Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.

In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well-preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

Development of the isolated dual perfused rat liver model as an improved reperfusion model for transplantation research.

The Isolated Perfused Liver (IPL) model is a widely used and appreciated in vitro method to demonstrate liver viability and metabolism. Reperfusion is performed in a controlled setting, however, via the portal vein only. To study transplant related questions concerning bile and transport of bile, the in vitro Isolated dual Perfused Liver model is revisited. The IdPL is an in vitro reperfusion model, using both portal vein and hepatic artery. Livers from 12 Wistar rats were flushed with University of Wisconsin-organ preservation solution, procured and reperfused in either the conventional IPL-model (n = 6) or the new IdPL-model (n = 6). Liver injury, assessed by the release of aspartate amino transferase and lactate dehydrogenase, showed similar levels during both IPL and I dPL reperfusion, only alanine amino transferase showed an improvement. Cumulative bile production showed an improvement: 176.3 +/- 8.4 in the IdPL compared to 126.1 +/- 12.2 microg/g-liver in the IPL (p < 0.05). Clearance of phenol red (PR) and taurocholic acid (TC) remained similar. At 90 minutes reperfusion the PR clearance showed 0.11 +/- 0.01 and 0.11 +/- 0.02 mg/30min/g-liver and the TC clearance 1.01 +/- 0.10 and 1.01 +/- 0.07 micromol/ml/30min/g-liver in the IPL and IdPL, respectively. Increasing the reperfusion time beyond the normally used 90 minutes resulted in a significant increase in transaminases and LDH and a decrease in bile production, liver morphology remained intact and glycogen content was appropriate. In conclusion, the IdPL-model showed similar or better results than the IPL-model, but the liver could not endure an extended reperfusion time using the IdPL.

Hypothermic machine perfusion of the liver and the critical balance between perfusion pressures and endothelial injury.

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.

Cytogenetic analysis of the mature teratoma and the choriocarcinoma component of a testicular mixed nonseminomatous germ cell tumor.

We karyotyped two histologically distinct components with different metastatic behavior of a testicular nonseminomatous germ cell tumor. The two components showed an almost identical chromosomal pattern. These almost identical karyotypes of the two components with different metastatic potential suggest that the difference in biologic behavior might result from subtle differences (on microscopic or submicroscopic level) in chromosomal pattern or that these differences are predominantly epigenetically determined and depend primarily on the lineage of differentiation of the tumor component. Trophoblastic differentiation results in an aggressive, angioinvasive tumor but in development of teratoma in a tumor with low malignant potential.

Cytogenetic analysis of the mature and immature teratoma components of a metastatic testicular nonseminomatous germ cell tumor.

A case is described in which the mature and immature teratoma components of metastases of the same testicular nonseminomatous germ cell tumor were karyotyped. The highly similar karyotypes of both components suggest that the phenotypic difference is predominantly epigenetically determined.

Cytogenetic abnormalities and clinical stage in testicular nonseminomatous germ cell tumors.

To study the impact of chromosomal abnormalities on the clinical behavior of testicular nonseminomatous germ cell tumors (TNSGCTs), we compared the chromosomal constitution of primary tumors of patients who initially presented and remained without metastases to those with metastatic disease. Furthermore, the chromosomal pattern of primary TNSGCTs was compared to ploidy and the clinicopathologic risk factors histology and small-vessel invasion. The modal chromosome number and the ploidy were in agreement. No correlation was found between the modal chromosome number and histology, presence of vascular invasion, or clinical stage. No correlation was found between structural chromosome abnormalities, like the number of copies of the i(12p) chromosome, and clinical stage. No obvious differences were found in chromosomal constitution of metastatic and non-metastatic tumors. The results of the present study suggest that in TNSGCTs differences in clinical behavior are not associated with gross chromosomal differences.