Microprobe multi-sensor for graft viability monitoring during organ preservation and transplantation

Low cost disposable sensor device for organ viability monitoring. A minimally invasive microprobe measuring tissular temperature and bio impedance has been designed, fabricated, validated and improved up to its present pre-industrial stage. It uses a Si-based multi-micro sensor needle as core element. Other sensoring devices such as pH and/or K+ sensors can be also integrated in it. In its present version, it follows biocompatibility, electrical safety and other directives allowing its use in experimental and clinical practice. Usefulness for organ viability monitoring has been demonstrated in diverse experimental works on pig heart, liver and kidney. It has been also used in rat kidney to measure renal ischemia and preconditioning effects. Laplacian electrogram can be also derived from the measures performed. Other applications in many different fields are currently under study. The micro probe can be easily fabricated using standard microelectronic technologies, which implies high reliability, low costs and the possibility to integrate other sensors and electronics on the same probe.

A portable instrumentation system capable of monitoring bioimpedance and other parameters was developed. It makes use of the bioimpedance probes evaluated during the project and its digital core and user interface is based on a PDA. Its main target application is to monitor the preservation and transport of grafts. Its main features are: - Portable (on the Cool Box lid); - Parameters: bioimpedance (module and phase at 1 kHz) and temperature; - Displays all the measured data and generates warnings if any threshold is reached; - Creates a log file containing all the data. The term PDA (Personal Digital Assistant) defines a set of advanced computing devices characterised by their small size (pocket size) that are usually used as personal organisers. Due to their interaction capabilities with desktop computer systems they are also referred as the 'portable window of your personal computer'. Concerning the digital design and the user interface, the PDA choice offers many advantages compared to other alternatives (custom digital electronics solutions or PC based solutions) in terms of capabilities and developing time.

It has been suggested that electrical bio-impedance can be a good marker of cell viability. The changes in bio-impedance of ischemic liver tissue, in vivo and ex vivo, at room temperature have been quantified (references). An increase in bio-impedance for all frequencies during the first two hours after removal of liver has been detected (Hemmilich). The changes are stronger at low frequencies (+180% at 1khz). This increase has been attributed to a decrease of extra-cellular space, cell swelling and closure of gap junctions (Hae 02, Konishi 95). After two hours, the bio-impedance decrease, probably owing to membrane breakdown. Electric bio-impedance is defined as the electrical resistance offered by a biological material to conduct the electricity. The tissue is composed by the cells (cytosol surrounded by plasma membrane) and extra-cellular medium, which is also an ionic solution. In electrical terms, the plasma membrane, as a lipid by-layer membrane, has a capacitive behaviour. Therefore, an electric current will cross easier the extra-cellular medium than a cell, being the resistance through the cell higher. Under ischemic conditions the cells become swollen (by edema), and therefore the space occupied by cells, that means cytosol plus PM, increases with respect to the extra-cellular medium. Hence, the impedance should increase after ischemia. There are few investigations into cold preservation of liver (2-4 degrees C with preservation solution). Some researches on rats show significant differences in bio-impedance evolution for several temperatures (Koisichi 95). Also, a stable behaviour of impedance after 6 hours of cold preservation (4 degreesC) has been detected (Raicu 00). However to date there is not a commercially available effective system to monitor the impedance. Therefore, we have been developing recently a sensor, an instrumentation system that is suited to our experimental conditions and viable for future clinical application. Furthermore, the first objective is to demonstrate that bio-impedance is able to follow the changes in the quality of an organ for transplant. To this end, livers were well procured and preserved, well procured and badly preserved or badly procured and preserved, and the impedance was monitored during the whole preservation period. In this way we induced increasing degree of lesion in the livers. The results of the present study confirm this hypothesis. In all the livers the impedance increased progressively during preservation. Moreover, the impedance increased much more as more badly the livers were procured and preserved. Therefore, the most injured livers showed the highest impedance values during preservation. On the other hand, ATP and EC levels as indicators of the cellular energy status also demonstrated differences between the study groups. Energy Charge gradually decreased during the preservation period, but well-preserved livers showed always-higher EC levels. In contrast UA, which is the final metabolite in the degradation pathway of ATP, accumulated more in the worst livers. In addition XDH-XO is an important mechanism inducing ischemic lesion via superoxide radical production. This enzyme catalysis the production of xanthine from hypoxanthine and so the ratio Xanthine/hypoxanthine is an index of this activity. We previously have demonstrated that this index correlates with the viability of the liver graft and how the xanthine levels after warm ischemia also indicates the degree of ischemic lesion. Accordingly in the current study, the ratio X/HX was significantly higher in the worst livers during preservation. Taking into account all these lesion parameters, if the initial cause of edema is the membrane imbalance promoted by ATP and EC depletion, they would inversely correlate with the degree of edema and to some extent with the raise of impedance. Effectively in the present study, the energy charge content inversely correlated with impedance. Therefore as more depleted is EC (ATP), more edema is produced and as a consequence impedance goes up. It suggests that at the end impedance measurement is an effective marker of the degree of injury, and more specifically it reflects the degree of edema as the cause of edema by ischemia correlates with impedance increase. The electrical impedance techniques allow an early detection of irreversible tissue changes. Therefore, the method might be used in the future to monitor donor tissues before organ transplantation.

Two kind of a measurement system have been developed, the first one to be used in clinical environment and the other one for in-vivo experimentation. Electronics for "on line" sensor characterization have been also developed. Developed instrumentation allows it to display, to measure and to store data from needle sensors inserted on the organ under study. A LabVIEW based software looking like a virtual measurement instrument have been developed. It allows it to display, store and control the data send from the different modules connected to an ADC board. The features of the system include: - Tissue impedance meter (Four-electrode method) with 16 channels, range of frequency from 100 Hz to 125 kHz and applies currents<10 microApp; - Tissue temperature meter (Four electrode method) with 16 channels and applies currents<40microA dc; - Two ISE (Ion Selective Electrode) measurement modules to measure pH and k+ with 16 channels each one and another module able to measure pH and k+ (or any other ions) using ISFETs has been implemented and tested but, it has not been used in vivo because of the lack of ISFET needles suitable for tissue insertion. Size, power consumption and telemetry have been optimised to perform the clinical use version.

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