HLA is the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Finding compatible platelets for those patients is a major obstacle in platelet transfusion therapy. The primary objective of this proposal is to develop new approaches for overcoming the immunological refractoriness to transfusion of random donor platelets. For one approach, we will establish a precise quantitative assay to measure the concentration of each specific HLA on platelets. At the beginning, monoclonal antibodies (MoAb) against monomorphic epitopes of HLA molecules will be developed for this assay by immunizing mice with synthetic peptides containing conserved amino-acid sequences. The precise quantitation of each specific HLA on platelets will be achieved by measuring the total HLA on a platelet with an established enzyme- linked immunoassay and by determining the relative quantity of each specific HLA on platelets using IEF-PAGE, anti-HLA MoAbs and immunoblots. The results can then be used to calculate the exact concentration of each specific HLA. This assay will enable us to study the potential benefit of transfusing alloimmunized patients with platelets from donors who have a low expression of one or two mismatched HLA antigens. It can also be used to compare the variable expression of specific HLA between platelets and lymphocytes, to study the genetic regulation of quantitative expression of each specific HLA on cells, and to investigate the linkage of variable expression of specific HLA to disease susceptibility and immune responsiveness. For another approach, we will initiate experimental studies on the use of platelets pretreated with 8-methoxypsoralen and UV-A irradiation to inactivate contaminating leukocytes for the prevention of primary allosensitization against class I MHC antigens in mice. Mice are chosen for their well characterized H- 2 system and the availability of inbred strains with well defined H-2 haplotypes. The results of this proposed study not only will provide us with important information for a future clinical trial but also will establish an experimental system that can be used to study the mechanisms by which the humoral immune response to MHC antigen is triggered by donor leukocytes. Further research in this direction will contribute to a greater understanding of the immunobiology of tissue and organ transplantation.
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