The prerequisites and kinetics of HLA-antibody-induced 14C-serotonin release from platelets were investigated with five HLA-specific antisera. By studying the influence of incubation time, temperature, pH, platelet count, stirring and extent of platelet labeling, optimal results were obtained after 30 (to 60) min of incubation at 37 °C, pH 7.5-8.0, under constant stirring (400 rpm). Optimal platelet counts varied between 160,000 and 400,000/µ1 depending on the antiserum applied. A comparison of the amount of 14C-serotonin released by different antisera from washed platelets and from platelets in platelet-rich plasma suggests that plasma components - most likely complement - may additionally influence the results. Based on these experiments, a standardized microtechnique of HLA-antibody-induced 14C-serotonin release is recommended.

Three HLA-specific antisera were comparatively analyzed by platelet microcomplement fixation and 14C-serotonin uptake and/or 14C-serotonin release studies with platelets. In general, there was a good correlation of HLA antibody specificity exhibited in all 3 test systems. Quantitative differences in the response of platelets to HLA antisera were observed and accounted for by differences in antigen density and different types of antibodies involved. Platelet autoantibodies could not be detected by means of 14C-serotonin release from platelets in plasma.

To evaluate the relevance of HL-A-specific antibodies in nonhemolytic transfusion reactions, sera from 72 patients with transfusion reactions were examined using platelet microcomplement fixation and microlymphocytotoxicity. Lymphocytotoxic antibodies were found in 24 and platelet complement-fixing antibodies in 18 patients. Thirteen positive sera were studied by absorption and elution with platelets. A total of 103 eluates (mean 7.9 eluates per serum) was tested serologically for antibody specificity. Results were statistically analyzed for x^2-association with known HL-A antigens. All sera investigated contained HL-A-specific antibodies. Antibody specificities determined were directed against 7 antigens (1, 2, 3, 9,10,11 and W28) of the first series and 10 antigens (5, 7, 8, 12, W5, W10, W15, W17, W21 and W27) of the second series of the HL-A system. By means of antibody concentration in the eluates, all sera revealed crossreacting HL-A-specific antibodies. The clinical importance of buffy coat-free red blood cells for the prevention of transfusion reactions is stressed.

The preconditions and kinetics of absorption and elution of HL-A specific antibodies from platelets, their antibody binding capacity (‘activity’) and the stability of HL-A receptors on platelets were studied. It was found that the acid elution technique is the only useful method as compared to the ether or heat elution. The optimal ratio of absorbed serum to eluate volume, platelet number, incubation temperature and time were evaluated. At pH 3.0-3.5 (range tested between pH 1.5 and 5.0) the strongest eluate activity was obtained. The fixation of HL-A specific antibodies to platelet receptors was not reduced by repeated washings with buffered saline, hypo- and hyperosmolaric salt solutions (range 0.077 to 2.464 mol NaCl). The capacity of antigenic receptors of platelets to bind complement-fixing HL-A antibodies remained unaltered after treatment with distilled water, hypo- and hyperosmolaric salt solutions (range as above) and incubation between pH 5 and 9. Freezing (-20 °C), heating (56 °C) and short incubation at pH 10 moderately altered HL-A receptors on platelets, which clearly deteriorated at pH 3 or after incubation at 100 °C. A standardized elution procedure is recommended.