Cold antibodies

[LAURAA80]

Are cold antibodies always seen at immediate spin? We have had some non-specific reactivity when using PeG and IgG, that always goes away with pre-warm. I don't think that these are PeG reactors, as they are not panagglutinins. But I am hesitant to call them cold antibodies for fear we are to miss something clinically significant.

[David Saikin]

Have you run a cold panel? I like to run my screening cells, auto ct, and 2 group O cord bloods at IS, 5'@rt and 5' @4C. This way I can see if there is something that reacts at these temps. I don't believe you can say there is a cold ab unless you have proved it.

[LAURAA80]

Thank you for the information. We currently do not have a procedure for a 'cold screen'...nothing in writing anyway. If we have reactivity on the screen at immediate spin, we automatically run a prewarm screen and if reactivity disappears, it is a cold antibody. What do you use as controls for your cold screen procedure? Would you be willing to share an outline of your procedure or where you found this information? This is something I think I need to get a process for and get into writing.

[Marilyn Plett]

This site has a good thread on prewarming. Search for CLiaa & Prewarming Procedures. I agree that you should know what you're dealing with before pre-warming.

[David Saikin]

I pretty much gave you my procedure. The auto is the control. If everything is positive including the auto I call it a cold autoagglutinin. If the cords are negative or less reactive than the adult cells I call it anti-I (I know - I'm not r/o Pr but that is a moot point for insignificant colds) - if the cords are more positive than the adults, I call it anti-i. I cannot give you a reference, this is how I have worked up colds for many years. Sometimes there will be an anti-M in the mix, but hopefully you have an M negative cell in your screen or are able to recognize a dosage effect. I only use 5 minutes in the cold so I don't activate everything that might be there, only something that might be giving me the problem.

[hati]

SUBJECT:

Blood Bank Mini Cold/Pre-warm Technique

NO:

707-0116

Policy Procedure Protocol/Pre-Printed Order Other:

Effective Date

12/08/2009

Date of Electronic Distribution

12/09/2009

Dept. Manager

Medical Director/ CAH Oversight

Administrative

Policy Committee

Committee

Other

Audit Review: Initials:

Date:

PRINCIPLE:

The tube method includes an immediate spin phase that detects antibodies at room temperature. Most saline reactive antibodies are considered clinically insignificant antibodies.

A mini cold panel can be performed when positive reactions are observed in the immediate spin phase with weaker reactions observed at 37C and negative anti-human globulin (AHG) reactions.

If the mini cold panel confirms the presence of a cold antibody, the pre warm technique can be performed.

The Pre-warm technique may be useful to minimize or reduce cold reactive auto- antibodies interfering with the testing phase reading. This test is particularly useful for testing sera of patients with these cold-reactive antibodies (cold agglutinins) that may mask the simultaneous presence of clinically significant antibodies.

The technique should be used with caution since it may decrease reactivity of some potentially significant antibodies and weak antibodies can be missed.

SPECIMEN:

Fresh plasma (EDTA specimen) that complies with pretransfusion specimen requirements (≤72Hrs old)

REAGENTS, SUPPLIES AND EQUIPMENT:

Test Tube Rack 37o C Heating Block

12 X 75-mm Test Tubes Disposable Plastic Pipettes

Centrifuge Pre-warmed (37o C) Saline in Dispenser Bottle

Panoscreen III Cell Screen Check Cells

Donor Units 4°C Refrigerator or 4°C slush bath

four tubes as MINI COLD PROCEDURE:

1. Set up and label follows:

Auto Control (patient cells with patient plasma) and screen cells I, II, and III.

2. To each of the above tubes, add 2 drops of patient’s plasma and 1 drop of the

appropriate red cells.

3. Gently mix tubes and place in the 4°C Blood Bank refrigerator for 15 minutes.

4. After the 4° C incubation, centrifuge tubes for the calibrated spin time.

5. Do not read the tubes immediately, but gently replace the tubes back into 4°C refrigerator for 5 additional minutes.

6. Read and record reactions on the Blood Bank log.

Interpretation of results:

If the reactions read 1+ or greater, the antibody can be determined to be a cold and the Technologist can proceed to the pre warm technique.

PRE WARM PROCEDURE:

If the presence of a cold antibody has been determined follow the instructions below:

1. Pre-warm a bottle of saline to 37o C. Place the bottle in the micro incubator for 1 hour.

2. Label one 12x75 tube for each reagent or donor sample to be tested in accordance with test requirements (crossmatch, antibody screen, reverse group, etc.).

3. Add one drop of 2-5% suspension of red blood cells to each appropriately labeled tube.

4. Place the tubes into the 37o C heat block and incubate for 5-10 minutes. This includes a tube containing a volume of the patient’s plasma adequate for performing all the necessary testing,

5. Transfer 2 drops of the prewarmed plasma into each tube containing prewarmed red blood cells. Mix tubes without removing the tubes from the heat block.

6. Incubate at 37o C for 30 - 60 minutes. Also warm an aliquot of IgG anti human globulin in a tube in the heat block.

7. Remove tubes from the incubator and quickly fill each one with prewarmed saline. Wash the cells 4 times by hand (do not use cell washer). Blot extra saline from each tube after the last wash.

8. Add two drops of pre-warmed anti-human globulin (IgG Coombs) to each tube and centrifuge for the calibrated spin time. Examine for agglutination using the magnifier viewer. Record your reactions.

9. Add one drop of Check cells to each negative tube and centrifuge for the calibrated spin time. Examine for agglutination. Failure to show any agglutination in this phase of testing is to be regarded as a QC failure, and the test must be repeated.

10. Discard the warm saline and wash the saline bottle thoroughly before filling with saline to avoid bacterial growth.

REPORTING RESULTS:

1. If the Pre-warmed technique elicits a negative result, report as follows:

“Probable cold antibody, clinically insignificant”. Additional referral testing performed upon physician request.

2. Blood warmers are not routinely used unless there is a potent cold antibody (3+ or greater reaction.)

NECESSITY OF CROSSMATCHED UNITS:

In the event a crossmatch is ordered for a patient with a cold antibody, a full AHG crossmatch using the pre-warm technique needs to be performed.

PROCEDURE NOTE:

Immediate spin and 37 C are not part of pre-warm technique.

This procedure will not detect alloantibodies that agglutinate at 37o C or lower and are not reactive in the antiglobulin phase.

REFERENCE:

Local policy

American Association of Blood Banks Technical Manual Current Edition and 8th edition.