Are laboratories following the requirement to use complement-coated RBCs for validating negative direct antiglobulin tests (DATs) performed with anti-C3b, -C3d reagents?

A colleague in Michigan wonders if the e-network forum participants in the United States are aware of the requirement to use complement-coated RBCs to validate negative direct antiglobulin tests (DATs) performed with anti-C3b, -C3d reagents. This requirement appeared several years ago in the product circular of at least one manufacturer of anti-C3b,-C3d, and has recently been incorporated into the CAP Inspection Check-List as a Phase II item, TRM.40210, to encourage laboratories to follow the reagent manufacturer's instructional insert and to validate that negative test results are truly negative, and not due to a technical failure.

The Michigan colleague would like to know if laboratories (including those outside the USA) are indeed validating negative anti-C3 DATs. If they are, what cells and what procedures are they using? Are they using commercially prepared cells or do they prepare them in-house; if the latter, what method are they using? Also, the Michigan colleague wonders if tests with anti-C3 provide any additional clinically useful information that could not be obtained in DATs with polyspecific and anti-IgG reagents, plus testing serum and eluates using indirect antiglobulin tests.

Editor's NOTE: While the new checklist item TRM.40210 does state that "When performing an antiglobulin test with anti-C3 antiglobulin reagents, are C3-coated red blood cells used in all negative antiglobulin tests?", the checklist item's commentary (which can be viewed via a link from the CAP home page) makes the following qualification: "If a licensed system is used that does not allow the use of C3-coated cells, an appropriate quality control system must be followed, as recommended by the manufacturer." So it would seem that one should read the product circular/instructional insert to know what QC and/or test validation is actually needed and/or required by the reagent manufacturer.

The following responses were received.

ADDENDA Mar. 12, 2003

1. W. John Judd, FIBMS, MIBiol, Professor of Immunohematology in the Department of Pathology at the University of Michigan Medical Center (name and institution used with permission) reports that based on his review (verbatim) "Two manufacturers' product circulars of anti-C3 reagents require validation of negative tests with C3-coated RBCs. Gamma stipulates use of C3b-coated RBCs, and Immucor says to use C3-coated RBCs. Ortho makes no requirement. The product circular for C3d-coated RBCs says they can be used for daily QC, but there is no mention of their use for validation of negative tests."
   
   John adds, "We find that Gamma's C3d-coated RBCs do not validate negative tests with Immucor's anti-C3b, -C3d reagent. Nor do in-house prepared C3b/C4b coated cells. Yes there is some reactivoty, that one could probably confirm microscopically, but the agglutinates are so fragile and difficult to discern when diluted with the uncoated cells. We will do more when we receive C3b coated cells from Gamma, at the end of next week."
   
   John also graciously contacted a few clinical hematologists for their clinical impression.

   Hematologist #1: "If the patient was IgG surface positive and had failed other therapies, I would recommend splenectomy regardless of complement presence. I do not care if the anti-C3 is no longer tested, as long as your report would comment on the presence or absence of a cold agglutinin."

   Hematologist #2: "I am not aware of any prospective studies that compare outcomes and/or treatment response on the basis of IgG alone or IgG+complement. I think that there is a clinical bias that dual positivity is somehow associated with a more aggressive disease process or an underlying disorder like lupus, but I don't think that is an evidence-based impression. Maybe we should look at this information prospectively or retrospectively. I would not treat patients differently on the basis of single or dual positivity. I would treat clinically (even in the absence of a positive DAT but clinical evidence of a hemolytic anemia consistent with immune destruction). For aggressive hemolysis, that means steroids. If the patient does not respond, or relapses, then I recommend splenectomy. With mounting evidence that rituxan is effective, that is an option prior to splenectomy, or if splenectomy is contraindicated."

2. A colleague who works at a large tertiary care hospital in Arkansas reports that at his facility, they routinely use complement-coated cells to validate their C3 direct antiglobulin tests. At this time they use Gamma C3b-coated cells and have found them to work just fine when using Immucor C3b,C3d reagent. To his knowledge, there is no way to validate negative tests when using Ortho C3d reagent; one can perform daily QC on the reagent using C3d-coated cells (they tried Gamma's), but as John Judd pointed out (see above), those cells do not work well as "check" cells.
   
   The Arkansas colleague would also appreciate any comments regarding whether other transfusion services actually try to distinguish between the type of complement coating patient cells (C3b vs. C3d). Is saying there is complement coating the cells enough, especially when testing EDTA samples?

ADDENDA Mar. 13, 2003

3. According to the supervising medical technologist at both the USC University and USC Norris hospitals, the following reagents are used for direct antiglobulin testing
   • Ortho's Anti-Human Globulin Bioclone Anti-IgG, -C3d; polyspecific
   • Immucor's Anti-Human Globulin Anti-C3b, -C3d
   • Gamma's Complement Coombs Control (EC3b) cells
   Both of the AHG reagents undergo daily QC with the C3b-coated cells. Reaction strengths are usually in the neighborhood of 1-2+ for the polyspecific AHG and 1-3+ for the anti-C3 AHG. In the experience of those laboratories, variability results primarily from variation in incubation time and technologist technique, i.e. it appears that AHG lot-to-lot potency variation and age of the C3b-coated cells are not a significant factor in variability in those labs. Negative DAT results using the polyspecific AHG are checked with IgG-coated cells. Negative DAT results using the anti-C3 AHG are checked with C3b-coated cells. The procedures seem to work fine in those labs. In other words, negative anti-C3 AHG tests validate when C3b-coated cells are added to the negative test mixtures.
   
   Astute individuals might ask how is it possible that Ortho's Anti-IgG, -C3d poly reagent reacts with C3b-coated cells. It turns out that the name of Ortho's reagent can be a little misleading because it reportedly also contains anti-C3b reactivity. The reagent is prepared from a pool of rabbit anti-IgG and mouse monoclonal anti-C3b and anti-C3d. The rabbit anti-IgG portion is obtained by injecting rabbits with purified human gamma globulin. The mouse anti-C3b and anti-C3d portions are obtained by injecting certain mice intraperitoneally with an anti-C3b secreting hybridoma, and other mice with an anti-C3d secreting hybridoma.
   
   
4. A colleague in the Pacific Northwest reports that they use anti-C3d, -C3b along with anti-IgG and polyspecific AHG for their direct antiglobulin tests. They validated both the Gamma C3d and C3b coated cells. The Gamma C3d-coated cells are intended to be used as a QC reagent for the antisera - not as a control cell for AHG wash. The C3b-coated cells are intended to be used to control the AHG wash phase. They performed a validation study to see if they could use either the C3b- or C3d-coated cells as a control for the AHG wash. This would also serve as a QC check on the reactivity of the reagent as well as provide evidence of an adequate wash in the AHG test. The validation testing demonstrated that the C3b-coated cells are superior to the C3d-coated cells when used as a control for AHG wash. The C3b-coated cells demonstrated acceptable reactivity when fresh and near expiration.

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Ira A. Shulman, MD
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