Optical multichannel (optimul) platelet aggregometry as an alternative to traditional light transmission aggregometry: potential utility in clinical settings

Melissa Chan1, Paul Armstrong1, Francesco Papalia1, Rachit Singhal1, Nicholas Kirkby2, Timothy Warner1. 1The William Harvey Research Institute, Barts & the London School of Medicine and Dentistry, Queen Mary University of London, EC1M 6BQ, London, United Kingdom, 2Cardiothoracic Pharmacology, National Heart and Lung Institute, Imperial College, SW3 6LY, United Kingdom.

Platelet reactivity testing is important for diagnosis of bleeding disorders, and increasingly to optimize anti-platelet therapy. Traditional light transmission aggregometry (LTA) is still considered the gold standard. However, LTA methodologies and outputs vary widely between laboratories. We have therefore assessed the potential of a 96-well plate-based method to deliver standardized optical detection of platelet aggregation.

Individual wells of half-area 96-well plates were coated with gelatin and then 8 concentrations each of arachidonic acid (AA, 0.03-1mM), adenosine diphosphate (ADP, 0.005-40μM), collagen (0.01-40μg/ml), epinephrine (EPI, 0.0004-10μM), TRAP-6 amide (0.03-40μM) and U46619 (0.005-40μM), before being lyophilized, vacuum-sealed, foil packed and stored at room temperature for up to 12 weeks. Blood was taken from healthy volunteers by venepuncture into 3.2% sodium citrate and centrifuged (175xg, 15min) to obtain platelet rich plasma (PRP). To measure platelet aggregation, 40µl PRP was added to individual wells of the plates, which were then incubated (37°C) for 16min in a kinetic plate reader (Tecan Sunrise); or for 5min in a high-speed (1200rpm) thermoshaker (BioShake IQ) before determination of light transmission by a standard 96-well plate reader. Percent aggregation was calculated by reference to the absorbances of PRP and platelet poor plasma. In parallel studies, release of ATP/ADP from platelets was determined by luminescence and release of thromboxane (TX) A2 by ELISA for its stable metabolite TxB2. Some experiments were conducted in the presence of aspirin (30µM) or prasugrel active metabolite (PAM, 3µM). Data are presented as area under the concentration-response curve (mean±s.e.m.; log[drug].%aggregation) and compared using one-way ANOVA.

The Optimul method detected concentration-dependent platelet aggregation, ATP/ADP release and TXA2 production to all agonists in both the kinetic plate reader and thermoshaker assays. The effects of PAM were readily detected as reductions (p<0.05) in platelet responses to AA (66±15 to 12±3), ADP (34±5 to 6±2), collagen (33±5 to 8±1), EPI (30±7 to 6±1), TRAP-6 (70±10 to 27±4) and U46619 (107±8 to 34±8), while the effects of aspirin were detected as inhibition of responses to AA (8±4), collagen (14±4) and EPI (18±6; n = 4-7 for all).

We conclude that this Optimul method offers a viable alternative to LTA for testing of platelet aggregation and release reactions. The centralised production of these plates coupled with the ability to read them in kinetic 96-well plate readers, or standard 96-well plate readers as an endpoint assay after a simple incubation step, would permit thorough platelet function analysis without the need for expensive or dedicated equipment.