Case 6 -

Harvest Injury (Ischemia-Reperfusion/Preservation Injury) Julia C. Iezzoni

Clinical History The patient, a 56-year-old Caucasian male, underwent orthotopic liver transplantation for end-stage liver disease due to alcohol abuse. The liver donor was a 50-year-old Caucasian male who had suffered a sudden, massive cerebral vascular accident. After declaration of brain death, the donor remained hemodynamically stable, and vasopressor support was not necessary to maintain organ perfusion. Organ harvest, transport, and transplantation were uneventful, with a total cold ischemia time of approximately 12 hours. As per protocol, a post-perfusion biopsy of the liver was performed immediately upon restoration of the allograft's blood supply once in the recipient (Figure 19 – H&E). For the first day after transplantation, the patient's liver function tests were characterized by markedly elevated aminotransferases and a high total bilirubin. By post-transplant day 7, these values had diminished only slightly, and a liver biopsy was performed. Diagnosis: Harvest Injury (Ischemia-Reperfusion/Preservation Injury) Case 6 - Figure 1 - H&E Case 6 - Figure 2 - H&E Case 6 - Figure 3 - H&E Pathologic findings: Sections of the post-perfusion biopsy of the liver allograft show early harvest injury, characterized by scattered clusters of necrotic hepatocytes with associated neutrophils. The biopsy 7 days after transplantation demonstrates the typical findings of moderate-to-severe harvest injury, including centrilobular hepatocellular ballooning degeneration, cholestasis, and bile ductular proliferation, which has periductular acute inflammation ("acute cholangitis pattern"). Discussion Harvest injury is the nonimmunologic damage to the liver allograft due to the mechanical and physiologic consequences inherent in the transplantation procedure. This damage results from two major insults, ischemia and reperfusion, and these two injuries occur at different steps in the process of transplantation. First to occur is the ischemic injury. The liver allograft is subjected to ischemia from the time it is disconnected from its blood supply in the donor to when its blood supply is restored in the recipient. To reduce this ischemic injury, the organ is kept cold, and this time period is called the cold ischemia time. Immediately upon restoration of the blood supply, the liver is subjected to further damage as the result of the initial reperfusion with warm, oxygenated blood. This reperfusion injury (also known as "warm injury"), both exacerbates the injury already caused by the ischemia and introduces additional mechanisms of damage. Despite advances in organ preservation and surgical technique, a certain degree of harvest injury of the allograft remains an inevitable consequence of liver transplantation and results in variable degrees of allograft damage and clinical dysfunction . As such, harvest injury is a common diagnostic consideration for the pathologist evaluating liver transplant biopsies. A basic appreciation of the transplant procedure is useful to understand this type of liver injury. In brain dead, heart-beating donors, preservation of the allograft is initiated by infusion of the liver with cold preservation medium prior to its extraction. The preservation medium used at most institutions is UW solution (so-called because it was developed at the University of Wisconsin), designed specifically to counter potential mechanisms that contribute to ischemic injury. The liver is removed from the donor, often flushed again with cold preservation medium, placed in a sterile plastic bag that is filled with the medium, and then fully immersed and stored in an ice slush maintained at approximately 0 °C. Prior to revascularization of the liver in the recipient, the cold preservation fluid in the allograft is flushed out with blood, lactated Ringer's solution, or a medium formulated specifically to minimize reperfusion injury. After the vascular anastomoses are in place and the blood supply is restored, a "post-perfusion" biopsy of the liver is obtained. This biopsy provides a "time zero" data point of the histologic extent of the harvest injury incurred as a result of the transplantation procedure. Several studies have demonstrated that the extent of damage present in the post-perfusion biopsy correlates directly with the degree of subsequent harvest injury-induced allograft dysfunction. As such, the post-perfusion biopsy is a valuable tool