Prewarmed testing can cause a laboratory to miss detecting a clinically significant red cell alloantibody

A blood bank technologist in New Mexico is concerned that her laboratory might be overusing prewarmed testing and as a consequence, missing some significant red cell alloantibodies. She recalls that in the past that they would "prove" that they were dealing with a cold reacting antibody before they would perform a prewarm test. However, she would prefer not to go back to the 'ancient times' when labs would identify cold antibodies by running a "mini-cold panel", using cord blood cells, group A, B, and O red cells, as well as an autocontrol. Her lab currently performs most of their testing using gel technology, so they are not typically looking at direct agglutination reactions at room temperature or 37 C. However, she has noticed that some of the technologists in her lab will do a prewarmed test in tubes whenever an initial antibody panel is performed to see if the antibody goes away. This is why the inquiring colleague is trying to come up with a protocol so they have to "prove" a cold reacting antibody is present before going to the prewaming procedure. She remembers from years ago that they used to run a cold screen first before any prewarming of cells, but she cannot find any current literature on this topic.

Editors' note: The data discussed in Leger R, Garratty G. Weakening or loss of antibody reactivity after prewarm technique. Transfusion. 2003 Nov;43(11):1611-4, and the articles listed below are germane to any policy development for the use of prewarmed testing in the blood bank laboratory.

• Mallory, D: Controversies in transfusion medicine. Prewarmed tests: pro - why, when, and how - not if. Transfusion 1995 35: 268-270.
• Judd, WJ: Controversies in transfusion medicine. Prewarmed tests: con. Transfusion 1995 35: 271-275.

The following comments have been received.

1. A retired SBB who is now a resident physician at a pathology training program in Colorado comments that in his years of experience as a blood bank tech, the main problem with the prewarm technique is the decreased sensitivity of the method, compared to enhanced methods. He has observed many technologists use the prewarm technique as a first line method to "get rid of weak antibodies", because they presume that what they are detecting is merely a cold reacting clinically insignificant antibody. The responding physician's impression is that this practice is more common than it is not. While he has not personally collected data on this issue, he can comfortably say that he has seen many antibodies with clinically significant specificities that have been "prewarmed away" at antibody screen and crossmatch. As a first line test after the antibody screen, the prewarm technique is inadequate, particularly since the hypothesis for using it this way is that the antibody screen is a false positive. He is sure this introduces a technical bias. Nevertheless, he believes the prewarm technique is a valuable tool to keep in our arsenal. He adds that the majority of the technologists that he has worked with are generalists and he has used the following approach to develop policy even though it can be argued that antibodies can still be missed in the setting of a cold plus a weak significant antibody: If the technologist suspects a positive antibody screen is the result of a cold agglutinin, they should first "prove" a cold is present. This doesn't mean they should identify the cold. They should, however, show that it's there by repeating the antibody screen by tube method with an immediate spin or room temperature phase prior to utilizing the prewarm technique.

2. A technologist for a large blood collecting organization with 20 years of experience in the reference lab writes:
   
   Testing a "cold panel" does nothing to "prove" that the reactions seen in Gel are due to a cold antibody. It would simply indicate that there is a cold antibody present at that particular phase of testing.
   
   Performing a cold autoadsorbtion (discussed in the AABB Technical Manual,16th edition, 2008, pp 492, 512, 922-923), would yield a negative Gel test if the antibody reactivity was due to a cold autoantibody. Also, testing for anti-IH in the proper could do "prove" the presence of a cold antibody reacting in Gel.
   
   Gel is an anti-IgG based test system that does not routinely detect classic cold antibodies. Like it or not, due to the sensitivity of the Gel test, weak variable reactivity is not uncommon. This may be due to a number of reasons including the presence of an emerging warm autoantibody, a weak"HTLA" antibody or jacked-up immune response in a recently transfusedPatient (see AABB Technical Manual, 16th edition, 2008, pp. 485, 490-491 and emergence of autoantibody in the recently transfused patient in Immune Hemolytic Anemias, 2nd edition, 2004, pp 338-340).
   
