Fig 1.
The effect of wavelength and sample volume on absorbance measurements.
Experiments were conducted at 37°C. Results in figure A-C is from unstimulated samples and measurements are performed without shaking. a) Shows the ratio between optical density in platelet-rich plasma (PRP) and optical density in platelet-poor plasma (PPP) when comparing wavelengths for absorbance measurements. Results are mean and SD from triplicate samples from three persons). b) Shows the ratio between optical density in PRP and optical density in PPP dependent on sample volume. Results are mean and SD from triplicate samples from three persons. c) Shows the effect of platelet count in PRP on absorbance measurements at different sample volumes. Results are mean of duplicates on a pool from three persons. d) Shows platelet aggregation results from samples with platelet counts ranging 50–300 x109/L constructed from isolated platelets suspended in autologous PPP and stimulated with 6.4 μM adenosine disphosphate, n = 5. * indicates p<0.05. Results are mean and SD. Group comparisons were done with the t-test.
Fig 2.
The effect of platelet adhesion and plate shaking on absorbance readings.
Experiments were conducted at 37°C and when relevant 10 minutes of shaking was used. a) Platelet adhesion in wells is detected by absorbance readings in wells emptied after platelet aggregation. It is compared to absorbance readings in wells previously containing unstimulated platelet-rich plasma (PRP) or platelet-poor plasma (PPP). Platelet adhesion in wells pre-coated with gelatin (+) is compared to wells without gelatin (-), results are mean and SD, n = 9 (triplicate samples from three persons). * indicates p<0.05. Group comparisons were done with the t-test. ADP, adenosine diphosphate; OD, optical density; TRAP, thrombin receptor-activating peptide; AA, arachidonic acid. b) The effect of shaking on absorbance readings in PRP. Measurements before and after 10 minutes of shaking is compared. Wells pre-coated with gelatin and without gelatin is compared, n = 9. Group comparisons were done with the t-test, none had p<0.05.
Fig 3.
Effect of time of shaking on platelet aggregation results.
The effect of time of shaking on platelet aggregation results using different agonists. Except for time settings, the experiments were conducted with the final protocol at 37°C. Each graph represents mean platelet aggregation for one agonist. The curve within a graph represents platelet aggregation as a function of shaking time at different agonist concentrations, n = 3 persons per test condition. Same persons for all agonists. ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide; AA, arachidonic acid; PAR-4, protease-activated receptor-4.
Fig 4.
Platelet aggregation in healthy individuals and the effect of aspirin.
Platelet aggregation was measured after 10 minutes according to the final protocol at 37°C. Platelet aggregation was tested in 10 healthy individuals, same individuals for all agonists. Median platelet aggregation at a given agonist concentration (circles) and interquartile ranges (whiskers) are shown. Samples from three individuals were pre-incubated with aspirin 40 μM. The graph shows median platelet aggregation (squares) and ranges for this group. * indicates p<0.05. Group comparisons were done with the Mann-Whitney U test. ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide; PAR-4, protease-activated receptor-4.
Fig 5.
Platelet aggregation in microtiter plates stored at -80°C.
The platelet aggregation was evaluated in microtiter plates pre-coated with agonist or buffer, sealed and stored for up to 15 days at -80°C, n = 4 samples. Platelet aggregation was conducted following the final protocol with 10 minutes of shaking at 37°C. ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide; AA, arachidonic acid, PAR-4, protease-activated receptor-4.
Table 1.
Coefficient of variation (intra-serial CV%).