Quantitative analysis of platelet surface HLA by W6/32 anti-HLA monoclonal antibody

Abstract

Class I molecules of human major histocompatibility complex (HLA) are the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Platelets with reduced expression of a specific type of HLA antigen may escape specific anti-HLA antibody-mediated destruction. By using 125I-labeled Fab fragments of W6/32 anti-HLA monoclonal antibody and competitive protein binding assays, we measured the range of total HLA concentrations on platelets. In 12 individuals examined, the mean number of HLA-A, B, and C molecules per platelet was 81,587 ± 20,016 (mean ± SD); its range was between 54,782 to 116,185 molecules per platelet. After treatment with chloroquine, 79.9 ± 7.0% (mean ± SD, n = 6) of HLA antigens were removed from platelets as determined by binding of 125I-W6/32 Fab. A similar result was obtained when HLA antigens on chloroquine-treated platelets were evaluated with immunofluorescence flow cytometry. In contrast, chloroquine treatment did not remove integral membrane protein such as PI(A1) antigens on platelets. The presence of HLA antigens in the chloroquine eluate of platelets could be demonstrated to contain HLA antigens similar in mol wts to intact class I molecules by an immunoblotting technique. These data suggest that 70% to 80% of platelet HLA antigens are adsorbed and that such HLA antigens are not proteolytic products of integral membrane class I molecules. The origin of the adsorbed platelet HLA-antigens remains to be determined.