A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ~2mm vein in six normal, eight naïve hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naïve VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naïve hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naïve hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders.

In this second of a two part series, we present pilot clinical data demonstrating Acoustic Radiation Force Impulse (ARFI) ultrasound for monitoring the onset of subcutaneous hemostasis at femoral artery puncture sites (arteriotomies), in vivo. We conducted a randomized, reader-blinded investigation of 20 patient volunteers who underwent diagnostic percutaneous coronary catheterization. After sheath removal (6 French), patients were randomized to treatment with either standard of care manual compression alone or, to expedite hemostasis, manual compression augmented with a p-GlcNAc fiber-based hemostatic dressing (Marine Polymer Technologies, Danvers MA). Concurrent with manual compression, serial ARFI imaging began at the time of sheath removal and continued every minute for 15 min. Serial data sets were processed with custom software to (1) estimate the time of hemostasis onset, and (2) render hybrid ARFI/B-Mode images to highlight displacements considered to correspond to extravasted blood. Images were read by an observer blinded to the treatment groups. Average estimated times to hemostasis in patient volunteers treated with manual compression alone (n = 10) and manual compression augmented by hemostatic dressing (n = 9) were, respectively, 13.00 ± 1.56 and 9.44 ± 3.09 min, which are statistically significantly different (p = 0.0065, Wilcoxon two-sample test). Example images are shown for three selected patient volunteers. These pilot data suggest that ARFI ultrasound is relevant to monitoring subcutaneous bleeding from femoral arteriotomies clinically and that time to hemostasis was significantly reduced by use of the hemostatic dressing.

Acoustic radiation force (ARF) ultrasound is a method of elastographic imaging in which micron-scale tissue displacements, induced and tracked by ultrasound, reflect clinically relevant tissue mechanical properties. Our laboratory has recently shown that tissue viscoelasticity is assessed using the novel Multi-Push (MP) ARF method. MP ARF applies the Voigt model for viscoelastic materials and compares the displacements achieved by successive ARF excitations to qualitatively or quantitatively represent the relaxation time for constant stress, which is a direct descriptor of the viscoelastic response of the tissue. We have demonstrated MP ARF in custom viscoelastic tissue mimicking materials and implemented the method in vivo in canine muscle and human renal allografts, with strong spatial correlation between MP ARF findings and histochemical features and previously reported mechanical changes with renal disease. These data support that noninvasive MP ARF is capable of clinically relevant assessment of tissue viscoelastic properties.

The world wide prevalence of cardiovascular disease leads to over seven million annual percutaneous coronary catheterization procedures, the majority of which exploit femoral artery access. Femoral puncture sites (‘arteriotomies’) can be associated with severe vessel complications after sheath removal if hemostasis is not properly achieved. Hemostasis onset is routinely determined by examination for bleeding at the skin puncture; however, clotting along the puncture path can obscure subcutaneous bleeding, and therefore hemostasis is blindly assessed. We hypothesize that hemostasis assessment can be un-blinded by Acoustic Radiation Force Impulse (ARFI) ultrasound. In this first of a two-part series, we present in vivo ARFI hemostasis imaging data obtained in relevant canine models of femoral artery puncture. Above arteriotomies, ARFI-induced displacements were large (3.5 to > 5.0 μm) relative to surrounding soft tissue soon after needle removal, which was consistent with our expectation for pooled extravasated blood. ARFI-induced displacements above arteriotomies decreased in magnitude (to ∼ 2 μm) some time after needle removal and suggested the onset of hemostasis. This preclinical investigation served as proof of concept and justification for a pilot human study, which is presented in part two of this series.

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents in preclinical animal models and in patients with inherited bleeding disorders, such as von Willebrand disease (VWD) and hemophilia A, or with acquired bleeding disorders such as those resulting from medications or disease processes. In this review, we discuss current methods for assessing hemostasis in vivo and the associated challenges. We also present ARFI-Monitored Hemostatic Challenge; a new, potentially alternate method for in vivo hemostasis monitoring that is in development by our group.