for the prediction of ensuing harvest injury in the early post-transplantation period. Clinically, harvest injury presents 1 to 3 days after transplantation. It is characterized by a sharp elevation in transaminases, a sustained high total bilirubin level, and in cases with more severe injury, an elevated prothrombin time. Diminished bile flow also may be noted. The time frame for functional and histologic resolution of harvest injury depends on the severity of the damage. Minimal-to-mild harvest injury usually is associated with good organ function, with functional and histologic reversion to normal as soon as one week after transplantation. In cases of modest harvest injury, complete functional and histologic recovery occurs in most cases, but it may require up to 6 weeks to normalize. In severe cases, abnormalities may persist for up to 6 months post-transplantation; sometimes, severe harvest injury may result in permanent damage to the allograft. Overall, most cases of harvest injury resolve within 3 weeks after transplantation. The most severe clinicopathologic manifestation of harvest injury is primary nonfunction (PNF). Primary nonfunction is characterized by the immediate and irreversible metabolic failure of the newly implanted allograft. Clinically, PNF is characterized by rapidly rising serum transaminases, lack of bile formation, and severe coagulopathy. The clinical course progresses quickly to hypoglycemia, hepatic encephalopathy, acute renal failure, disseminated intravascular coagulation, and death unless urgent re-transplantation is performed. Despite advances in preservation technique, PNF occurs in 5% to 15% of liver transplant patients. Several factors are associated with an increased risk of the development of harvest injury in the liver allograft. These include cold ischemia time exceeding 10 hours, donor episodes of hypotension (especially if treated with vasopressors), increased donor age (especially when greater than 50-years-old), and severe macrovesicular steatosis of the liver allograft. Of particular relevance to the pathologist is the risk factor of macrovesicular steatosis, because the pathologist is called upon routinely to assess the extent of macrovesicular fatty change in a liver being considered for transplantation. Typically, this evaluation is performed on a frozen section of a biopsy of the potential allograft. Several studies have documented that donor livers with severe macrovesicular steatosis (>60%) are more susceptible to harvest injury, including PNF, than those devoid of or with mild (<30%) macrovesicular steatosis. The outcome of livers with moderate macrovesicular steatosis (30-60%) is variable and may depend on the presence of additional risk factors. As a general rule, donor livers with greater than 50% macrovesicular steatosis are considered unsuitable for transplantation. When assessing a potential donor liver for fatty change, it is essential to evaluate only the macrovesicular, not microvesicular, steatosis. As will be discussed below, microvesicular steatosis is a common finding in ischemic allografts, and while studies are pending, currently microvesicular steatosis has not been shown to be irreversibly deleterious to the allograft. Since available histochemical stains for fat (such as Oil red O) are frought with technical difficulties and inter-observer variability, we and others recommend that the extent of macrovesicular steatosis be assessed on routine H&E stained tissue sections. This approach helps to avoid the misintrepretation of microvesicular steatosis as macrovesicular steatosis, which potentially could result in discarding a useful allograft. Mechanistically, harvest injury is a complex, multi-factorial cascade of pathophysiologic processes that involves various cell types (e.g. hepatocytes, sinusoidal endothelial cells, activated Kupffer cells, neutrophils), platelets, and a diverse variety of bioactive molecules, some of which are cytotoxic (e.g. reactive oxygen intermediates, acute reactant cytokines, proteases, prostaglandins). While a detailed description of these mechanisms is beyond the scope of this discussion, it is notable that the ischemia and reperfusion induced injury of the sinusoidal endothelial cells is a central step in the chain of events that results ultimately in the clinical and histological manifestations of harvest injury. In addition to neutrophil margination and platelet activation, this sinusoidal endothelial cell injury