(P-CB-11) HLA Specific Antibodies Determines Platelet Survival By Regulating Apoptosis

Platelet transfusion refractoriness (PTR), a serious clinical problem, resulting in adverse outcomes and increased health care costs. PTR caused by HLA antibodies accounts for the majority of immune cases and is commonly managed by transfusing HLA-matched platelets. Currently, searching for suitable platelets is based on possessing a large pool of platelet donors and does not guarantee full matching. Moreover, scientific evidence is still not sufficient to elucidate the biological mechanisms of PTR after platelet transfusion in patients with HLA alloantibodies. The aim of this project was to characterise the effects of HLA antibodies with different concentrations on platelets.

Study

Design/Methods:

A batch of HLA antibodies (anti-HLA-A02, anti-HLA-A11 and anti-HLA-A24 antibodies) were diluted to a series of concentrations and tested titer by HLA antibody detection kit (One Lambda) using Luminex assay, then grouped according to the mean fluorescence intensity (MFI) levels. Different concentrations of antibodies were incubated with matching platelets (2.5×10⁸/ml) at 37℃ for 0 h, 1 h, 18 h, 24 h and 30 h. Flow cytometry was used to analyse whether these HLA antibodies were capable of inducing platelet apoptosis and evaluated apoptosis degree at different concentrations and different action times by Annexin V kit (BD Biosciences).

Results/Findings:

HLA antibodies were divided into several groups with MFI values around 0, 1000, 3000, 10000, respectively. The group with MFI value equal to 0 was used as control. The concentrations of anti-HLA-A02 antibody are 0, 25, 68, 550 μg/ml; anti-HLA-A11 antibody are 0, 18, 52, 300 μg/ml; anti-HLA-A24 antibody are 0, 7, 23, 90 μg/ml, respectively. Over the course of 18 h after incubation with HLA antibodies, the phosphatidylserine (PS) exposure of platelets rose between 0 μg/ml and 550 μg/ml for anti-HLA-A02 antibody (from 56.26±6.72% to 82.93±0.73%), increased by 35.18% at 300 μg/ml anti-HLA-A11 antibody, enhanced from 63.63±2.07% to 90.15±3.41% between 0 μg/ml and 90 μg/ml for anti-HLA-A24 antibody. At different concentrations of these HLA antibodies incubated with platelets for 24 h, only 90 μg/ml anti-HLA-A24 antibody could markedly induced platelet apoptosis compared with the untreated group (from 62.78±2.71% to 92.74±1.89%). After treated with HLA antibodies for 30 h, the anti-HLA-A02 antibodies of 25, 68, 550 μg/ml exhibited a increased trend in apoptosis rate, increased by 15.66%, 19.27% and 20.61%. Meanwhile, anti-HLA-A11 antibodies (both 52 μg/ml and 300 μg/ml) could significantly cause platelet apoptosis, increased by 29.46% and 32.72%. However, there was no significant difference in anti-HLA-A24 antibodies-treated platelets compared with the untreated platelets at 30 h.

Conclusions:

HLA antibodies (anti-HLA-A11, anti-HLA-A24 and anti-HLA-A02 antibodies) induced apoptosis and accompanied PS exposure in platelets.