Future directions in liver transplantation

Future Directions in Liver Transplantation Marie Csete and Andre De Wolf

N O R M O U S PROGRESS has been made in 'liver transplantation such that it is now the accepted therapy for end-stage liver disease. However, along with advancements, additional challenges have come requiring the develepment of new transplantation techniques and intraoperative methods. The following techniques are discussed in this article: reduced-size liver transplantation, artificial liver support systems, gene therapy, and xenotransplantation. Developments in the management of pulmonary hypertension and bleeding during liver transplantation are also included.

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REDUCED-SIZE LIVER TRANSPLANTATION

During the last few years, the shortage of cadaver donor livers for transplantation has become critical. More patients are being placed on transplant waiting lists. However, there has been no further increase in the supply ofcadaveric organs, despite improved organization of the organ procurement system and supportive legislation. It has been especially difficult to find sufficient organs for pediatric patients, because of the need for a donor-recipient size match and the limited number of pediatric donors. As a result, as many as 25% of pediatric candidates for liver transplantation die before they can be transplanted.1 New techniques have been developed to address these problems, and creative approaches have been used to increase the supply of organs. 2-3 Three techniques will be discussed in this section: reduced-size cadaveric transplantation, split-liver transplantation, and partial grafts from livingrelated donors. Reduced-size cadaveric liver transplantation makes more organs available to pediatric recipients by eliminating the size discrepancy, but this technique does not alleviate the overall shortage of organs. 2'4-5 Split-liver transplantation allows one organ to be used in two recipients. 2'67 In living-related liver transplantation, the organs for transplantation are obtained from a living-related donor, creating a new source of organs for transplantation. 2'8 The surgica! techniques used in reduced-size liver transplantation are based on similar principles, namely Seminars in Anesthesia, Vo114, No 2 (June), 1995: pp 93-109

the surgical size reduction of the organ while maintaining organ function, hopefully without severe complications. Surgical Techniques

Most experience has been with reduced-size cadaveric liver transplantation. This technique was first described by Bismuth and Houssin (Fig 1) and Brolsch et al in 1984. 4,9 Dissection of the hepatic parenchyma is performed on the back table, in accordance with the anatomic principles of Couinard, and either a right lobe graft, left lobe graft, or left lateral segment graft can be prep a r e d Y In general, a recipient to donor size discrepancy of 1:2 can be overcome by a fight lobe graft; a 1:4 discrepancy by a left lobe graft; and a 1:8 discrepancy by a left lateral segment graft. 2 Although some modifications may be required for hepatic venous outflow (size-reduction of donor inferior vena cava, or the use of a piggy-back technique) vascular anastomoses are end-to-end. The bile duct is anastomosed in a Roux-en-Y to the recipient jejunum. With split-liver transplantation, which was first performed by Pichlmayr et al in 1987,1~ the donor liver is split so that the parts can be transplanted into two recipients. 2,6-7,11 The right lobe contains the portal vein, hepatic artery, common bile duct, and inferior vena cava. The left lobe uses the left lobar branches of vessels and bile ducts. The right lobe is transplanted similarly to a whole organ, with standard vascular anastomoses, but with the left lobe, interposition grafts are frequently required for vascular reconstruction. Venous outflow of the left lobe is obtained by anastomosis of the left hepatic vein to the retained vena cava From the Department ofAnesthesiology, School ofMedicine, University of California-Los Angeles, CA; California Institute of Technology, Pasadena, CA; and the Department of Anesthesiology and Critical Care Medicine, University of Pittsburgh; and Presbyterian University Hospital, Pittsburgh, PA. Address reprint requests to Marie Csete, MD, Department ofAnesthesiology, UCLA School of Medicine, 10833 LeConte Avenue, Los Angeles, CA 90024-1778. Copyright 9 1995 by W.B. Saunders Company 0277-0326/95/1402-000455.00/0 93

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vasculature (determined by angiography), and absence of psychological contraindications (determined by a psychiatrist). 2

Complications

Roux en Y loop Fig 1. Anatomic reconstruction for o reduced-sized adult liver graft orthotopicolly transplanted. IVC, inferior vena cova; PV, portal vein; HA, hepatic artery; CBD, common bile duct. Reprinted with permission. 4

of the recipient (piggy-back technique). The most difficult aspect of this technique is the appropriate separation of arterial and biliary systems. Living-related liver transplantation has been used since 1989. 8 It not only increases the organ availability for small children, but also allows for these procedures to be done on an elective basis before the development of severe complications of liver failure. 2 In addition, the donor organ will be of excellent quality without significant ischemic injury, and the procedure may have immunological advantages. However, there is potential risk to the donor, and careful psychological evaluation has to be done to address the ethical considerations.2'12 Improvements in surgical technique have allowed left lateral segmentectomy without surgical complications or blood transfusion. 2 Eligibility criteria for donation include normal liver function, good overall medical condition, ABO compatibility, acceptable size of the left lateral segment (determined by computed tomography scan), appropriate hepatic arterial

In general, complications are seen more frequently in reduced-size liver transplantation than in whole organ transplantation, with split-liver transplantation being associated with the highest complication and mortality rates. 2-3 Because all of these techniques result in a graft with a large raw surface, increased blood transfusion requirements have been reported by most institutions. 2'13 In addition, failure to ligate all biliary ducts at the cut surface margin may lead to bile leakage or seroma formation.14 Reduced-size liver transplantation was initially associated with an increased incidence of portal vein thrombosis, probably related to positioning problems of the graft. ~3 This can be prevented by slight rotation of the graft counterclockwise in the abdominal cavity. ~3 On the other hand, hepatic artery thrombosis seems to occur less frequently with the use of reduced-sized grafts, probably because of the larger extra- and intrahepatic donor blood vessels.5-6' 13-16

More specifically, split-liver transplantation has been associated with a higher incidence of biliary complications, especially in the earlier series. 7 However, Heffron et al more recently reported that the incidence of biliary complications did not increase significantly with the use of reduced-size liver transplantation. 17 Complications that were frequently encountered in the early series have decreased with changes in operative techniques. These changes include interposition of arterial grafts to the recipient's aorta, a tendency to discard the left median segment (because of frequent necrosis), repositioning of the hepatic vein anastomosis of the left lateral segment grafts to a long anterior vena cava venotomy rather than to the orifice of the recipient left hepatic vein, and shortening of the graft bile ducts to the greatest extent possible to assure adequate vascular supply. 2'7 Cholangiogram and arteriogram are recommended before hepatic division to detect anatomic variations and avoid extensive dissection that may endanger bile duct vascularity. 6'18 Recipients of living-related donor organs have no more complications than recipients of whole organs, with the possible exception of increased

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION

intraoperative blood loss and biliary tract complications. 2'8 The incidence of hepatic artery thrombosis has decreased with the use of aortic inflow through a saphenous vein segment, instead of through the recipient's common hepatic artery. 8 Similarly, stretching of the portal vein has to be avoided, and may require the use of an inferior mesenteric vein graft. 8 Surgical complications have been seen in living-related donors, including splenic injury requiring splenectomy, biliary duct injury, and intraoperative blood loss requiring blood transfusion. The incidence of these complications may decrease with the use of a left lateral segmentectomy instead of a full left hepatectomy. 2'8 However, many transplant surgeons consider the morbidity and possibly mortality in the living-related donors ethically unacceptable and do not perform this procedure.

