Platelet Function Assay Using Dielectric Blood Coagulometry

Figures

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  Figure 1. Schematic of platelet function assay in whole-blood coagulation process by dielectric blood coagulometry. The total coagulation ability can be tested without an agonist, as in the regular DBCM recalcification assay, in which naïve platelets at the initial stage of the measurement are activated by thrombin through the blood coagulation process. Such platelets fully contribute to clot retraction and increase the peak value of the dielectric permittivity peak at 1 MHz. The baseline can be determined with cytochalasin D or TRAP-6, where platelets are fully inhibited or consumed, respectively, and therefore, no contribution to clot retraction or the dielectric permittivity peak is expected. Each platelet activation pathway was tested with the corresponding agonist, and the antiplatelet drug effect was reflected by the ratio of preserved platelets that were not activated by the agonist. The preserved platelets were activated by thrombin and contributed to clot retraction and the dielectric permittivity peak.

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  Figure 2. Change in dielectric permittivity at 1 MHz associated with blood coagulation reaction restarted by calcium readdition to citrated blood samples. The inset shows the dependence of peak permittivity on platelet number.

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  Figure 3. Changes in dielectric permittivity at 1 MHz. Time 0 was defined as the time when blood was injected into the cartridge. The blood sample was then stirred for approximately 3 min and 30 s (black broken line), and dielectric measurements were performed. When cytochalasin D was added to inhibit platelet aggregation, the peak value was significantly reduced (red) compared to a control without the addition of cytochalasin D and a platelet activator (black). Furthermore, even when platelets were activated to the maximum level by TRAP-6, platelets were aggregated and consumed at the time when fibrin gel was formed, resulting in a significant decrease in the peak value (blue).

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  Figure 4. Changes in dielectric permittivity at 1 MHz. Time 0 was defined as the time when blood was injected into the cartridge. The blood sample was then stirred for approximately 3 min and 30 s (black broken line). Compared to that for the control without the platelet activator (black), the peak value was significantly reduced with the addition of AA (solid blue line), similar to the case of TRAP-6 (Figure 2, blue). By contrast, when AA was added in the presence of aspirin (dashed blue line), the decrease in peak value was suppressed, and the value was close to that of the control.

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  Figure 5. Box plot of Panr platelet index. The control without any agonist is shown in black; samples with collagen, AA, and ADP added as agonists are shown in red, blue, and green, respectively. One-sided Student’s t-tests were used to evaluate whether increases in Panr were observed because of the presence of antiplatelet drugs. (*: p < 0.01, **: p < 0.001, ***: p < 0.0001, ****: p < 0.00001).