Currently, more than 16,000 candidates are listed with the United Network for Organ Sharing awaiting liver transplantation. Nevertheless, only 4800 cadaveric liver transplants are performed annually in the United States. Due to this discordance between organ demand and supply, it is estimated that approximately 10% of patients in the waiting list will die before obtaining an organ. As a result, novel strategies to expand the donor pool have been explored. With the exception of live donor liver transplantation, the remaining strategies involve the use of older cadaveric grafts, allografts with mild steatosis or even donors with evidence of past hepatitis B or C infection . This is why, one of the major obstacles to be tackled, is the development of clinically significant ischemia and reperfusion injury, which is even more important for "marginal" grafts. Every progress towards understanding the molecular events following not only cold storage but also cold and warm reperfusion of the graft could have a significant impact on the current transplantation practices. Indeed, recent data suggest that following I/R there is a balanced apoptosis and occasionally necrosis of hepatocytes translating into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy . Furthermore, when these events become predominant, they can lead, at least in animal models, to the development of calcifications as observed in livers of rabbits infected with rabbit haemorrhagic virus .
In the present case report, we have shown that these events can take place in human subjects following liver transplantation. To our knowledge these are the first reported cases in the literature of liver calcification following liver transplantation, presumably secondary to I/R injury. Not only did both patients have biochemical evidence of severe graft I/R injury, they also had biopsy proven I/R induced injury associated with the development of calcifications. Furthermore, both succumbed to the sequelae of this injury. Both recipients received grafts from donors with normal serum biochemistries and no evidence of hepatic trauma or steatosis. Both donors had no evidence of crystal deposition or storage disease, and although we did not perform any donor liver biopsies, the grafts appeared macroscopically normal and perfused well with UW solution. Corroborating to this remains the fact that all four kidneys (from both donors) were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination.
Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification .
Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage , as well as synthetic HA formed in serum , the intracellular precipitation of HA within hepatic cells is unique and has not been reported from other physiological and pathological tissues.
The observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver.
In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant.