Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing

Platelets. 2011;22(7):485-94. doi: 10.3109/09537104.2011.592958. Epub 2011 Aug 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspirin / pharmacology
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism
  • High-Throughput Screening Assays
  • Humans
  • Platelet Activating Factor / pharmacology
  • Platelet Aggregation Inhibitors / pharmacology
  • Platelet Aggregation* / drug effects
  • Platelet Function Tests / instrumentation*
  • Platelet Function Tests / methods*
  • Reproducibility of Results

Substances

  • Platelet Activating Factor
  • Platelet Aggregation Inhibitors
  • Aspirin