Light transmission aggregometry using pre-coated microtiter plates and a Victor X5 plate reader

Abstract

Background: Light transmission aggregometry (LTA) can be performed with microtiter plates (96-well LTA). When conducting LTA, an agonist is added to platelet-rich plasma and the sample is shaken for minutes after which absorbance readings are done. Platelet aggregation is detected as decrease in absorbance. However, the classical method is cumbersome and therefore microtiter plates can be used for concomitant testing of multiple samples. Furthermore, it would be convenient to prepare the plate in advance of platelet aggregation testing. Aim: The aim of the present study was to establish a simplified 96-well LTA protocol, where plates were pre-coated with agonists and stored at -80 C until use. Results: We developed and validated a protocol for 96-well LTA using a Victor X5 plate reader and pre-coated microtiter plates. The minimum requirement of platelet-rich plasma was 45 μL per sample and the sample platelet count should not be below 100 x109/L. Optimal absorbance reading was 595 nm wavelengths. Platelet aggregation results were higher at 37C than at room temperature. Platelet adherence to wells after stimulation was observed; it was not avoided by pre-coating of the wells with gelatin. A range of up to 7 concentrations for each agonist (collagen, arachidonic acid, adenosine diphosphate, thrombin receptor-activating peptide and protease-activated receptor-4) was tested concomitantly. A transient rise in platelet aggregation was observed after 2 minutes of shaking in some samples with low agonist concentration, and platelet aggregation was optimal after 10 minutes of shaking for samples with high agonist concentration. Plates could be stored at -80C for 15 days without significant change in the platelet aggregation results. Conclusion: The 96-well LTA is suitable for platelet aggregation testing and a range of agonist concentrations can be concomitantly tested.