Our initial prototype consisted of a high-precision impedance measurement chip (AD5940, obtained from Analog Devices, Inc.), with a four-electrode measurement configuration (diagram shown in Figure 1A, picture of device shown in Figure 1B). In this configuration, one pair of electrodes applies current across the tissue, while the other pair of electrodes is responsible for detecting the resulting changes in voltage. The voltage-sensing electrodes must be positioned between the current leads on the region of interest to increase the specificity of the measurement and protect the leads from electrode polarization. From the voltage and current readings, the impedance of the tissue can be calculated using Ohm’s law. The AD 5940 chip is capable of applying frequencies between 0.015 Hz and 200 kHz.

The chip was interfaced with the material of interest with the help of 3M Red Dot electrodes. The chip was also connected to a laptop and interfaced with the graphic user interface tool SensorPal, which provided real-time measurements and plots of both the magnitude and phase of the impedance being measured. This data was exported to Microsoft Excel for further analysis.

To determine if EIS can detect changes in extracellular fluid, the four electrodes in our first prototype were connected to a sausage. A frequency sweep was performed where the impedance was measured in increments of 5 kHz from 5 kHz to 95 kHz. This frequency range let us test what settings would be optimal in being able to best isolate the extracellular fluid impedance needed to diagnose compartment syndrome. A sausage was used as a simplified version of a muscle compartment. Our reasoning was that if our device was to be able to measure impedance within the complex physiology of the human leg, it would need to be able to measure impedance in a simplified model. Baseline measurements with no saline were taken before measuring the impedance of the sausage with 1 mL and 2 mL of injected saline solution, and the results are shown in Figure 2.

Also, this test shows this device is sensitive enough to detect the impedance differences due to saline injections, which helps build towards the goal of detecting extracellular fluid volumes. A limitation of this experiment was that the saline itself is conductive, so injecting the saline into the sausage affected the impedance results accordingly. Studies have suggested that osmolarity has a greater impact on impedance than fluid volume itself. Therefore, it’s difficult to distinguish changes in impedance due to conductivity versus changes in impedance due to increased sausage volume. This shows that a sausage is not an ideal simplified model of a muscle compartment in a human leg. Despite this, however, it’s still encouraging that our device was able to detect even small injections of saline to a statistically significant degree.

The second, and more important, test involves measuring the impedance of the anterior and lateral compartments in two subjects, for 95% of CECS cases occur in one of those two compartments (diagram of compartments shown in Figure 2). For each subject, the lower leg on the subject's dominant side was taken before and after power-walking and with and without compression. The data of the two subjects’ pre-exercise impedance and post-exercise impedance were converted to a percent impedance change to normalize the data. Then, the subjects’ data were then averaged, and the standard deviations were calculated and shown in Figure 4.

The post-exercise percent impedance change between the non-compression tests and the compression tests were different to a statistically significant degree, with a p-value of 9.75*10^-7. Also, it was observed that the impedance rose as pressure rose. This shows that the constriction of the legs changed the extracellular fluid volume and blood flow volume in the leg, which then changed the impedance we measured. The reason it shows these changes is because the constriction of the leg doesn’t allow extra blood flow through the muscles during exercise, and it also doesn’t also the fascia surrounding the muscles to expand to increase the extracellular fluid volume. This is crucial since this is the main indicator of detecting if someone has compartment syndrome, so being able to measure the fluid volume demonstrates the potential of our device. We suspect that the changes in impedance in the compressive condition at low frequencies is more due to extracellular fluid while changes at high frequencies is more due to changes in cell membrane capacitance.