causes microcirculatory blood flow disturbances, which leads to severe alterations and impairments in local tissue oxygenation. In conjunction with the reperfusion-induced activation of Kupffer cells, this impaired oxygenation results in hepatocellular damage. Depending on the severity of the damage, this injury causes variable degrees of allograft dysfunction or even nonfunction. Pathology The morphologic features of harvest injury differ relative to the time interval post-transplantation - i.e. the amount of time since the insult. The post-perfusion (or "time zero") biopsy shows the immediate damage; biopsies thereafter demonstrate the residual sequelae of and recovery from this injury. As such, liver allograft biopsy interpretation should be performed in conjunction with knowledge of the post-transplantation interval. In addition, the greater the degree of harvest injury, the longer time is necessary post-transplantation for histologic (and functional) recovery. Time zero At time zero (i.e. the post-perfusion biopsy), harvest injury is characterized by several morphologic features, which vary in extent relative to the degree of severity of the injury. The characteristic morphologic changes are: 1) intrasinusoidal neutrophils; 2) microvesicular steatosis; and 3) hepatocellular necrosis (varies from necrosis of individual or small clusters of hepatoctyes to zonal necrosis, depending on the severity of the harvest injury). Virtually all allografts, even those with minimal harvest injury, demonstrate at least mild intrasinusoidal neutrophils and focal microvesicular steatosis at time zero. These features are more extensive in cases with greater degrees of harvest injury. Intrasinusoidal neutrophils are rarely, if ever, present in biopsies taken before reperfusion, and they are probably of recipient origin. Microvesicular steatosis likely represents a hepatocellular mitochondrial response to ischemia, but it may also be related to donor management. As described above, while primary allograft dysfunction has been associated with severe macrovesicular steatosis, microvesicular steatosis currently has not been shown to be irreversibly deleterious to the allograft. Hepatocellular necrosis is present at time zero in allografts with moderate-to-severe harvest injury. With moderate injury, this manifests as necrosis of individual or small clusters of hepatocytes; these punctate necrotic foci often are distended with neutrophils. With severe injury, zonal hepatocellular necrosis, usually involving the centrilobular zone, is seen. This zonal injury has the appearance of "infarct-type" necrosis, and it may be due to end-arteriolar vasospasm, or alternatively due to microcirculatory blood flow disturbances caused by sinusoidal endothelial cell injury. Zonal necrosis is seen particularly in allografts from donors maintained on vasopressors for prolonged periods. Post-transplantation Days 1-21 Biopsies from days 1-21 show a somewhat different constellation of findings from those at time zero. As before, however, these features vary with the severity of the injury. During this period, allografts with minimal-to-mild degrees of harvest injury routinely show: 1) hepatocellular ballooning degeneration (centrilobular); 2) cholestasis (centrilobular); 3) individual hepatocyte necrosis; and 4) persistent microvesicular steatosis. Hepatocellular ballooning degeneration in the transplant setting is believed to result either from dilatation of the rough endoplasmic reticulum and/or impairment of the secretory capacity of the injured hepatocytes, which in turn causes intracellular fluid accumulation. As a result, the ballooned hepatoctyes are swollen and enlarged (two times normal size), with "diluted" cytoplasm, which appears finely granular or reticulated. The ballooning change usually affects hepatocytes in the centrilobular region. Cholestasis, either intracellular or intracanalicular, often occurs, and it also has a centrilobular predominance. In addition, individual hepatocyte necrosis ("acidophilic bodies") may be seen, either scattered throughout the lobule or mainly centrilobular in distribution. Persistent microvesicular steatosis usually is present. Liver allografts with moderate-to-severe degrees of harvest injury are characterized by: 1) extensive hepatocellular ballooning degeneration; 2) extensive cholestasis; 3) lipopeliosis; 4) bile ductule proliferation with periductular acute inflammation ("acute cholangitis pattern"); and 5) focal and/or zonal hepatocytic necrosis. With increasing severity of harvest injury, the hepatocellular ballooning degeneration becomes more extensive, and in severe cases, it may be panacinar in extent. Intracellular and intracanalicular cholestasis may be severe, and there may be associated bile duct plugging. Lipopeliosis, a condition in which the sinusoids become engorged by rounded fat globules, which follow the release of fat from necrotic hepatocytes, occurs in approximately 5% of liver allografts and is a manifestation of harvest injury. Bile ductule proliferation with acute inflammation ("acute cholangitis" pattern), morphologically may mimic extrahepatic biliary tract obstruction or ascending cholangitis. This bile ductular proliferation likely is a response to ischemic injury of the bile duct epithelium. Focal or zonal hepatocellular necrosis often is present and is generally centrilobular in location. Differential diagnosis The entities in the differential diagnosis are included not only for their histologic similarities to harvest injury, but also because they typically occur in the early post-transplantation period, as does harvest injury. 1) Pathologically documented cases of hyperacute (antibody-mediated, humoral) rejection are vanishingly rare. On a statistical basis alone, therefore, this diagnosis is highly unlikely. Hyperacute rejection occurs due to the presence within the allograft recipient of preformed antibodies directed against donor antigens. Clinically, patients with hyperacute rejection experience a relentless rise in serum aminotransferases, coagulopathy, and fulminant hepatic failure within 1-2 weeks after transplant. Biopsies from post-operative day 1 show focal hepatocellular necrosis and intravascular thrombi. Immunohistochemical or immunofluorescence studies may demonstrate sinusoidal, venous, or arterial deposition of immunoglobulins and complement. Biopsies taken 2-3 days after transplantation demonstrate extensive coagulative and hemorrhagic necrosis and numerous sinusoidal neutrophils. Thereafter, the organ undergoes progressive hemorrhagic infarction, and the entire scenario occurs over a period of 7-10 days. Severe harvest injury initially may resemble hyperacute rejection due to the hepatocellular necrosis and the sinusoidal neutrophils. However, harvest injury will not show intravascular thrombi or progressive infarction. 2) Hepatic arterial thrombosis, alone or in combination with portal vein thrombosis, occurs in a minority (<5%) of patients after liver transplantation. Arterial thrombosis often occurs within several days of transplantation. Depending on the severity and extent of the occlusion, hepatic artery thrombosis may cause ischemia or infarction of part or all of the allograft. As such, the biopsy findings are highly subject to "sampling effect" and may show normal tissue, centrilobular ballooning with cholestasis, irregular zonal necrosis or complete infarction. These histologic features are very similar to those of harvest injury. Because of the highly variable extent and distribution of the histologic findings in hepatic artery thrombosis, this diagnosis is one of exclusion and requires radiologic confirmation of the vascular occlusion. References Angelescu M, Hofmann W, Zapletal C, Bredt M, Kraus T, Herfarth C, Klar E. Histomorphological analysis of preservation injury as determinant of graft quality in clinical liver transplantation. Transplant Proc 1999;31:1074-1076. Canelo R, Braun F, Dopkens K, Ramadori G, Ringe B. Characterization and influence of risk factors on initial liver function after transplantation. Transplant Proc 2000;32:60-61. Cha IC, Bass N, Ferrell LD. Lipopeliosis: An immunohistochemical and clinicopathologic study of five cases. Am J Surg Path 1994;18:789-795. Gaffey MJ, Boyd JC, Traweek ST, Ali MS, Rezeig M, Caldwell SH, Iezzoni JC, McCullogh C, Stevenson WC, Khuroo S, Nezamuddin N, Ishitani MB, Pruett TL. Predictive value of intraoperative biopsies and liver function tests for preservation injury in orthotopic liver transplantation. Hepatology 1997;25:184-189. Markin RS, Wisecarver JL, Radio SJ, Stratta RJ, Langas AN, Hirst K, Shaw Jr BW. Frozen section evaluation of donor liver before transplantation. Transplantation 1993;56:1403-1409. Selzner M, Clavien P-A. Fatty liver in liver transplantation and surgery. Sem Liver Disease 2001;21:105-113. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury. Am J Surgery 2001;181:160-166.