Abstract Number: PB0678
Meeting: ISTH 2020 Congress
Background: ITP is a common autoimmune bleeding disorder in which anti-platelet autoantibodies target one’s own platelets. Most ITP autoantigens have been localized on αIIbβ3(GPIIbIIIa) integrin and GPIbα. We were among the first to demonstrate ITP mediated by anti-GPIbα-antibodies is distinct from anti-αIIbβ3 (Blood. 2006); many anti-GPIbα antibodies mediate Fc-independent ITP, (Nat.Commun. 2015, Blood. 2018) leading to resistance of first-line therapies. The gold standard for ITP-autoantibody detection is the monoclonal antibody immobilization of platelet antigens (MAIPA) assay. However, MAIPA is rarely clinically utilized due to the necessity of a large amount of patient blood, fresh donor platelets and three different monoclonal antibodies, furthermore, MAIPA takes 3-4 days to perform, lacks standardization and is prone to false positives/negatives. Thus, leaving clinicians to rely on empirical treatment.
Aims: Development of functional αIIbβ3- and GPIbα- bound monolayer coatings that can be applied to magnetic-silica beads for the rapid detection of ITP mediating autoantibodies to improve the diagnosis and treatment of ITP.
Methods: Surface- and bio-chemistry synthesis.
Results: Both coatings are based on a self-assembling-monolayer which facilitates oriented (binding-site upward) covalent linkage of either αIIbβ3 or GPIbα and is resistant to non-specific protein binding. These two coatings were synthesized on 0.6-3µm silica beads. Immobilized GPIbα bound both conformational and linear-epitope specific anti- GPIbα mAbs. αIIbβ3 coated surfaces bound fibrinogen and both conformational and linear-epitope specific mAbs. Furthermore, αIIbβ3 coated beads were incorporated into platelet aggregates. Using these beads, we developed a flow cytometry-based assay for anti-platelet autoantibodies. Our assay produced a limit of detection one order of magnitude more sensitive than MAIPA, while only requiring ~6 h to complete.
Conclusions: Our facile flow cytometry assay is more rapid and sensitive than MAIPA, likely due to the higher antigen density than on a platelet surface. A study evaluating our assay against MAIPA with ~50 ITP patient samples is ongoing.
To cite this abstract in AMA style:Neves M, Arad A, Wang Y, Zhu G, Galant R, Thompson M, Ni H. Development of a Flow Cytometry-based Assay for the Detection of ITP Autoantibodies [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/development-of-a-flow-cytometry-based-assay-for-the-detection-of-itp-autoantibodies/. Accessed February 27, 2024.
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