Measurement of Whole Blood Platelet Aggregation by Multiplate® Aggregometry Before and during Aspirin Treatment.
Authors: Pedersen Susanne B. , Kristensen Steen D. , Hvas Anne-Mette 
Published: October 2019

The inhibition of platelet aggregation by aspirin (ASA) is fundamental in treatment of ischemic heart disease (IHD). Several studies report findings of normal platelet aggregation despite ASA treatment in some individuals, referred to as ASA resistance (AR). It has been hypothesized that AR increases the risk of a future ischemic event. We evaluated a new impedance method for measurement of platelet aggregation, Multiplate® aggregometry (MA), and compared this method to light aggregometry ad modum Born (OPA), with reference to repeatability and detection of AR. Blood samples from 43 IHD patients and 21 healthy individuals treated with ASA 75 mg daily were analyzed in duplicate by MA and OPA on 4 consecutive days. An additional blood sample was obtained prior to ASA treatment in the group of healthy individuals. Compliance was confirmed by measurements of thromboxane B2 in serum. MA was performed with arachidonic acid (AA) in concentrations of 0.25 mM, 0.50 mM and 0.75 mM, and with adenosine diphosphate (ADP) in concentrations of 7.5 μM and 15 μM. OPA was performed with AA-concentrations of 0.5 mM, 1.0 mM and 1.5 mM, and with ADP-concentrations of 5 μM and 10 μM.

Table 1. Area under the curve (AUC) measured by MA in patients and in healthy individuals before and during ASA treatment. Agonist AUC, aggregation units · min Healthy Before ASA HealthyDuring ASA PatientsDuring ASA Median Range Median Range Median Range AA, mM 0.25 520 402–999 38 12–83 41 8–110 0.50 574 461–976 51 20–112 56 17–187 0.75 551 434–889 68 21–333 98 18–418 ADP, μM 7.5 474 272–859 422 195–816 472 126–720 15 503 328–922 479 262–995 525 172–834

In healthy individuals, the AA-induced AUC was reduced significantly by ASA at all concentrations (88–93%, p=0.0001). The reduction of AUC was small and insignificant when using ADP (5–11%, p≥0.06). There was a trend towards a higher median AUC measured in patients than in healthy individuals during ASA (p=0.07).

Table 2. Coefficients of variation (CV) of double measurements determined by MA and OPA in healthy individuals prior to ASA treatment and during ASA treatment. AA, mM MA AA, mM OPA CVBefore ASA, % CVDuring ASA, % CVBefore ASA, % CVDuring ASA, % 0.25 8 46 0.5 48 25 0.50 10 40 1.0 5 20 0.75 12 41 1.5 5 21

The CV of OPA was generally lower. The reference method was OPA with AA 1.0 mM and AR was defined as a residual platelet aggregation ≥ 20%. According to this definition 7 participants (16%) had AR. A receiver operating characteristics (ROC) analysis showed a sensitivity of MA using AA 0.75 mM of 100% at an AUC cut-point of 94 aggregation units (AU) · min, 71% at 135 AU · min and 29% at 212 AU · min. The specificity was 60, 81 and 93%, respectively. The area under the ROC-curve was 0.79 (95% CI 0.66–0.92). In conclusion, the large ASA-induced reduction in AUC of healthy individuals indicated that MA measures the effect of ASA efficiently when using AA. ADP seems less suitable, as the AUC was only slightly reduced by ASA. The CV of MA was high during ASA treatment, indicating that platelet aggregation during ASA was low and difficult to measure precisely with MA. The area under the ROC-curve was moderately satisfying, but of uncertain correctness due to the rather small number of observations.