Effect of high-intensity focused ultrasound on whole blood with and without microbubble contrast agent

Abstract

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm². Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 μL CA per mL whole blood at an intensity of 2360 W/cm² for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 μL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm² where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitro and may lead to clinically significant hemolysis in vivo.

Introduction

High-intensity focused ultrasound (HIFU) is currently being studied as a noninvasive method for inducing hemostasis Delon-Martin et al 1995, Hynynen et al 1996a, Hynynen et al 1996b, Vaezy et al 1997, Vaezy et al 1998. The treatment goal is to use diagnostic ultrasound to locate the site of bleeding, and then use HIFU to induce hemostasis. Continuous wave (CW) HIFU has been used at intensities of 500 to 3100 W/cm² for approximately 2 min to successfully halt bleeding in exposed rabbit liver (Vaezy et al. 1997) and in punctured blood vessels (Vaezy et al. 1998). Gas-filled ultrasound contrast agents (CA) are frequently added to the blood to enhance the images on diagnostic ultrasound devices and are also currently being studied as a means to locate hemorrhage (Schmiedl et al. unpublished observations). The acoustic generation of microbubble boluses in a specific artery has also been suggested as a means of locating hemorrhage (Ivey et al. 1995). In the context of these studies, we investigated the effect of HIFU on hemolysis in whole blood and whole blood containing CA. Extensive hemolysis can cause significant increases in morbidity and mortality. If HIFU increases hemolysis in conjunction with CA, the parameters used for hemostasis may need to be altered to avoid clinically significant hemolysis.

Our goal was to use a static in vitro system to model the conditions that flowing blood will encounter when a vessel is exposed to therapeutic HIFU. To accomplish this, we: 1. varied the exposure time from 10 ms to 1 s to simulate the time that blood in different vessels would be exposed to HIFU (i.e., venous to arterial vessels); 2. varied the exposure intensities from 560 to 2360 W/cm² spatial average, similar to the range of intensities used in previous HIFU research; and 3. used clinically relevant concentrations of CA in whole blood to cover the typical range of CA concentrations found in well-mixed blood. A 1.1-MHz transducer was chosen, instead of higher frequencies used in other acoustic hemostasis studies Delon-Martin et al 1995, Hynynen et al 1996a, Hynynen et al 1996b, Vaezy et al 1997, Vaezy et al 1998, in anticipation of the eventual application of HIFU to achieve transcutaneous hemostasis.

In prior studies on the effect of ultrasound (US) exposure on red cells, hemolysis has been enhanced by using dilute solutions of red cells Miller and Williams 1992, Miller and Thomas 1993, by rotating tubes (Williams and Miller 1989), or by bubbling gases through the blood to increase the number of cavitation nuclei Brayman and Miller 1997, Carstensen et al 1993. In our study, anticoagulated whole blood was drawn and used immediately to simulate whole blood in vivo.

One acoustic mechanism that can cause hemolysis is cavitation, which is the expansion and compression of gas bubbles caused by the applied acoustic field. The violent implosion of a bubble can lead to the production of shock waves, high-velocity liquid jets, free radical species, and strong shear forces that can damage blood cells Leighton 1994, Miller and Thomas 1993, Miller et al 1996. Therefore, we studied the correlation between the amount of hemolysis and the amount of cavitation that occurred in whole blood and in whole blood with CA added. In our model, we assumed CA had circulated for several min and was uniformly distributed throughout the circulatory system, resulting in a maximum CA concentration of 4.2 μL per mL of whole blood. This concentration is significantly less than those used in previous in vitro experiments (Brayman et al. 1995) and in vivo experiments (Dalecki et al. 1997).