Fig 1.
Acoustic Radiation Force (ARF) based clot stiffness measurement.
(a) The ARF based measurement setup used a 10 MHz ultrasound transducer to apply ARF to blood plasma in which beads were suspended. A microscope with 10 X objective and video camera had a mutual focus with the ultrasound transducer so application of ARF to the sample was imaged. (b) To assess blood plasma stiffness: 1) Video microscopy of the blood plasma sample was collected during application of ARF, 2) The bead motion due to applied ARF was tracked by an ImageJ program, 3) Displacements during the ARF pulse interval were determined, and 4) The displacement data was analyzed for the sample’s viscoelastic parameters.
Fig 2.
Ultrasound transducer pressure field and bead displacement in a viscous fluid.
(a) The pressure field of the 10 MHz focused ultrasound transducer at the focal length (19mm) was simulated in Field II software. (b) Hydrophone measurements of the acoustic pressure produced by the ultrasound transducer were used to determine the -6 dB lateral beam width (400 μm) and the -6 dB axial beam length (5 mm) at the focus. The microscope field of view is shown. In b & c, the y-axis is expanded in the interest of clarity. (c—e) The microscope field of view was positioned on the beam focus by centering over the largest bead displacements. The ARF induced displacement of 15 μm beads was analyzed in the S6 viscosity standard (8.743 cP). Bead displacement was measured during the 0.6 s ARF pulse interval, Δt = tf-ti where maximum displacement Dm was determined at t f.
Fig 3.
The effects of attenuation and pulse repetition frequency (PRF) on bead velocities and force magnitude.
(a) Bead terminal velocities under applied ARF at 7.5 kHz PRF in the viscosity standards S6 (8.743 cP) and S3 (4.063 cP), and in citrated platelet rich plasma were measured. Standard error is shown with n = 10 beads. (b) Applied ARF magnitude at 7.5 kHz was determined using Stokes’ law and terminal bead velocities. Distinct acoustic attenuations between the fluids account for much of the difference in the applied force in the fluids. At 7.5 kHz the force applied in citrated blood plasma was estimated to be 147 pN. Standard error is shown with n = 10 beads. (c) Bead displacement after 0.3 s of pulsing due to applied ARF increased linearly with increasing PRF in the three fluids. Standard error is shown with n = 9 beads.
Fig 4.
Characteristic bead displacement due to applied ARF at 7.5 kHz in clotting platelet poor plasma.
(a) At t = 1 min the bead displaced 40 μm during a 0.5 s pulse interval and recoiled after the pulse stopped. (b) At t = 15 min the bead displaced 0.9 μm during the pulse interval and recoiled after the pulse stopped. (c) At t = 18 min the bead displaced 0.5 μm during the pulse interval and recoiled after the pulse stopped. Optical noise was reduced by smoothing with a 3 data point moving average filter.
Fig 5.
Characteristic mechanical properties of clotting platelet rich (PRP) and platelet poor plasma (PPP) as assessed by application of ARF at 7.5 kHz.
(a) Clot stiffness (S), S(t) = Fplasma/Dmavg, of clotting PRP and PPP was determined. Bead displacements (n = 5) were averaged to compute stiffness, which is displayed with 60% quartiles (***, p<0.0005 between PPP and PRP from t > 12 min). (b) In the Kelvin-Voigt model, which predicts the displacement D(t) response of viscoelastic materials to applied force F(t), a viscous element (dashpot) is in parallel with an elastic element (spring). The parameters k and μ describe the elastic and viscous properties respectively. (c-e) The Kelvin-Voigt model was fit to bead displacement data in clotting PPP (c) where displacement decreased abruptly between 6–15 min. (d) From 15–24 min the model was fit to bead displacement data and displacement continued to decrease. At all time points, in contrast to measurements in the viscous fluids, the displacement approached a maximum. Standard error is shown with n = 5 beads per time point. (e-f) Kelvin-Voigt viscous (μ) and elastic (k) parameters of PPP and PRP were determined. Standard error is shown with n = 5 beads per time point (*, p<0.05)(**, p<0.006)(***,p<0.0005). (e) During clotting in the PPP sample the Kelvin-Voigt viscous (μ) and elastic (k) parameters increased 1107 fold and 6.9 fold respectively. (f) During clotting in the PRP sample the average fold difference of viscous (μ) and elastic (k) parameters of the clot as compared to PPP between 15 and 21 min were 1.3 fold and 11 fold larger, respectively.
Fig 6.
Sensitivity of optical ARF clot stiffness assessment to strength of kaolin clotting stimulus.
Clot stiffness of PRP stimulated with 0.5, 5, and 50 μg/mL kaolin was measured by optical ARF. Optical ARF detected kaolin concentration dependent kinetics of clot stiffness. Standard error is shown with at least n = 5 beads per time point.