BIOLOGIC AND THERAPEUTIC ADVANCES IN HEART TRANSPLANTATION I

A. Nikaein¹, N. El-awar², J. Hunt^3
1Tansplant Immunology, Texas Medical Specialty, Inc., Dallas/UNITED STATES OF AMERICA, ², One Lambda, Inc., Los Angeles/CA/UNITED STATES OF AMERICA, ³, Medical City Dallas Hospital, Dallas/TX/UNITED STATES OF AMERICA

Body: Introduction: With the advent of solid phase immunoassay, it has been possible to identify both class I and class II HLA antibody specificities. However, more investigation is required to assure that these methods assign clinically significant anti-HLA antibodies. El-Awar has shown that single antigen beads (SAB) by flow and microarray test lists both HLA and natural HLA-specific antibodies. Therefore, it is important to be able to distinguish these antibodies in order to decrease the rate of unnecessary exclusion of patients who would otherwise receive transplants. The aim of our study was to determine if clinically significant HLA antibody specificity analysis would be possible by determining either a cut-off value for mean fluorescence intensity (MFI) reactivity, crossmatching with surrogate donors or by denaturing antigen with elution buffer. Methods: Forty one sensitized heart or renal transplant candidates were included and 125 crossmatches were performed with surrogate donors using different sera for each patient. HLA antibody identification was performed using SAB by flow cytometry and Luminex before and after denaturizing HLA antigens. To denature HLA antigens on beads elution buffer was used which splices the B2 microglobulin and peptides from heavy chain. Results: Compared to virtual crossmatch, 13/41 patients had false negative crossmatches (T cells, B cells or both). 9/13 false negative crossmatches were class I and 4/13 were class II HLA antibody related. Most patients were found to have natural antibodies to heavy chain with or without alloantibodies. Most antibodies with MFI>5000 resulted in positive crossmatches. However, 3 heart transplant candidates with LVAD were found to have natural antibodies to epitopes of denatured heavy chain or cryptic epitopes of class I HLA antigens. Some natural antibodies had high MFI values of 6000-16000. Flow crossmatches with surrogate donors each expressing different unacceptable antigens were negative for T cells and half were positive for B Cells. The latter may be due to autoantibodies which were not performed. Based on these results, we decided not to list any unacceptable antigens for these patients. Presence of natural antibodies was not limited to heart transplant candidates. Many of renal transplant candidates had different levels of natural antibodies in addition to HLA antibodies. This method has helped to distinguish the alloantibodies which bind to intact HLA molecules from those directed to denatured heavy chain of HLA or cryptic epitopes. Conclusion: We have shown for the first time that most patients may have natural HLA-specific antibodies with or without allo-specific antibodies to intact molecules. It is impossible to differentiate these antibodies by MFI values only and without additional testing. Therefore, assignment of antibodies must be carefully analyzed, especially in cardiac transplant candidates with LVAD as these antibodies with no apparent clinical significant increases rate of an unnecessary exclusion of patients who would otherwise receive transplant.

Disclosure: All authors have declared no conflicts of interest.