Fig. 1

Details of the results sheet of the single antigen HLA antibody test before depletion (day −7, a) and after depletion (day +15, b) are shown. The anti-A2 antibody columns are indicated with thick black arrows. All highly positive reactions against HLA-A2 (a) became negative (b). In contrast, antibodies towards bystander HLA specificities that were co-expressed on the transfused platelets, such as HLA-B18 and B50, were only partially reduced, whereas third party specificities not expressed on the platelets, such as HLA-B78 and B51, remained at a high level. The incremental effect of each of the first five platelet transfusions and the respective antigen is shown in c. The two transfusions given just before the hematopoietic cell transplant were not included since the data would be skewed by the high number of A2-positive cells within the transplant. We observed a stable MFI reduction for the HLA-A2 DSA, whereas the bystander antigens of the HLA-B locus were less effective. To investigate the specificity of the HLA-A2-expressing platelets, the anti-HLA antibodies of the day −7 serum were depleted using either HLA-A2 positive (n = 3) or negative (n = 3) platelets as follows: 20 µl of recipient serum were incubated with 120 µl of a platelet suspension containing 1 × 10⁹ platelets/ml in 0.9% NaCl for 30 min at 37 °C. Subsequently, the suspension was centrifuged for 5 min at 12,000 × g. The supernatant was removed and depleted two more times as described, before being analyzed in the solid-phase antibody test. Serum treated with PBS was used as control. To prove that the depleted antibody was adsorbed by the platelets, the centrifuged platelets were subjected to an acid elution using a commercial elution kit according to the manufacturer’s instructions (BAG-Elutions-Kit, BAG Health Care GmbH, 35423 Lich, Germany) and the eluate was analyzed for anti-HLA antibodies. The results of three independent runs with different platelet preparations are shown in (d), as the mean ± s.d. Whereas depletion with HLA-A2-negative platelets had no effect on the level of the anti-A2 antibody, it disappeared after depletion with A2-positive platelets. In contrast, elution of antibodies from the respective platelets demonstrated that the anti-A2 antibody could be recovered from the A2-positive platelets but not the A2 negative. This shows that depletion of the anti HLA-A2 antibody was a specific effect of the A2 expressing platelets