Outcome Although 1-year survival rates are better after living-related liver transplantation (89.5%) than split-liver transplantation (67%), overall results are similar to those after full-size transplantation (1-year survival rates of 72% to 85%). Rates are 76% to 77% with reduced-size liver transplantation. 2'5'7'19 Higher retransplantation rates have been reported with split-liver transplantation (32%) than with full-size liver transplantation (27%). 2 In contrast, the retransplantation rates for reduced-size liver transplantation and livingrelated liver transplantation were 20% and 17%, respectively.2 The relatively high retransplantation rate for full-size organs in pediatric series has been attributed to the high rate of vascular complications and the tendency to accept a small infant donor regardless of the donor condition. 2 The lower retransplantation rate with reducedsize organs may be caused by better quality of donor organs, and the decreased incidence of hepatic artery thrombosis. 2'14 Large experience with reduced-size liver transplantation and split livers has also been obtained at the University of Nebraska. 2~ The investigators reported that the use of reduced-size liver transplantation, including split livers, in mostly pediatric patients with endstage liver disease has resulted in good outcomes (1-year survival of 68%), and that there has been a reduction in the number of children dying awaiting transplantation (2.6% v 13%). 2o

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Split-liver transplantation has rarely been used in adults, and its outcome has been disappointing. 2-3'6-7These poor results may be related to the extreme urgency of the procedure in critically ill patients. Therefore, split-liver transplantation in adults remains a very controversial procedure. 2'7 In general, most of the initial reduced-size liver transplantations have been performed in critically ill patients who urgently required transplantation. However, because results with reduced-size liver transplantation have been almost as good as those with whole organ transplantation in pediatric patients, it seems justified to use these techniques (especially split-liver transplantation) in elective cases as well.

Implications for the Anesthesiologist There has been considerable concern regarding excessive bleeding from the raw surface of the liver. However, this has not proven to be a major problem, as perioperative blood loss has only increased slightly.13 The use of split-liver transplantation and living-related liver transplantation requires significant surgical, anesthetic, and nursing staff manpower. However, living-related liver transplantation is performed electively, and therefore should allow for sufficient preparation time.

Summary In the centers where reduced-size cadaveric liver transplantation is used, it has reduced global mortality in children with end-stage liver disease by increasing the donor pool, and thereby significantly decreased preoperative mortality. 2'3'5'14 This reduction in pretransplantation mortality results in an improved overall mortality rate, because survival after reduced liver transplantation is as good as after whole organ transplantation. 3,5,14 However, this has been accomplished at a cost of increased surgical complexity and more postoperative complications. 3 The increased morbidity may be related in part to the emergent nature of the procedure in critically ill patients. Performing the procedure in patients before they are terminally ill, and further refining the surgical techniques may reduce the incidence of complications and improve the outcome. Finally, although the ethical issues have not been completely resolved and morbidity or possible mortality in the donor remains theoretically

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problematic, living-related liver transplantation provides organs of excellent quality. ARTIFICIAL LIVER SUPPORT SYSTEMS

Artificial liver support systems are potentially beneficial in patients with fulminant hepatic failure. If medically treated, only 20% of patients with fulminant hepatic failure survive. 21 Despite the good overall results of liver transplantation ( 1-year survival rates of 6 5%) in patients with fulminant hepatic failure, there continues to be a high mortality rate, mainly because many patients die while waiting for a donor organ. 22 Only about 10% of patients with fulminant hepatic failure will receive a liver transplantation, 23 highlighting the need for nonsurgical support to improve outcome. Because the liver destruction in fulminant hepatic failure is potentially reversible, the native liver does not always need, to be removed and not all such patients require liver transplantation. Thus, if transplantation is performed too early, patients with potential for spontaneous recovery are exposed to the unnecessary risks of the liver transplant and committed to lifelong immunosuppression. If liver transplantation is performed too late, the patient may develop irreversible complications (especially neurological) resulting in death. 21 In other words, it is not always possible to wait for spontaneous recovery, because other organ systems may be irreversibly affected, and the results of liver transplantation are worse when performed in patients with severe hepatic coma and multiorgan failure. Therefore, the use of artificial liver support systems in acute fulminant liver failure that is potentially reversible or in fulminant liver failure as a bridge to liver transplantation has been proposed. Only patients who failed to recover from fulminant hepatic failure would receive a liver transplant, saving money and organs. Artificial liver support systems were described as early as the 1950S. 24-27 However, early techniques did not gain clinical acceptance because only temporary improvements in neurological condition were obtained, without longterm gain. In addition, the technical complexity was a significant burden. The pathophysiology of fulminant hepatic failure is very complex. Besides loss of hepatic function (synthetic, metabolic, and secretory), cytokines and other toxins may be released by

the necrotic liver into the circulation, contributing to multiorgan failure. Therefore, simple artificial liver support systems (without active hepatocytes) as a bridge to transplantation or to spontaneous recovery are likely to fail. 23 Indeed, several techniques, such as charcoal hemoperfusion, hemodialysis, and plasmapheresis have not been successful, mainly because there was inadequate support of all the essential hepatic functions. However, the use of ex-vivo liver perfusion has been reintroduced recently, 28-29 and the use of a bioartificial liver has received significant attention. 3~ These techniques have resulted in improvements in hepatic encephalopathy and have served as a bridge to transplantation in a limited number of patients. It has also been proposed that by providing liver function to the patient, the circumstances for spontaneous regeneration of the patient's liver are improved. On the other hand, the continued presence of the diseased liver means that there may be continuous leakage of toxins and mediators into the systemic circulation. It has been speculated that the presence of a necrotic liver, releasing many toxins and cytokines, may worsen the patient's condition, and therefore has to be removed. 33 Artificial liver support could then be considered during a prolonged anhepatic state, while waiting for a liver transplant. 33 The two main artificial liver support techniques are ex-vivo liver perfusion and bioartificial liver use. It is unclear which technique is most promising. Furthermore, although both techniques may temporarily improve the condition of the patient, there have not been any randomized clinical trials to objectively assess the efficacy of these artificial liver support techniques on outcome, and therefore it is too early to recommend routine use of these techniques. 34

Ex-vivo Liver Perfusion In this technique, heparinized blood from the patient is pumped through an ex vivo autologous, or more frequently, a heterologous (pig) liver, usually after passing through a membrane oxygenator and heat exchanger (Fig 2). 2829 Techniques for preservation of the liver are identical to those used for liver transplantation. The patient's femoral and internal jugular veins are cannulated. The cannulas are connected to a venous circuit that contains a centrifugal pump, a

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION

97 Liver

Fig 2. The venovenous circuit for ex vivo pig-liver perfusion. Blood was drawn from the body through the catheter in the left femoral vein into the venovenous circuit and returned through the catheter in the right internal jugular vein. Bile was collected by placing the catheter in the common bile duct. A pump circulated blood outside the body and a membrane o x y g e n a t o r - h e a t exchanger warmed the blood to 3 7 ~ Important features of the circuit were the liver and circuit bridges, which allowed blood to circulate several times through the pig liver without returning to the patient's body. Reprinted by permission of The N e w England Journal of Medicine? 8

membrane oxygenator, and a heat exchanger. The liver is connected to the venous circuit using sterile techniques, and is perfused through the portal vein 28 or through both the portal vein and hepatic artery. 29 The duration of the procedure is limited by the function of the liver, with an increase in portal perfusion pressure and a decrease in bile production indicating imminent failure. 28 Neutrophil accumulation in the liver may contribute to early liver injury, In addition, deposits of platelets and fibrin have been observed. Platelet transfusion has been required during liver perfusion, because platelets were lost in the membrane oxygenator. 28 Potential complications of this technique include the consequences of anticoagulation with heparin. Because of the short duration of perfusion, multiple livers are frequently needed. In the future, baboon or transgenic animal livers may be used to hopefully increase the lifespan of the perfused livers.

