Supplementary Material for: In vitro generated megakaryocytes for the detection of human platelet antigen-specific alloantibodies

Introduction: Serologic characterization of anti-human platelet antigen (HPA) alloantibodies is crucial in fetal neonatal alloimmune thrombocytopenia (FNAIT). The gold standard MAIPA assay requires fresh platelets from HPA-genotyped donors, which is challenging for some labs. Megakaryocytes express HPA epitopes and offer an alternative source for detecting anti-HPA antibodies. The objective of this study was to assess the efficacy of a novel assay called monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) for detecting anti-HPA antibodies.
Methods: CD34+ cells from buffy coats were differentiated into megakaryocytes in vitro. The performance of the MAIMA assay was evaluated using WHO reference reagents for HPA-1a, HPA-3a, and HPA-5b, along with sera samples from patients who had well-characterized anti-HPA antibodies.
Results: The WHO anti-HPA-1a reference reagent showed similar binding to megakaryocytes and platelets in MAIMA and MAIPA, respectively. On the other hand, OD values for the WHO anti-HPA-3a reference reagent were lower in MAIMA than in MAIPA. Anti-HPA-5b antibodies were not detectable in MAIMA. Patients’ sera containing anti-HPA-1a antibodies were successfully detected in MAIMA in all clinical samples. Moreover, OD values in MAIPA and MAIMA showed high correlation (r=0.96, p< 0.001). MAIMA was reactive for samples with anti-HPA-3a as well as anti-HPA-3b, however, OD values were lower compared to MAIPA. Interestingly, all patient samples with anti-HPA-5b antibodies were tested negative in MAIMA.
Conclusions: In vitro generated megakaryocytes can be used to detect anti-HPA-1a alloantibodies. However, despite this potential, they may be less suitable for the detection of alloantibodies against other HPAs such as HPA-5b.