   I'd recommend that you first try to identify the reactivity seen in Gel using the Gel test and if that fails to yield a firm result change your testing to LISS, PEG or even saline techniques.
   
   A blood bank technologist should never assume that the observed reactivity is due to a cold antibody without first trying to determine if a clinically significant alloantibody(ies) is present. Test results which yield reactivity in all cells tested can easily be due to the presence of multiple alloantibodies, warm autoantibody, a high incidence antibody or an antibody directed at an antigen that can by variably expressed from one person to another. If pursuing in Gel is unsuccessful, the next step would be change your method of testing; go to LISS, PEG, albumin or saline tube methods, or even test an enzyme-treated panel. The Technical Manual even states: "The technique should be used with cautionand not used to eliminate unidentified reactivity." (pp.902)
   
   He concludes: NEVER go directly to a prewarm technique. While it can be debated whether the prewarm technique should be used at all, using the prewarm technique to eliminate your initial panel results is not an acceptable practice.
   
   
3. A Transfusion Service Manager at a Childrens Hospital reports that in her laboratory, they only perform the prewarm test as a last resort, and only after testing shows the presence of a cold reacting antibody. Therefore, if their initial testing reveals a possible interfering cold antibody, they do a "cold mini-panel" first by incubating plasma with screen cells and an autocontrol for 15min at 4C. Only if this comes up positive, and they are unable to rule out clinically significant alloantibodies, do they proceed with a prewarm screen/panel. They also use a GEL technology, so sometimes they are able to get a compatible crossmatch using the GEL and avoid a prewarm crossmatch using a tube system. They do not do anything further to identify a cold agglutinin unless specifically requested by the patient's physician and/or the transfusion service Medical Director (anti-I/IH etc).

4. A technologist for a large blood collecting organization with 20 years of experience in the reference lab writes:
   
   Testing a "cold panel" does nothing to "prove" that the reactions seen in Gel are due to a cold antibody. It would simply indicate that there is a cold antibody present at that particular phase of testing.
   
   Performing a cold autoadsorption (discussed in the AABB Technical Manual, 16th edition, 2008, pp 492, 512, 922-923), could yield a negative Gel test if the antibody reactivity was due to a cold autoantibody and the adsorption was successful. In addition, testing for anti-IH in the proper setting could help "prove" the presence of a cold antibody reacting in Gel.
   
   Gel is an anti-IgG based test system that does not routinely detect classic cold antibodies. Like it or not, due to the sensitivity of the Gel test, weak variable reactivity is not uncommon. This may be due to a number of reasons including the presence of an emerging warm autoantibody, a weak "HTLA" antibody, multiple antibodies or jacked-up immune response in a recently transfused patient (see AABB Technical Manual, 16th edition, 2008, pp. 485, 490-491 and emergence of autoantibody in the recently transfused patient in Immune Hemolytic Anemias, 2nd edition, 2004, pp 338-340).
   
   A blood bank technologist should never assume that the observed reactivity is due to a cold antibody and perform a prewarm test without first trying to determine if a clinically significant alloantibody(ies) is present. If pursuing in Gel is unsuccessful, the next step should be to change your method of testing; go to LISS, PEG, albumin or saline tube methods, or even test an enzyme-treated panel. In regards to the prewarm test, the AABB Technical Manual even states: "The technique should be used with caution and not used to eliminate unidentified reactivity." (pp.902)
   
   He concludes: NEVER go directly to a prewarm technique. While it can be debated whether the prewarm technique should be used at all, using the prewarm technique to eliminate your initial panel results is not an acceptable practice.
   
