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MASKING OF HLA ANTIBODIES BY THYMOGLOBULIN
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Howard M. Gebel Ph.D. 1, Mike Bunce Ph.D. 2, Ian Crosby Ph.D. 2, John David L. Nolen M.D.,Ph.D. 1 and Robert A. Bray Ph.D. 1. 1 Pathology, Emory University Hospital, Atlanta, GA, USA and 2 Dynal Biotech, LTD, Bromborough, Wirral, United Kingdom .
The use of Thymoglobulin as an induction and/or rescue agent has increased, particularly among patients treated with IVIG and plasmaphereisis to eliminate donor reactive antibodies. These patients are at high risk for antibody mediated rejection. Since Thymoglobulin contains antibodies to human class I and class II HLA antigens, we speculated its presence in patient sera would interfere with HLA antibody detection. In the following flow cytometric based studies, we documented that the presence of a therapeutic dose (100 ng/ml) of Thymoglobulin in patient serum masked HLA antibody detection in both screening assays and lymphocyte crossmatches. When Dynabeads coated with goat-anti-rabbit immunoglobulin were used to adsorb Thymoglobulin , both class I and class II HLA antibodies became detectable. Furthermore, crossmatches with T and B lymphocytes expressing the relevant HLA antigens converted from negative to positive after Thymoglobulin was removed. Importantly, restoration of antibody detection was dependent upon complete adsorption/removal of Thymoglobulin . Therefore, we incorporated a reference standard into every adsorption procedure to validate assay performance. These data have important clinical implications. Specifically, consider patients already receiving Thymoglobulin who present with symptoms of acute rejection: If they are monitored for HLA antibodies without Thymoglobulin removal, they would be considered to be antibody negative and the therapeutic strategies to treat these patients would not include antibody depletion. In conclusion, the presence (or absence) of HLA antibodies in sera containing Thymoglobulin can only be reliably ascertained upon its complete removal.