Transplantation Technologies & Research

Background: Currently, severed limbs after combat or traumatic injuries are preserved with cold ischemic storage. However, this method maintains limb viability for no more than 12 hours. In this study, a new device, referred to as the Universal Limb Stasis System for Extended Storage (ULiSSES™), was used to maintain rodent skeletal muscle viability for 24 hours, after 4 hours of ambient temperature ischemia.

Methods: Hind-limbs from 5 Sprague Dawley rats were recovered and allowed to lie on a counter for approximately 4 hours at room temperature (19-24°C) to simulate delayed limb recovery. Limbs were then perfused for 24 hours using room temperature Krebs Henseleit solution. Arterial and venous pressure, flow, PaO₂, PvO₂, perfusate pH, and temperature were recorded hourly.

Results: Ambient ischemia time was 3.4 ± 0.5 hours. Perfusion pressure was 8.2 ± 2.0 mmHg with a mean flow to the limbs of 9.5 ± 5.0 ml/min. The pH and temperature of the KH perfusate were stable throughout preservation at 7.38 ± 0.05 and 23.7 ± 0.5ºC, respectively. Oxygen consumption reached a plateau of 0.28 ± 0.04 ml O₂/min/100 g by 17 hours with vascular resistance hovering around 1.0 ± 0.2 mmHg/ml/min initially, then declining by about 50% after 18 hours. Mean limb weight gain was 37.7 ± 31.8%.

Conclusions: ULiSSES™ appears to open the potential for stabilization and preservation of avulsed limbs for 24 hours or longer, leading to the feasibility of costeffective transport from any recovery site to any re-plantation site with minimal tissue deterioration.

Background: We have discovered and validated a microarray-based test that analyzes blood gene expression profiles (GEP) as an indicator of immune status in liver transplant recipients with stable liver function.

Methods: Analytical performance studies to characterize stability of RNA in blood during collection and shipment, analytical sensitivity (input RNA concentration), analytical specificity (interfering substances) and assay performance (clinical validity, and intra-assay, inter-assay, inter- laboratory reproducibility).

Results: Total RNA extracted from whole blood specimens collected in PAXgene Blood RNA tubes was stable up to 3 days at room temperature (stable RNA yield). Under routine ambient shipping conditions, storage and shipping temperatures did not affect results. However, specimen shipments exposed to temperatures >400°C or to ambient temperatures for >3 days were unacceptable for processing. Analytical sensitivity studies demonstrated tolerance to variation in RNA input (50 to 400 ng per 3’ IVT (in vitro transcript] labeling reaction). Specificity studies using genomic Jurkat DNA spiked into 3 ’IVT reactions at 10-20% demonstrated negligible assay interference. The test was reproducible across operators, runs, reagent lots, and laboratories. External validation demonstrated that the TruGraf Liver blood test accurately classified patients in 84% of 155 samples.

Conclusions: The previously published biomarker is the first non-invasive test to be demonstrated to have clinical utility in assessing immune status of LT recipients with stable liver function and shows promise as a reasonable and necessary tool supporting personalizing immunosuppressive therapy.