Bioartificial Liver A bioartificial liver consists of hepatocytes in suspension, on flat plates, or in a hollow-fiber cartridge. The hollow-fiber dialysis cartridge that contains living, functioning hepatocytes is used most frequently. In order to maintain a steady supply of cells, hepatocytes can be derived from

Bile collection

~ne oxygenator tt exchanger

a well-differentiated human hepatoblastoma cell line; 3~ alternatively, pig hepatocytes have also been used. 32 Hepatocytes are brought into the extracapillary space of the device, and are allowed to grow for several weeks, while supported by the appropriate medium. Maturity of the cartridge is determined by glucose use and albumin production. Some systems use whole blood partly anticoagulated with heparin (to an activated coagulation time of twice normal), whereas other systems use plasma perfusion. Despite mild anticoagulation, clotting of the cartridge may occur, requiring it to be replaced. Anticoagulation is not required when plasma is used; however, platelet count and fibrinogen levels decrease with each treatment. Even then, perfusion is limited to 6 to 7 hours by hepatocyte viability. It is still unclear whether these devices can provide sufficient metabolic support, because they contain significantly less hepatocytes (+_200 g) than a normal liver. In addition, there are no bile-duct epithelial cells, Kupffer's cells, or endothelial cells. Also, m a n y of the larger molecules (fibrinogen) are unable to pass through the membrane and there is a concern that if the membrane is damaged, hepatoma cells m a y enter the patient's circulation. Despite these shortcomings, t e m p o r a r y improvements in encephalopathy have been achieved.

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A major advantage of the bioartificial liver is the membrane, which acts as a barrier between the hepatocytes and the host's immune system, allowing this procedure to be performed without immunosuppression, even if hepatocytes from a different species are used. Another advantage is that functional cartridges may be stored while being perfused with an appropriate medium, and therefore are available whenever needed.

Auxiliary Partial Orthotopie Liver Transplantation This technique obviously does not provide artificial liver support, but is described here because its indications are the same as those for artificial liver support. The procedure consists of partial resection of the recipient's liver (usually a left lobectomy or left trisegmentectomy), followed by the placement of a reduced-size or small whole liver graft in the space created by the resection. This provides temporary support until the patient's own liver recovers. The technique was originally described by Bismuth and Houssin. 35 The orthotopic position has several advantages over the heterotopic, including better portal venous inflow and hepatic venous outflow. 36-38Also, heterotopic transplantation may create space problems in the abdomen, leading to increased intraabdominal pressure and difficult or impossible wound closure. If the native liver does not recover, it may require resection. If the native liver recovers, immunosuppression can be discontinued, and the graft can be removed or allowed to atrophy.3638 With recovery of the native liver, the patient is not obliged to lifelong immunosuppression.

Summary In conclusion, artificial liver support systems are now rapidly progressing and may successfully support patients until spontaneous recovery of the liver after fulminant hepatic failure. In other patients, artificial support may serve as a bridge to transplantation while preventing severe complications of fulminant hepatic failure. PULMONARY HYPERTENSION

Primary pulmonary hypertension is more common in patients with end-stage liver disease (0.73%) than in the general population (0.13%).39 With an increasing number of patients under-

going liver transplantation, more liver transplantation candidates are expected to have pulmonary hypertension. Although liver transplantation can be performed successfully in patients with mild pulmonary hypertension (mean pulmonary artery [PA] pressure between 25 and 35 mmHg),4~ performing this procedure in patients with moderate (mean PA pressure between 35 and 50 mmHg) to severe (mean PA pressure >50 mmHg) pulmonary hypertension carries an extremely high perioperative mortality r a t e . 41-43 Very few patients with significant pulmonary hypertension have survived liver transplantation. 43 One of these survivors continued to have pulmonary hypertension after liver transplantation, 44 but the PA pressures of the other two patients normalized between 12 and 22 months after liver transplantation.45-46 The pathophysiology of the disease is unclear. Portal hypertension (but not necessarily cirrhosis) is a prerequisite. 47 Recurrent thromboembolism may play a role, but many patients do not have pathological evidence of pulmonary thromboembolism on autopsy. 39 In fact, the pathological defect, plexogenic pulmonary arteriopathy, is identical to that seen in primary pulmonary hypertension. 48 Chronic pulmonary vasoconstriction caused by vasoactive substances (eg, kinins, serotonin, histamine, and elastase) not cleared by the liver may result in medial hypertrophy and intimal fibrosis. 39'48"49 The pulmonary hypertension is often resistant to all vasodilators, including nitroglycerin, sodium nitroprusside, beta agonists, calcium channel antagonists, and p r o s t a g l a n d i n E 1 o r I2.4t'43'45 A few patients had some response to prostaglandin E1 or I2 .44,46 Preliminary data from several institutions suggest that inhaled nitric oxide is also not effective in reducing the pulmonary hypertension in patients with portal hypertension. Furthermore, significantly elevated endothelin levels in arterial blood were found in one patient with this condition (C. S. Gandhi, personal communication, October 1994). These findings show that there is probably not a reduction of endogenously produced nitric oxide, but there may be an unresponsiveness of the pulmonary vasculature to nitric oxide, or an increase in production of endothelin with decreased systemic sensitivity to endothelin. Clinical signs and symptoms of pulmonary hypertension include dyspnea on exertion, syn-

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION

cope, chest pain, and fatigue.48 Hypoxemia is usually not present. On physical examination, a loud second heart sound and a systolic murmur are frequently observed. 48 At least 75% of patients have electrocardiographic or roentgenographic evidence of right ventricular hypertrophy with right bundle branch block, cardiomegaly, and prominent pulmonary arteries. 48 This diagnosis is confirmed by PA catheterization, showing elevated pressures in the absence of increased PA occlusion pressure. Secofidary (usually mild) pulmonary hypertension, caused by intravascular volume overload, left ventricular dysfunction, or mitral valve disease, has to be excluded. However, hypovolemia may lead to an underestimation of the severity of pulmonary hypertension because of a decrease in cardiac output. Therefore, to determine the true degree of pulmonary hypertension, attempts should be made to make the patient normovolemic when the right heart catheterization is performed. Quite often, the diagnosis of pulmonary hypertension is not suspected before transplantation, but is made only after arrival in the operating room. In these patients, liver transplantation should be postponed until right ventricular function is assessed, and the patient has been fully informed of the significantly increased perioperative risks. Echocardiography is very important in the determination of right ventricular function, and right ventricular dysfunction is usually a contraindication to liver transplantation unless the pulmonary hypertension is mild and the patient has no other risk factors. Patients with moderate pulmonary hypertension (mean PA pressure between 35 and 50 mmHg) should only be transplanted if right ventricular function is excellent. Patients with severe pulmonary hypertension (mean PA pressure >50 mmHg) carry the highest risk, and should be considered for alternative therapies (eg, simultaneous liver-lung or liver-heart-lung transplantation, or the use of a right ventricular assist device). Combined liver-lung or fiver-heartlung transplantation has been reported to be successful in two patients (one had pulmonary hypertension caused by a patent ductus arteriosus), but both are radical approaches with significant risks.5~ l Intraoperative management should be directed to avoid factors that further increase PA pressures, such as hypercarbia, hypoxia, acidosis, and

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increases in catecholamine levels. Lung volumes should be kept close to normal, and function of the right ventricle should be supported by optimizing the preload, maintaining perfusion pressure (-- arterial pressure), and avoiding myocardial depressant drugs. Right ventricular volumes and function should be monitored with transesophageal echocardiography or a right ventricular ejection fraction PA catheter. An increase in preload should be avoided because it may increase oxygen consumption of the right ventricle. Although most patients are resistant to pulmonary vasodilators, usually one of these drugs is infused intraoperatively in an attempt to avoid further exacerbation of pulmonary hypertension. Inotropic agents (eg, dobutamine) may improve fight ventricular function. Frequently PA pressures decrease during the anhepatic stage, probably related to a decrease in cardiac output. However, after graft reperfusion, the pulmonary artery pressure may increase dramatically, possibly related to toxins or vasoactive substances released by the newly perfused liver, frequently resulting in acute fight ventricular failure and cardiac arrest. Exacerbation of pulmonary hypertension may continue into the postoperative period, and acute fight ventricular failure and sudden death may occur in the first few weeks after liver transplantation in the intensive care unit.

Summary Management of the care of patients with pulmonary hypertension is a major clinical challenge. Understanding of the pathophysiology is still incomplete, and it is still unclear whether these unfortunate patients should undergo liver transplantation alone or in combination with lung or heart-lung transplantation. Furthermore, the reversibility of the disease after successful transplantation has not been firmly established because of the very small number of liver transplant survivors with severe pulmonary hypertension. PHARMACOLOGICAL ANTIFIBRINOLYTIC THERAPY FOR INTRAOPERATIVE BLEEDING

The last decade of liver transplantation has been marked by significant improvement in the amount of blood products required during surgery. In part, this decrease in transfusion require-

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ments has been caused by pre-emptive treatment of coagulopathy early in the surgical procedure. 52 For example, though a patient's prothrombin time may be only slightly prolonged at the start of surgery, experienced transplant anesthesiologists aggressively treat the inevitable dilution of clotting factors, which occurs during dissection. Continuous monitoring of coagulation, either by thrombelastography53 or observation of clotting with the Lee-White clotting time, 54 in addition to standard coagulation asgays, has also aided real-time interpretation of bleeding diatheses in the operating room. There are no controlled studies to support the benefits of continuous coagulation monitoring and early, aggressive transfusion of plasma products, but there is general agreement that these practices (along with improved surgical technique) have decreased transfusion requirements during surgery and the immediate postoperative period. Nonetheless, adult patients undergoing transplantation often require one blood volume in banked blood products, therefore, efforts to decrease transfusion requirements must continue. Future liver transplantation will most certainly benefit from improvements in understanding of antifibrinolytic therapy, and by the introduction of oxygen-carrying blood substitutes into clinical practice.

Epsilon Aminocaproic Acid The mainstay of pharmacological therapy for intraoperative bleeding during liver transplantation has been epsilon-aminocaproie acid (EACA). 55 EACA exerts specific antifibrinolytic activity by inhibiting plasminogen activator proteins and, to a lesser extent, by inhibiting plasmin activity. The effectiveness of EACA during liver transplantation is likely caused by the effect of the drug on tissue plasminogen activator (tPA). Because tPA is normally cleared in the liver, tPA levels increase during the anhepatic period, 56 initiating a primary fibrinolytic process. Furthermore, the natural inhibitor oftPA is synthesized in the liver, and therefore, the increase in tPA activity cannot be adequately counterbalancedY The immediate postreperfusion stage of liver transplantation is often complicated by further, sometimes massive increases in tPA release. The timing of this process suggests that the donor graft itself (from endothelium) is the source of the tPA 58 and may be an inevitable consequence of

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preservation injury. These assumptions about the source of tPA release and its subsequent activity have been supported by studies of pigs, which have shown that increases in severity of fibrinolysis are a direct consequence of increased preservation time. 59 Furthermore, heterotopic pig transplants (with a native liver present to clear tPA) are characterized by less fibrinolysis than orthotopic transplants. 59 Although EACA is used in almost all transplant centers to deal with this tPA release, the pattern of drug use varies tremendously from center to center. For example, the University of Pittsburgh group often uses small bolus doses in the range of 250 mg of EACA to respond to a thromboelastographic reading suggesting fibrinolysis. In contrast, the UCLA group uses a continuous infusion of the drug starting in the anhepatic period until transfer to the intensive care unit, with a dose (as recommended in the EACA package insert) of 5 g loading dose and 1 g per hour infusion. To date, no carefully controlled studies have been performed to directly correlate EACA drug dose with assays of fibrinolysis in cirrhotics or in patients undergoing liver transplantation. In order to maximize the efficacy of the use of this drug, these studies will be necessary in the future. It is possible, for example, that both groups may be underusing EACA, or that some intermediate dose is optimal. Large doses of EACA, in the range of 24 g per day for 3 days, have been associated with increased bleeding time in patients with intracranial aneurysms;6~ in this setting, EACA seems to interfere with normal platelet to blood vessel wall interactions.6~ In vitro, EACA also inhibits plasminogen binding to adenosine diphosphate (ADP)-activated platelets. 61 The threshold dose for such an antiplatelet function side-effect of EACA is not known, nor has the antiplatelet effect been studied in liver transplant patients. Again, further studies must be performed in order for anesthesiologists to be able to optimally administer this agent. A final question in the use of EACA relates to its ability to induce generation of thrombin in vitro. 62 In clinical practice, EACA is commonly avoided when secondary fibrinolysis (disseminated intravascular coagulation) is the cause of the bleeding diathesis. This practice is based largely on rare case reports of thromboses asso-

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ciated with the use of E A C A . 63-64 On the other hand, EACA has been used safely in settings known to be complicated by disseminated intravascular coagulation (DIC). 65 Whether DIC is an important contributor to the coagulopathy of end-stage liver disease is controversial, though laboratory parameter abnormalities of DIC are commonly found in the setting of liver disease. 66 Patients with end-stage liver disease are certainly at risk for secondary processes such as infection, which in turn are associated with DIC. Despite this theoretical potential for thrombotic complications of EACA in the presence of DIC, liver transplant patients receiving EACA very rarely experience thromboembolic events. Furthermore, in the rare series reporting thromboses during transplantation, EACA has not been implicated. 6v Once again, the issue of thrombin generation or DIC as a function of EACA administration has not been rigorously investigated in liver transplant recipients. One small retrospective report suggests that "prophylactic" EACA is not effective in reducing blood transfusion requirements during transplantation. 68 Another well-designed, double-blind prospective trial of EACA (5 g loading dose, 1 g continuous infusion) conducted at the University o f Nebraska suggested that a significant reduction in transfusion requirements was afforded by EACA. 69 However, this study has not been published as a full manuscript. In summary, EACA is widely used during liver transplantation as an adjunct to the treatment of intraoperative coagulation disturbances, specifically for its inhibiting effect on tissue plasminogen activator activity. Although the literature supporting this assumption is scant, transplant anesthesiologists generally accept its usefulness in reducing transfusion requirements. Future studies should be directed at pinpointing the optimal dosing regimen and characterizing side effects, especially of large doses.

Tranexamic Acid Tranexamic acid, like EACA, inhibits fibrinolysis by binding to plasminogen and plasmin and blocking fibrin breakdown. 7~The drug is not as often used during liver transplantation as EACA, perhaps because its effective half-life is considerably longer than that of EACA.71 Because it has been shown to have potent antifibrinolytic

I01

effects in other settings such as cardiac surgery, 72 this drug also deserves further study as a possible addition to the treatment for bleeding during liver transplantation.

Aprotinin The procoagulant activities of aprotinin are more complicated than those of EACA and tranexamic acid. The drug is a nonspecific serine protease inhibitor. The antifibrinolytic effects of aprotinin are primarily mediated through antiplasmin and antikallikrein actions. 73 In addition, aprotinin inhibits urokinase-type plasminogen activator (uPA). TM The precise role of this enzyme in the fibrinolysis of orthotopic liver transplantation is unclear. One study suggested that uPA levels are not increased during liver transplantation, 56 whereas another study showed significantly increased levels. 75 Aprotinin does not directly inhibit tissue-type plasminogen activator] 5 Until recently, aprotinin was not available for clinical use in the United States. Currently its approved indications do not include treatment of bleeding during liver transplantation, but based on European findings, it seems inevitable that the drug will be studied in the United States for this indication. Early small series indicated that aprotinin was effective in reducing transfusion requirements during liver transplantation, TM especially in the neohepatic period. 77 Several other small series have confirmed these early findings.78"81 However, these studies must be viewed with caution, because none included large numbers of patients, and after attempts at prospective trials of aprotinin, some of these studies used historical controls. 8~ Because liver transplant centers characteristically use less blood products over time, retrospective studies that include blood product use as an endpoint are difficult to interpret. Furthermore, some of the studies excluded patients based on specific diagnoses, although it has never been clearly shown that blood product use can be predicted based on the specifics of the underlying liver disease. Finally, when taken together, the dose of aprotinin used in the studies varied widely (sometimes by design, to test different doses). Aprotinin may have other effects that can be used to advantage in the liver transplant setting. In an animal model, hypothermic activation of

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proteases was inhibited by aprotinin administration, translating into increased survival in treated animals. 82 In another study, aprotinin pretreatment of donor rat livers prolonged the ischemic tolerance time of the grafts,s3 but another similar study did not show this protective effect. 84 However, these studies do raise the intriguing possibility of donor pre-treatment to reduce fibrinolysis in the recipient's circulation. In summary, although there has been more of an attempt to study aprotihin in a prospective controlled manner than has been made with EACA, the weakness of the published literature should prompt a large-scale prospective study to examine the efficacy of aprotinin on blood loss during liver transplantation. Furthermore, the adverse effects of this drug should also be characterized, because a cautionary note was delivered in one report of thrombosis associated with the use of aprotinin during liver transplantation, s5

Artificial Hemoglobins Early hemoglobin preparations were made from blood lysates. These crude solutions were nephrotoxic (from stroma, vasoactive substances, or from filtration of the hemoglobin) and caused coagulation abnormalities. Later, the "stromafree" preparations still caused adverse reactions, probably because of low-level contamination. 86 Pure hemoglobin solutions can be produced with recombinant DNA techniques (using bacteria as factories) or perhaps in transgenic animals. These genetically engineered hemoglobins have several problems: the oxygen affinity in the absence of 2,3-diphosphoglycerate (DPG) is too great such that oxygen unloading is limited, and dissociation of the molecules yields potential renal damage. Nonetheless, researchers persist in trying to perfect these substances because animal studies clearly show that infused hemoglobin is life-saving in extreme hemorrhagic states. H u m a n recombinant hemoglobin has been recently produced that does not dissociate into the dimers that cause renal toxicity.87 One such recombinant hemoglobin has undergone phase I trials at the University of Utah, and is in early phase II trials in anesthetized surgical patients. At this point, it seems that hemoglobins are, in general, more likely than perfluorocarbons 88 to influence clinical practice, and may be particularly important

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in reducing banked blood requirements during liver transplantation. GENE THERAPY

A major challenge to the future of liver transplantation is the critical shortage of donor livers. Various alternative techniques are being introduced to reduce the dependence on cadaveric donors including artificial liver support systems, xenotransplantation, living-related donors, splitliver grafts for two recipients, and most recently, gene therapy technology. Gene therapy is the introduction or alteration of genetic sequences in living cells for diagnostic or therapeutic purposes. Gene therapy protocols are under the regulation of the Food and Drug Administration (FDA), like any other biological product. The FDA defines somatic-cell therapy as the "administration to humans ofautologous, allogeneic, or xenogeneic living somatic cells that have been manipulated or processed to change their biologic characteristics. ''89 A variety of somatic cell gene therapy protocols are under development around the country for the treatment of cancers and various inherited metabolic deficiencies. Although the number of patients with a particular inherited metabolic deficiency leading to liver failure is small, when taken together metabolic deficiencies are a relatively common indication for pediatric liver transplantation. The deficiencies, which are likely to be most amenable to gene therapy, include Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, Wilson's disease, tyrosinemia, alpha-l-antitrypsin deficiency, and lysosomal storage diseases. Many methods are available to introduce genes into cells, in the hopes of having the target cell express the protein product of the inserted genes. Physical methods include particle bombardment with DNA projectiles, receptor-mediated gene transfer with polylysine or other links to the target cell, electroporation to disrupt the target cell membrane, liposome-mediated gene transfer, and even little single-cell gene guns. 89 These methods have all been successful in specific settings, but generally do not yield good efficiency. In other words, only a small percentage of cells are successfully targeted by these methods and the number of gene copies transferred per cell is low. The best characterized methods for gene transfer are biological, the replication-deficient

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION

viral vectors. In order to create a safe virus to shuttle genes into cells, the viral genome is modified by deleting part of its sequence. Usually the sequences deleted have been those of structural proteins that allow the virus to rebuild itself for replication and the lytic cycle. After such a deletion, the viral vector is still able to attach to receptors on host cells, and insert its genome into the host cytoplasm, but it cannot replicate. In place of the snipped-out sequences in the viral genome, it is possible to insert other functional gene or genes (depending on size constraints). In this way the viruses act as vehicles to transfer a gene of interest into cells or into a whole organ. The efficiency of gene transfer using viral vectors depends on the density of the virus receptors on the particular target cell, and on the ratio of the number of viral particles to the number of target cells. Retroviral vectors have been widely used experimentally as vehicles for gene transfer. They have a simple genetic backbone, are easy to grow in the laboratory, and attach to a wide variety of cell types. The disadvantage of retroviral vectors is that their efficiency is dependent on active target cell replication, such that quiescent cells such as hepatocytes are not efficiently targeted. A theoretical advantage of these particular vectors is that the transferred genes are incorporated into host chromosome, such that expression of the gene product can be expected for the life of the cell. This characteristic is particularly appealing for the therapy of inherited deficiency disorders affecting the liver. However, in reality, the immortality expected of the host-incorporated sequences has not always followed successful transduction, 9~ and the need for repeated gene therapy may, in theory, initiate immune recognition responses with severe clinical consequences. Animal models of metabolic deficiencies provide hope that liver-directed gene therapy will offer a therapeutic alternative for patients with these unusual disorders. For example, the gene for human ornithine transcarbamylase has been successfully inserted into mouse hepatocytes in culture without significant damage to the hepat o c y t e s . 91 The human gene for alpha-1-antitrypsin has been successfully introduced into dog hepatocytes in culture and then used for autologous transplantation. The gene was expressed for a

