The relationship of acoustic emission and pulse-repetition frequency in the detection of gas body stability and cell death

The effect of pulse-repetition frequency (PRF) and number of exposures on membrane damage and subsequent death of contrast agent-attached phagocytic cells was examined. Phagocytic cells of a mouse macrophage cell line were grown as monolayers on thin Mylar sheets. Optison(R) microbubbles were attached to these cells by incubation. Focused ultrasound exposures (P-r = 2 MPa) were implemented at a frequency of 2.25 MHz with 46 cycle pulses and clinically relevant PRFs of I kHz, 100 Hz, 10 Hz, 1 Hz and 0.1 Hz in a degassed water bath. A 1-MHz receive transducer measured the scattered signal. The frequency spectrum was normalized to a control spectrum from linear scatterers. Photomicrographs of the cell monolayer were made before and after exposure, and a dye exclusion test (Trypan blue) was used to find the percentage of blue-stained cells indicating cell death, which was then related to acoustic emission. For 10 acoustic pulses and a high prerinse gas body concentration, there was less cell death and correspondingly lower change in the acoustic emissions at a PRF of I kHz than with PRFs of 100 Hz, 10 Hz, I Hz and 0.1 Hz (p < 0.001). The reduced effect at high PRF may be indicative of some evolution of the shelled microbubble that requires significant total exposure duration (> 10 ms, but < 100 ms).