<h2>Abstract</h2> In the design of experiments the methods for project initiation may involve a hypothesis formulation. Once identified, the hypothesis should be testable, internally consistent, repeatable, verifiable, and show dose dependency. Alternatively a project may begin after a screening study has been undertaken. In this case the hypothesis is formulated at end of the study and any observed positive "effect" can serve as a working postulate. <i>In vitro</i> cell exposure conditions can influence the end result, therefore experimental methodology should include a description of the cells lines and whether synchronised, in suspension, or a mono-layer of cells attached to vessel surface were used. Details of the medium should include the volume within the insonation chamber, its temperature and pressure, and whether it is aerated or contains nuclei for cavitation and whether radical scavengers are present. Important information about the ultrasound exposure would include the pulsing conditions, degree of focussing, presence of travelling waves, standing waves or stationary waves and internal reflections. An attempt should be made to identify the physical mechanisms of action, whether thermal, transient or stable cavitation, or other mechanical effects. Experimental design directly influences the interactive mechanism involved during <i>in vitro</i> exposures and is particularly important for non-thermal effects such as cavitation. Within an ultrasound beam passing through a cell suspension in a plastic tube, cells and bubbles are segregated by the presence of a stationary wave pattern such that cells collect in the pressure nodes whilst bubbles are held in the antinodes of the pressure wave. When the tube is rotated cells are swept in the fluid past arrays of bubbles trapped at the pressure maxima (Church & Miller, 1983) and lysis occurs at about 1 W/cm<sup>2</sup> SATA. However, if the tube remains stationary the cells are unaffected. The possibility of gas bubbles existing <i>in vivo</i> has not been resolved and needs further investigation. The importance of proper statistical interpretation of experimental results was stressed. Conclusions cannot be drawn from an <i>a posteriori</i> statistical analysis which can only lead to a hypothesis. Studies should start with a hypothesis of what is expected and the results analysed to test this hypothesis. Sound statistical analysis should be employed to avoid statements such as "the results were not significantly different, but a trend was noted." Finally, it was noted that safety of diagnostic ultrasound cannot be directly demonstrated by studies which only show absence of effects. The value of <i>in vitro</i> experiments to health and safety assessment is that they allow examination of the physical mechanisms of ultrasound interaction in biologically relevant systems.
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