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month after transplantation. 92 These are two of several examples of successful gene transfer via retroviral vectors in animal models, chosen because the disease processes are well-known in liver transplant centers. In fact, the era of liver-directed human gene therapy with these vectors has already begun with early clinical trials in patients lacking the gene for low-density lipoprotein receptor. In the past this disorder was treated successfully with liver transplantation, 93 although the native liver was not itself diseased. Rather, the lack of the receptor results in devastating hypercholesterolemia with cardiovascular complications. Liver transplantation represents one method for replacing the enzyme. Another approach was first attempted in the animal model of low-density lipoprotein receptor deficiency, the Watanabe rabbit. Hepatocytes from resected liver were placed in culture, then exposed to retroviral vectors packaged to contain the gene for the low-density lipoprotein receptor. The hepatocytes were then transplanted into the low-density lipoprotein receptor-deficient animals. Significant, long-term lowering of cholesterol levels was accomplished. 94 H u m a n trials have now begun using autologous, genetically-modified hepatocytes. A segment of the patient's liver is removed, enzymatically broken down into a single cell suspension, and the hepatocytes isolated and cultured. After retroviral introduction of the gene, the cells are re-infused into the patient via a portal venous catheter. The long-term outcome of this therapy has not yet been published. Implications of such gene therapy for the anesthesiologist are only speculative at this point. However, retroviral vectors are not the only biological vectors approved for human trials. Others of the replication-deficient viral vectors may be limited by preformed antibody to their wild-type counterparts. Early work with inhaled adenoviral vectors containing the cystic fibrosis transmembrane conductance regulatory gene (for treatment of cystic fibrosis) suggests that the local immune response to the virus may be difficult for some patients to tolerate. 95 In addition, we have noted an anaphylactic-like reaction in pigs when adenoviral preparations were infused into the portal vein (M. Csete, unpublished observations). Nonetheless, these vectors have distinct advantages. There is no known association of adeno-

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viruses and malignancies, and the viral genome is large enough to accommodate a large insertion. Furthermore, the time for gene transfer using these vectors is very quick (compared with retroviral vectors). In fact, in our UCLA laboratory we can efficiently transfer genes to hepatocytes in suspension with the adenoviral vectors without having to put the hepatocytes into c u l t u r e , 96 thereby minimizing the risk of contamination of the cells. Other viruses used to shuttle vectors for genes such as replication-deficient herpes viruses may also cause immune responses because of preformed antibody. It is also possible that the transduced proteins themselves could initiate an immune r e s p o n s e . 97 Injection of cells into the portal vein for liverdirected gene therapy may, in theory, cause acute or chronic obstruction of the portal vein, and perhaps portal hypertension.9v Long-term issues of malignant transformation caused by the retroviral gene transfer remain unknown. 97 For other gene therapy protocols, the fiver may be the site of infusion of modified cells, though the liver itself is not diseased. For example, pancreatic islets are currently transplanted into the liver in some centers. 9s In theory, geneticallymodified islets, transduced to produce immunologic mediators, could initiate a cytokine cascade with systemic symptoms. At this time gene therapy protocols seem far removed from everyday clinical practice, but with more than 50 approved human protocols now in progress, academic anesthesiolrgists will certainly be exposed to patients in these trials in the next few years. The clinician must be prepared for the major impacts of gene therapy, including acute immunologic responses or cytokine syndromes. Liver-directed gene therapy in the case of inherited metabolic deficiencies is theoretically wellsuited to intervention in utero, to prevent the consequences of the inherited disorders at an extremely early stage of development. It is now technically feasible to access the fetal liver for gene transfer (as well as for other surgical procedures). 99 So, the future no doubt holds the challenge of adequate anesthesia for the fetus and mother to enable successful genetic engineering of the fetal liver. XENOTRANSPLANTATION

Xenotransplantation, in which the organ is obtained from another species, has been proposed

as a solution to the shortage of human organs for transplantation. However, at this moment, there is not sufficient knowledge regarding the immunologic consequences ofxenotransplantation to make this a clinically successful procedure. This is illustrated by the two unsuccessful baboon-to-human liver transplantations recently performed at the University of Pittsburgh (June 1992 and January 1993) and by the hyperacute rejection of a pig-to-human liver xenografl at the Cedars-Sinai Research Institute. 100-103Despite the unfortunate outcomes, these procedures and many laboratory experiments have provided a wealth of information, and there is optimism that xenotransplantation will eventually become a clinical alternative to cadaveric human transplantation. 102,104

Immunologic Considerations When xenografts are transplanted in humans, hyperacute rejection occurs immediately. This phenomenon is caused by the binding of preexisting xenoreactive natural antibodies (mostly immunoglobulin M [IgM]) to the endothelial cells of the graft immediately after reperfusion, followed by activation of the complement system and of the endothelial cells. 1~176 Complement is activated and consumed to such an extent that it cannot be detected in the plasma, 1~ and complement activation seems to correlate with the degree of graft damage. 1~ When activated, endothelial cells reverse their anticoagulant role and become procoagulant, leading to thrombosis of the microvasculature in the xenograft with platelet activation and fibrin deposition. TM Leakage of cells (including polymorphonucleocytes) from the intravascular to the interstitial space also contributes to hyperacute rejection. 1~ Hyperacute rejection is more severe in xenotransplanration between discordant species (eg, pig to human) than between concordant species (eg, baboon to human). Traditional immunosuppression in previous attempts at xenotransplantation of kidneys and hearts to humans did not prevent hyperacute rejection, and was followed by antibody-mediated occlusive endothelitis of the graft microvasculature and parenchymal necrosis. 1~176 Immunosuppression used in the recent baboon-to-human liver transplantations consisted of tacrolimus (formerly FK 506), prednisone, prostaglandin E 1,

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION and cyclophosphamide.l~176 Cyclophosphamide was added to the commonly used cocktail, because it is an antimetabolite that targets the proliferation of B cells, 1~ in an attempt to diminish the antibody-mediated component of xenograft rejection. The prevention of this antibody-mediated reaction and the subsequent complement activation are necessary prerequisites for successful xenotransplantation.104 Other techniques to control xenograft rejection include the removal of the natural human xenoanfibodies against vascular endothelium from the recipient's blood by plasmapheresis or perfusion of blood through kidneys and livers of the donor species. 1~176 These techniques probably also reduce complement levels; 1~ however, their effect is only temporary, and therefore, the use of an antimetabolite (eg, cyclophosphamide) may be preferable. On the complement level, nafamostat mesilate, an anticomplement agent, has delayed the hyperacute rejection in a discordant liver xenotransplantation model.l~ 1 Other anticomplement agents such as K76 or F U T have also shown promising results. ~~ 109 Liver xenotransplantafion is a special form of xenotransplantation, because the liver is the main site of complement synthesis. Patients with endstage liver disease have decreased levels of complement factors C3 and C4, which may contribute to the decreased immunologic function in these patients. These findings may explain the decreased severity of hyperacute rejection in the two baboon-to-human liver xenotransplantations.106 However, it is speculated that the xenografts were still damaged, because 12 days after transplantation, biopsy of the xenografts showed deposition of IgM and immunoglobulin G (IgG) which was not observed 24 days after transplantation. 100.1o2 These immune complexes may have contributed to complications such as the denuded epithelium of the bile ducts, resulting in widespread biliary sludge in the biliary tree despite a seemingly satisfactory choledochojejunostomy. 1~ Possibly even more importantly, it has been discovered that recipient-type complement is required for hyperacute rejection. 112 Because the liver is the main site of complement production, the complement in the recipient gradually becomes that of the other species, and therefore should not react against the xenograft. Thus, anticomplement agents should be necessary only for a short

