Flow cytometryによる同種および自己抗ヒト血小板抗体の解析：Ⅰ. 酸処理後ホルマリン固定血小板を用いての抗体測定法の開発

Abstract

Flow cytometry (FCM) has recently been used to determine the epitopes of mouse monoclonal antibodies against platelets glycoproteins (GP). However, the utilization of this technique to detect the autologous anti-platelet antibody has been limited, because a high frequency of non-specific reaction has been observed in serum from healthy individuals. One reason for this has been thought to be related to the surface bound HLA antigens. To overcome this problem I have used acid treatment of platelets. The platelet pellets prepared by albumin density gradient separation were first treated with 0.123 M citric acid-Na₂HPO₄, pH3.0 for 10 min. at 0℃ followed by formalin fixation. Using these platelets, the percentage of fluorescence positive cells (%FPC) for HLA-A, B, C antigens markedly decreased from 97.0 to 21.3, whereas that for platelet surface IgG slightly decreased from 40.4 to 28.8. On the other hand, %FPCs of platelet GPⅠb and GPⅡb/Ⅲa complex remained almost unchanged, suggesting that the respective epitopes remain intact. The level of platelet bound IgG (PBIgG) of healthy adults and the multi transfused patients were determined. For healthy adults (n=10), the %FPC was 46.5±10.7 (mean±SD) before treatment, and 10.3±2.9 after treatment. Thus the %FPC after acid treatment were significantly decreased. In multi transfused patients (n=5), the %FPCs were almost unchanged before and after acid treatment, from 86.6±21.3 to 84.3±25.6. The average %FPC value in 50 healthy adults (25 males and 25 females) was 12.6±6.6 and the cut off value was 20.0. These results clearly indicate that acid treatment of platelets increases the availability and specificity for the detection of allo and autologous anti-platelet antibodies.