a sample from a hospital that is suspected of containing a "cold" auto-antibody.

The first thing we do (IN ALL CASES), after having performed an ABO, D, Rh phenotype and K typing, is to perform a standard 10 cell panel by column agglutination technology (in our case using DiaMed technology, or is it Bio-Rad technology now?) using straightforward red cells by IAT and papain-treated red cells by IAT. In addition, we would perform a differential DAT using monospecific anti-IgG, anti-IgM, anti-IgA, anti-C3c and anti-C3d, and an auto-control.

If we are unable to get an ABO and D group, we would then wash the red cells from the sample several times in warm (37oC) isotonic saline. It is a rare occurance, but not unknown, that we are unable to assign an ABO and D group after this (if we cannot assign these, we suggest the use of group O, D negative, K negative red cells for transfusion).

As far as the DAT is concerned, normally the red cells show agglutination with the anti-C3d alone, or the anti-C3d and the anti-IgM, but there are occasions when even this does not work. In this case, we would perform the DAT by tube, this time only using monospecific anti-IgG and anti-C3d (and saline as a negative control). I have never seen this not to work, but I'm sure that there must be occasions when it doesn't.

If the DAT is positive with the monospecific anti-C3d or the anti-C3d and the anti-IgM, we would test the patient's plasma against the panel cells by LISS tube direct agglutination at 30oC (Petz and Garratty state that, if the auto-antibody reacts at 30oC, it is clinically significant, and that if it doesn't, then it isn't, as long as the patient is kept warm - including extremities like toes, fingers, nose and ears).

We would also perform a pre-warmed, warm-washed LISS tube IAT at 37oC, using a monospecific anti-IgG reagent. This normally "does the trick" as far as the "cold" auto-antibody is concerned, and will reveal such antibodies as the anti-K mentioned in the post by KKidd.

Failing all of this working, we would then resort to alloadsorption at 4oC, and then testing the adsorbed plasma. Only if this did not work would we go on to use either RESt or treatment of the plasma with 0.01M dithiothreitol. This would allow us to identify underlying IgG alloantibodies.

Failing that, we suggest, again, transfusing group O, rr, K- blood, AND PRAYER!!!!!!!!!!!!!!!

:boogie::boogie:

[Bill Sinn]

Getting back to the original post.. for a cold agglutinin reacting in a gel Ab screen, we have had some success in "prewarming" the test system--the patient's plama, the gel card, and the screening cells pipeted into the gel card-- and then pipeting the plasma into the card. I know the card will cool down to room temp. during centrifugation , but sometimes this will work, and you can demonstrate a negatvie Ab screen.

Bill

[Mabel Adams]

When I first started using gel they had a procedure for prewarmed testing. When I changed jobs and asked Ortho tech support about it they said they did not have a procedure for it. As I recall, it was as you describe.