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time in liver xenotransplantation. In addition, infusion of human complement (eg, during blood transfusion) must be avoided after liver xenotransplantation, lO2,112Preparing complement-free blood components is possible and fairly easy to do. Mixed chimerism was observed in the baboonto-human xenotransplantation recipients. 1~176176 Interstitial (migratory or passenger) leukocytes did migrate in both directions, from the xenograft to the recipient, and from the recipient to the xenograft. Chimerism is believed to be the first step toward chronic acceptance and possibly donor-specific tolerance of allografts as well as xenografts.1~176176 Such chimerism also has been observed in long-term surviving liver and cardiac xenograft recipients in animals. 115 The Baboon as a Xenograft Donor

The baboon is attractive as a xenograft donor for several reasons. The main advantage is that it is a concordant species, and xenotransplantation between concordant species has to be resolved first before xenotransplantation between discordant species can be attempted. Another advantage is that the baboon liver is thought to be resistant to hepatitis B. Also, the metabolic function of the baboon liver mimics that of the human very closely, which is important because the hepatocytes in the xenograft retain their donor metabolic specificity. Therefore, the serum proteins including coagulation factors become those of the xenograft species. In addition, in the two baboon-to-human transplants, serum uric acid and cholesterol levels decreased toward those that are normal for the baboon. Albumin levels also decreased to levels observed in the donors preoperatively. The metabolic differences from human liver function were overall very small, and did not have adverse clinical consequences. 100-101 Liver regeneration followed a pattern similar to that seen in human grafts, indicated by the growth of the xenograft from 660 g at the time of transplantation to 1,555 g by day 24.1~ Finally, baboons are easily bred in captivity. 116 There are still some problems associated with the use of baboon organs for xenotransplantation.104 Baboons have a relatively long gestation time, and the organs are fairly small. The possible infection of baboons with viruses that may infect humans has not been completely resolved. 116

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There are no baboons with blood group O. Finally, some people, especially animal rights activists, may have ethical objections to the breeding of baboons as donors for humans. However, ethicists indicate that the painless sacrifice of a primate seems to be justified when the alternative to xenotransplantation is the death of a human being.~ 16 Human life is more important than the life of any nonhuman animal. ~17 Even so, the survivor of a xenotransplant may have to deal with the psychosocial cons~equences of such a procedure.

Summary In conclusion, advanced knowledge of rejection and immunosuppression will almost certainly lead to the clinical use of xenografts in the not too distant future. The immunosuppressants currently used may be sufficient to control rejection, but new drugs are rei:luired to prevent hyperacute rejection and graft damage. The use of animals with genetically altered endothelial cells may eliminate hyperacute rejection. ~~176 REFERENCES 1. Malatack J J, Schaid DJ, Urbach AH, et al: Choosing a pediatric recipient for orthotopic liver transplantation. J Pediatr 111:479-489, 1987 2. Thistlethwaite JR Jr, Emond JC, Heffron TG, et al: Innovative use of organs for liver transplantation. Transplant Proc 23:2147-2151, 1991 3. Broelsch CE, Emond JC, Whitington PF, et al: Application of reduced-size liver transplants as split grafts, auxiliary orthotopic grafts, and living related segmental transplants. Ann Surgery 212:368-377, 1990 4. Bismuth H, Houssin D: Reduced-sized orthotopic liver graft in hepatic transplantation in children. Surgery 95:367370, 1984 5. Otte JB, de Ville de Goyet J, Sokal E, et al: Size reduction of the donor liver is a safe way to alleviate the shortage of size-matched organs in pediatric liver transplantation. Ann Surg 211:146-157, 1990 6. Otte JB, de Ville de Goyet J, Alberti D, et al: The concept and technique of the split liver in clinical transplantation. Surgery 107:605-612, 1990 7. Emond JC, Whitington PF, Thistlethwaite JR, et al: Transplantation of two patients with one liver. Analysis of a preliminary experience with "split-liver" grafting. Ann Surg 212:14-22, 1990 8. Broelsch CE, Whitington PF, Emond JC, et al: Liver transplantation in children from living related donors. Surgical techniques and results. Ann Surg 214:428-439, 1991 9. BrNsch CE, Neuhaus P, Burdelski M, et al: Orthotope Transplantation yon Lebersegmenten bei Kleinkindern mit Gallengangatresien. (Orthotopic transplantation of hepatic

segments in infants with biliary atresia.) Langenbecks Arch Chir, 105-109, 1984 (suppl) 10. Pichlmayr R, Ringe B, Gubernatis G, et al: Transplantation einer Spenderleber auf zwei Empfanger (SplittingTransplantation)--Eine neue Methode in der Weiterentwicklung der Lebersegmenttransplantation. (Transplantation of one donor liver to two recipients (splitting transplantation)-A new method for further development of segmental liver transplantation.) Langenbecks Arch Chir 373:127-130, 1988 11. Bismuth HM, Morino M, Castaing D, et al: Emergency orthotopic liver transplantation in two patients using one donor liver. Br J Surg 76:722-724, 1989 12. Singer PA, Siegler M, Whitington PF, et al: Ethics of liver transplantation with living donors. N Engl J Med 321: 620-622, 1989 13. Esquivel CO, Nakazato P, Cox K, et al: The impact of liver reductions in pediatric liver transplantation. Arch Surg 126:1278-1286, 1991 14. Ryckman FC, Flake AW, Fisher RA, et al: Segmental orthotopic hepatic transplantation as a means to improve patient survival and diminish waiting-list mortality. J Pediatr Surg 26:422-428, 1991 15. Stevens LH, Emond JC, Piper JB, et al: Hepatic artery thrombosis in infants. A comparison of whole livers, reducedsize grafts, and grafts from living-related donors. Transplantation 53:396-399, 1992 16. Tan KC, Yandza T, de Hemptinne B, et al: Hepatic artery thrombosis in pediatric liver transplantation. J Pediatr Surg 23:927-930, 1988 17. Heffron TG, Emond JC, Whitington PF, et al: Biliary complications in pediatric liver transplantation. A comparison of reduced-size and whole grafts. Transplantation 53:391-395, 1992 18. Rat P, Paris P, Friedman S, et al: Split-liver orthotopic liver transplantation: how to divide the portal pedicle. Surgery 112:522-526, 1992 19. Br01sch CE, Stevens LH, Whitington PF: The use of reduced-size liver transplants in children, including split livers and living related liver transplants. Eur J Ped Surgery 1:166171, 1991 20. Langnas AN, Marujo WC, Inagaki M, et al: The results of reduced-size liver transplantation, including split livers, in patients with end-stage liver disease. Transplantation 53:387391, 1992 21. Riegler JL, Lake JR: Fulminant hepatic failure. Med Clin North Am 77:1057-1083, 1993 22. Mufioz SJ: Difficult management problems in fulminant hepatic failure. Semin Liver Dis 13:395-413, 1993 23. Cattral MS, Levy GA: Artificial liver support--pipe dream or reality? Editorial. N Engl J Med 331:268-269, 1994 (editorial) 24. Otto J J, Pender JC, Cleary JH, et al: The use of a donor liver in experimental animals with elevated blood ammonia. Surgery 43:301-309, 1958 25. Eiseman B, Liem DS, Raffucci F: Heterologous liver perfusion in treatment of hepatic failure. Ann Surg 162:329345, 1965 26. Abouna GM, Cook JS, Fisher LMA, et al: Treatment of acute hepatic coma by ex vivo baboon and human liver perfusions. Surgery 71:537-546, 1972

