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IntroductionThe safety criteria of ultrasound for live bodies are one of the important factors in the development of ultrasound imaging and therapeutic techniques using high-intensity ultrasound such as shear-wave elastography [1] and highintensity focused ultrasound therapeutic techniques [2]. These functional techniques enable the visualization of additional information and expected treatment effects. However, the local temperature rise and high stress caused by high-intensity ultrasound will induce biological effects. Several researchers have reported the effects on blood under high stress or highintensity ultrasound [3,4]. The effect on blood can be evaluated quantitatively by the amount of hemolysis, in which the cell membrane of red blood cells is ruptured by physical, chemical and biological factors, and hemoglobin included in the red blood cells flows out plasma [5]. Our group has developed an ultrasonic bubble filter for extracorporeal circulation and investigated its effect on blood [6,7], and it was found that ultrasound exposure at lower frequencies induced greater damage to blood at the same sound pressure level. These experimental results imply that the hemolysis is related to acoustic cavitation since the sound pressure threshold for cavitation generation increases with the ultrasound frequency. Acoustic cavitation increases the dose efficiency in ultrasound-triggered gene and drug delivery techniques [8], and the ultrasound pulse length is one of the important factors in cavitation generation and in determining the efficiency [9].In this paper, we focused on hemolysis caused by pulsed ultrasound and performed in vitro experiments using bovine blood. Temporal changes in the generation of cavitation were measured while changing the pulse repetition frequency (PRF) and pulse length, and the relationship between the hemolysis and the acoustic cavitation was evaluated quantitatively.

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To establish safety criteria of ultrasound for blood, we examined hemolysis induced by low-intensity pulsed ultrasound and performed in vitro experiments using bovine blood. Quantitative evaluation by microscopic observations and the relationship between the hemolysis and cavitation microbubbles were discussed. Hemolysis was evaluated by red blood ghost cell. The presence of red blood ghost cells in the plasma component implies that the cell membrane was broken and the internal hemoglobin flowed out to surrounding blood plasma. Pulsed ultrasound at 1 MHz with the maximum sound pressure amplitude of 200 kPa was irradiated for one minute, and the ratio of non-exposure time (0—80%) was changed. After ultrasound exposure, the blood samples were centrifuged and divided into two layers: blood cells and plasma components. 10 μL of the plasma components was sampled and the number of the red blood ghost cells was counted. The number of red blood ghost cells increased gradually with the decrease in the ratio of non-exposure time and decreased again at 0% (continuation wave). This result implies that the hemolysis is mainly caused by microbubble cavitation and the non-exposure time of ultrasound is one of the important factors for hemolysis in such low-intensity ultrasound field.