title: Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing.
abstract: Platelet reactivity testing is important for the diagnosis of bleeding disorders, and increasingly to optimise anti-platelet therapy. Traditional light transmission aggregometry is considered the gold standard, whilst 96-well plate aggregometry, founded on similar principles, provides a higher throughput screening method. Despite the widespread use of both, methodologies and outputs vary widely between laboratories. We report a methodological approach towards providing a standardised optical detection of platelet aggregation (optimul method) based upon 96-well plate aggregometry. Individual wells of half-area 96-well plates were coated with gelatine and one of seven concentrations of arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide or U46619, before being lyophilised, vacuum-sealed, foil-packed and stored at room temperature for up to 24 weeks. For platelet testing, 40 µl of platelet-rich plasma was added to each well. Platelet aggregation was determined by changes in light absorbance, release of ATP/ADP by luminescence and release of thromboxane (TX) A(2) by ELISA. Some experiments were conducted in the presence of aspirin (30 µM) or prasugrel active metabolite (PAM; 3 µM). Optimul plates stored for up to 12 weeks permitted reliable detection of concentration-dependent platelet aggregation, ATP/ADP release and TXA₂ production. PAM caused reductions in platelet responses to AA, ADP, collagen, EPI, TRAP-6 and U46619, whilst aspirin inhibited responses to AA, collagen and EPI. We conclude that the optimul method offers a viable, standardised approach, allowing platelet reactivity testing and could provide a broad platelet function analysis without the need for dedicated equipment.
authors.affiliation: The William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M6BQ, UK.
authors.forename: Melissa V
authors.initials: MV
authors.lastname: Chan
authors.forename: Paul C J
authors.initials: PC
authors.lastname: Armstrong
authors.forename: Francesco
authors.initials: F
authors.lastname: Papalia
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Kirkby
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Warner
mesh_terms: D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010979:Platelet Function Tests / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D001241:Aspirin