Direct antiglobulin testing and the evaluation of transfusion reactions

The blood bank manager at a hospital located in the MidWest comments that in her experience many transfusion service laboratories of similar size to her lab experience transfusion reaction work ups only occasionally. She adds that the College of American Pathologists' (CAP) Laboratory Accreditation Checklist seems to require that when working up a transfusion reaction, that a direct antiglobulin test be done, and that this testing examine for both IgG and complement coating on red cells. However, she wonders if it is really necessary to test for complement coating on red cells, considering that a check for hemolysis is routinely done, and there is a lack of required testing for complement in any other pre-transfusion testing. Furthermore, so called "complement-only" red cell antibodies are rare. In her experience, it is of no value to test for complement binding of red cells when doing a transfusion reaction work up. She would like to know if any institutions are NOT testing for complement coating of red cells when doing a transfusion reaction work up. For those that do test for complement coating, what QC is done for when using polyspecific AHG and when using anti-complement AHG, when working up a transfusion reaction? Do laboratories keep complement-coated QC cells for that once/month testing?

The following comments were submitted in response.

ADDENDA July 9, 2009

1. The Medical Director of a Blood Bank and Transfusion Service in New York reports that his blood bank does perform testing for complement coating of red cells when doing a direct antiglobulin test (DAT), not only as part of the work up for transfusion reactions, but also as part of a DAT profile upon request for patients suspected of having immune hemolysis. They do use complement-coated QC cells and participate in the CAP DAT survey which includes complement DAT.

ADDENDA Oct. 31, 2009

2. A colleague comments that the 26th Edition of Standards for Blood Banks and Transfusion Services indicates that a direct antiglobulin test (DAT) shall be performed as part of a Transfusion Reaction investigation. She wonders if the DAT must include the use of reagents that will detect red cell bound complement, or if using only anti-IgG anti-human globulin reagents will suffice.

ADDENDA Nov. 11, 2009

3. Professor John Judd, Emeritus Professor of the Department of Pathology at the University of Michigan comments that he is aware of at least one laboratory that does not use reagents containing anti-C3 for the direct antiglobulin test (DAT) when a routine investigation of a transfusion reaction is done. The rationale for use of anti-IgG alone in a routine transfusion reaction investigation is complicated, but largely due to the fact that anti-IgG rather than polyspecific AHG is used for pretransfusion testing.
   
   In Professor Judd's experience the DAT (whether done with polyspecific AHG or anti-IgG) has not been particularly informative when investigating transfusion reactions. In cases of a hemolytic reaction when the DAT has been positive, other tests have been positive; when DAT-negative, other tests have also been positive. Indeed, all three of the most profound hemolytic transfusion reactions he has encountered had a negative polyspecific DAT (one ABO incompatibility, one due to a C3-binding alloanti-C, and one associated with a bizarre autoantibody. Of course, all three were encountered at a time when were not required to validate negative tests with reagents containing anti-C3!
   
   If use of C3-coated RBCs is required to validate negative tests with reagents containing anti-C3 by wording in the product circular (and therefore by the FDA and accrediting agencies) then so be it. However, he thinks it odd that it is required by one manufacturer but not by another. Also, he remains of the opinion that use of IgG-coated RBCs to confirm negative tests with polyspecific reagents is adequate. If not, then the tests must be set up in duplicate to allow validation with both IgG- and C3-coated RBCs. Furthermore, requirements stipulating that negative tests with monospecific anti-C3 be confirmed using C3-coated RBCs has placed a burden both on transfusion services, to the extent that in some instances diagnostic DATs are now sent to reference laboratories, causing delay in patient diagnosis and treatment. Thus, he asks have we improved patient care, or is the downside of sending tests out mitigated by the test being performed in accordance with the edicts of the manufacturer and those who deign to regulate us?

ADDENDA November 14, 2009

4. Dr. George Garratty writes: "I believe that a DAT must always be performed when investigating a hemolytic transfusion reaction (HTR) [90% have a positive DAT, the remaining 10% usually being associated with ABO incompatibility; to cover the latter always looks for hemoglobinemia by simply looking at the color of the post-transfusion plasma (even 5 ml of RBCs destroyed by intravascular lysis will yield a pink plasma)]. My personal preference is to use anti-IgG and anti-C3 for investigating HTRs and autoimmune hemolytic anemia. Only anti-IgG is necessary for investigating hemolytic disease of the fetus and newborn (even when associated with ABO antibodies). I agree that a positive reaction with anti-C3, but not anti-IgG post HTR is unusual, but it does happen (remember, it is a sensitive indication for an ABO incompatible HTR, in addition to some Kidd and rare examples of Kell and Duffy antibodies). True hemolytic transfusion reactions, luckily, are unusual (about 1 in 10,000 units, or 1 in 2,000 patients), so I believe it is worth the extra effort/expense to cover all bases, when investigating the cause. With regard to the use of C3-coated control RBCs, you don’t have much choice if you want to satisfy CAP; but this is controversial. Some people do not think it is necessary, others would argue that if you follow the general rules of QC, then you should follow the same rules as using anti-IgG (i.e., use C3-coated RBCs to check activitity daily and to control that the RBCs in that tube have been washed adequately and that anti-C3 was added). This, of course, assumes use of tube DAT; gel DAT does not involve washing. I would add that some well-known immunohematologists do not even support the value of using commercial “check cells” for anti-IgG [(i.e., good QC would require weakly sensitized RBCs to detect contamination with minimal amounts of plasma; commercial “check cells” are too strongly sensitized) (see Garratty & Arndt, Evaluation of IgG sensitized RBCs used for controlling antiglobulin tests. Transfusion 1999;39:104S {abstr} )]. It should be noted that package inserts from both Ortho and Immucor/Gamma AHG reagents recommend using anti-IgG and anti-C3d when investigating HTRs. One other approach (but probably not acceptable by CAP) is to perform the DAT with polyspecific AHG, and only use IgG “check cells”, which would control that the AHG was added and that the RBCs in the tube were washed adequately [(as there is always much more IgG than C3 in any contaminating plasma but, would only control that the anti-C3 is working (or more accurately, “presumed to be working”), if the polyspecific AHG was positive and anti-IgG was negative)]."

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