   
5. The Transfusion Services Manager for a Children's Hospital in the Midwest writes:
   
   There seems to be a lot of controversy over whether or not (and when) to do prewarm testing. We only perform the prewarm as a last resort, and only after testing shows the presence of a cold reacting antibody. If our initial testing reveals a possible interfering cold antibody, we do a "cold mini-panel" first by incubating plasma with screen cells and an autocontrol for 15 min at 4 degrees. Only if this comes up positive, and we're unable to rule out clinically significant alloantibodies, do we proceed with a prewarm screen/panel. We also use gel, and sometimes after we have a positive cold screen and have ruled out other alloantibodies, we get a compatible gel crossmatch and are able to avoid a prewarm crossmatch. We do not do anything further to identify a cold agglutinin (such as anti-I or anti-IH) unless specifically requested by the physician or Transfusion Service Medical Director.

6. George Garratty, PhD, FRCPath., Scientific Director of the American Red Cross Blood Services in the Southern California Region writes the following: "The 'prewarming" technique (PWT) has been used for about 50 years in the serological differential diagnosis of the autoimmune hemolytic anemias where the classification is based on 'warm' versus 'cold' antibodies. It did not become popular in routine immunohematology until the 1970s; it was not in the 5th edition (1970) of the AABB Technical Manual, but appeared in the 6th edition (1977). At this time room temperature (RT) testing was commonly used and many 'cold' antibodies were being detected, which could carry through to the antiglobulin test. The PWT was, and should be now, used to determine if a cold antibody of known specificity (e.g., anti-P1, M, N, Lea, Leb, I) is reactive at 37C and thus potentially significant. This test was never intended to be used as a way of removing unwanted reactivity. Using it for this purpose can be unsafe. In 2003 (Transfusion 43:1611), we confirmed Judd's findings that some potentially clinically significant antibodies (e.g., Rh, K, Jka) were not detected or weakened using the PWT. We found that 47% of LISS antiglobulin tests were weaker following PWT and 14% no longer reacted if PWT was used. PLEASE do not use this method to make incompatibilities go away when there is no evidence for a cold antibody causing the problem. I have never understood the rationale of a 'mini cold panel'. One cannot assume that reactions by the AGT are due to a cold antibody because a 4C panel is positive, if there are no agglutinins detected at RT or above".
   
   
7. W. John Judd, FIBMS, MIBiol (Emeritus Professor of the Department of Pathology at the University of Michigan - attribution used with permission) suggests that readers look at his editorial in Transfusion 2006;46:324-6. Below is a summary provided by Dr. Judd, highlighting the salient points:

MANAGEMENT OF COLD AGGLUTININS

• Avoid their detection
  • No reading for direct agglutination, including immediate-spin tests and after 37°C incubation
  • No microscopic examination of tests
  • Use of anti-IgG instead of polyspecific AHG
• Appropriate Management if Detected
  • Resolve blood typing discrepancies
    • Warm-washed or 2-ME/DTT-treated RBCs
    • Autoadsorbed or group O adsorbed plasma
  • Differentiate auto- from alloantibodies
    • Especially auto-vs alloanti-I, anti-P, etc.
  • Exclude presence of concomitant potentially significant alloantibodies
    • Usually requires autologous adsorption
    • Do not use rabbit erythrocyte stroma (nonspecific adsorption of alloantibodies)
• Selecting Blood for Transfusion
  • Alloanti-M, -P1, -Le and HI (active at IAT)
    • Issue IAT-compatible units
    • Avoid use of acidic LISS reagents
    • No need to confirm antigen-negative status of compatible units
  • Anti-N
    • If black, patient’s RBCs may lack ‘N’ on glcyophorin B; N-U- RBCs will be compatible
  • Autoanti-I (active at IAT)
    • Confirm ABO types of donor units and intended recipient’s RBCs at time of assignement (reservation
    • Or, perform an immediate-spin crossmatch with autoadsorbed serum
    • Or, perform a saline-IAT crossmatch with unadsorbed serum (no enhancement medium)

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