Evaluation of an in-house bead-based assay using recombinant HPA 1a/b, -5a/b and -15a/b

Abstract

Human Platelet Antigens (HPAs) are epitopes on platelet membrane glycoproteins that play a crucial role in transfusion medicine and immune-mediated platelet disorders, such as Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) and Platelet Transfusion Refractoriness (PTR). The clinical significance of HPA-1a, HPA-5b, and HPA-15b antibodies makes their accurate detection essential for patient management. This study evaluates the performance of an in-house bead-based assay (BBA) designed to detect HPA-1, HPA-5, and HPA-15 antibodies. Using recombinant glycoprotein complexes, the assay offers a novel approach compared to gold-standard methods like the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) assay. The BBA demonstrated high specificity across all HPA antibodies (95.80% to 98.17%) with better performance for HPA-1a antibody detection (90.00% sensitivity and 98.17% specificity). However, sensitivity varied across different HPA specificities highlighting the need for further optimization. To address higher background issues, particularly with HPA-15, new beads (HPA15.001) were developed, resulting in significant improvements in specificity and reduction in false positives. This advancement not only improves the accuracy of HPA-15 antibody detection but also highlights the importance of continuous refinement in diagnostic testing methodologies.The findings suggest that this bead-based approach could streamline platelet antibody detection with high-throughput potential, offering an effective tool for diagnosing alloimmune platelet disorders. However, the potential for false negatives, especially for non-HPA-1a antibodies, suggests that it may need to be used in conjunction with other tests to ensure accurate diagnosis across all HPA specificities.