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION 27. Abouna GM, Fisher LM, Porter KA, et al: Experience in the treatment of hepatic failure by intermittent liver hemoperfusions. Surg Gynecol Obstet 137:741-752, 1973 28. Chaff RS, Collins BH, Magee JC, et al: Treatment of hepatic failure with ex vivo pig-liver perfusion followed by liver transplantation. N Engl J Med 331:234-237, 1994 29. Fox IJ, Langnas AN, Fristoe LW, et al: Successful application of extracorporeal liver perfusion: A technology whose time has come. Am J Gastroenterol 88:1876-1881, 1993 30. Sussman NL, Chong MG, Koussayer T, et al: Reversal of fulminant hepatic failure using an extracorporeal liver assist device. Hepatology 16:60-65, 1992 31. Sussman NL, Kelly JH: Improved liver function following treatment with an extraeorporeal liver assist device. ArtifOrgans 17:27-30, 1993 32. Rozga J, Podesta L, LePage E, et al: A bioartificial liver to treat severe acute liver failure. Ann Surg 219:538-546, 1994 33. Rozga J, Podesta L, LePage E, et al: Control of cerebral edema by total hepatectomy and extracorporeal liver support in fulminant hepatic failure. Lancet 342:898-899, 1993 34. Nyberg SL, Peshwa MV, Payne WD, et al: Evolution of the bioartificial liver: the need for randomized clinical trials. Am J Surg 166:512-518, 1993 35. Bismuth H, Houssin D: Partial resection of liver grafts for orthotopie or heterotopic liver transplantation. Transplant Proc 17:279-283, 1985 36. Moritz M J, Jarrell BE, Armenti V, et al: Heterotopic liver transplantation for fulminaiat hepatic failure--a bridge to recovery. Transplantation 50:524-526, 1990 37. Gubernatis G, Pichlmayr R, Kemnitz J, et al: Auxiliary partial orthotopic liver transplantation (APOLT) for fulminant hepatic failure: First successful case report. World J Surg 15: 660-666, 1991 38. Shaw BW Jr, Cattral M, Langnas AN, et al: Orthotopic auxiliary liver transplantation: The treatment of choice for acute liver failure? Hepatology 18:66A, 1993 (abstr) (suppl) 39. McDonnell PJ, Toye PA, Hutchins GM: Primary pulmonary hypertension and cirrhosis: Are they.related? Am Rev Respir Dis 127:437-441, 1983 40. Plevak D, Krowka M, Rettke S, et al: Successful liver transplantation in patients with mild to moderate pulmonary hypertension. Transplant Proc 25:1840, 1993 41. De Wolf AM, Gasior T, Kang Y: Pulmonary hypertension in a patient undergoing liver transplantation. Transplant Proc 23:2000-2001, 1991 42. Cheng EY, Woehlck HJ: Pulmonary artery hypertension complicating anesthesia for liver transplantation. Anesthesiology 77:389-392, 1992 43. De Wolf AM, Scott VL, Gasior T, et al: Pulmonary hypertension and liver transplantation. Anesthesiology 78:213, 1993 44. Csete Prager M, Cauldwell CA, Ascher NL, et al: Pulmonary hypertension associated with liver disease is not reversible after liver transplantation. Anesthesiology 77:375-378, 1992 45. Scott V, De Wolf A0 Kang Y, et al: Reversibility of pulmonary hypertension after liver transplantation: A case report. Transplant Proc 25:1789-1790, 1993 46. Yoshida EM, Erb SR, Pflugfelder PW, et al: Singlelung versus liver transplantation for the treatment of porto-

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pulmonary hypertension--A comparison of two patients. Transplantation 55:688-690, 1993 47. Cohen MD, Rubin L J, Taylor WE, et al: Primary pulmonary hypertension: An unusual case associated with extrahepatic portal hypertension. Hepatology 3:588-592, 1983 48. Robalino BD, Moodie DS: Association between primary pulmonary hypertension and portal hypertension: Analysis of its pathophysiology and clinical, laboratory and hemodynamic manifestations. J Am Coll Cardiol 17:492-498, 1991 49. Ilkiw R, Todorovich-Hunter L, Maruyama K, et al: SC-39026, a serine elastase inhibitor, prevents museularization of peripheral arteries, suggesting a mechanism of monocrotaline-induced pulmonary hypertension in rats. Circ Res 64: 814-825, 1989 50. Wallwork J, Williams R, Calne RY: Transplantation of liver, heart, and lungs for primary biliary cirrhosis and primary pulmonary hypertension. Lancet 2:182-184, 1987 51. Maurer JR, Winton TL, Patterson GA, et al: Single lung transplantation for pulmonary vascular disease. Transplant Proc 23:1211-1212, 1991 52. Starzl TE, Groth CG, Makowka L: In Clio Chirurgica: Liver Transplantation. Austin, Texas, Silvergirl, 1988, p 168 53. Goldman E, Yablok D, Tesi RJ, et al: Analysis of two thrombokinetic measurements (thrombelastograph and sonoclot) during liver transplantation. Transplant Proc 25:1820, 1993 54. Csete ME: Operative hemostatic changes and their treatment, in Busuttil RW, Klintmalm GBF (eds): Liver Transplantation: Principles and Practice. Philadelphia, PA, Saunders, 1995, in press 55. Kang Y, Lewis JH, Navalgund A, et al: Epsilon-aminocaproic acid for treatment of fibrinolysis during liver transplantation. Anesthesiology 66:766-773, 1987 56. Dzik WH, Arkin CF, Jenkins RL, et al: Fibrinolysis during liver transplantation in humans: Role of tissue-type plasminogen activator. Blood 71:1090-1095, 1988 57. Porte RJ, Bontempo FA, Knot EAR, et al: Systemic effects of tissue plasminogen activator-associated fibffnolysis and its relation to thrombin generation in orthotopic liver transplantation. Transplantation 47:978-984, 1989 58. Porte RJ, Bontempo FA, Knot EAR, et al: Tissue-type plasminogen activator-associated fibrinolysis in orthotopic liver transplantation. Transplant Proc 21:3542, 1989 59. Bakker CM, Blankensteijn JD, Schlejen P, et al: The effects of long-term graft preservation on intraoperative hemostatic changes in liver transplantation. A comparison between orthotopic and heterotopic transplantation in the pig. HPB Surg 7:265-280, 1994 60. Green D, Ts'ao CH, Cerullo L, et al: Clinical and laboratory investigation of the effects of epsilon-aminocaproic acid on hemostasis. J Lab Clin Med 105:321-327, 1985 61. Adelmar B, Rizk A, Hanners E: Plasminogen interactions with platelets in plasma. Blond 72:1530-1535, 1988 62. Sloan IG, Firkin BG: Effects of EACA on thrombin generation as measured by the chromagen $2238. Thromb Res 44:761-769, 1986 63. Gralnick HR, Griepp P: Thrombosis with epsilon aminocaproic acid therapy. Am J Clin Path 56:151-154, 1971

108 64. Pitts TO, Spero JA, Bontempo FA, ct al: Acute renal failure due to high-grade obstruction following therapy with e-aminocaproic acid. Am J Kidney Dis 8:441-444, 1986 65. Stahl RL, Henderson JM, Hooks MA, et al: Therapy of the Kasabach-Merritt syndrome with cryoprecipitate plus intra-arterial thrombin and aminocaproic acid. Am J Hematol 36:272-274, 199t 66. Cart JM: Disseminated intravascular coagulation in cirrhosis. Hepatology 10:103-110, 1989 67. Gosseye S, van Obbergh L, Weynand B, et al: Platelet aggregates in small lung vessels and death during liver transplantation. Lancet 338:532-534, 1991 68. McSorley MW, Taraporewalla KJ: Does prophylactic epsilon-aminocaproic acid improve blood loss and coagulation in liver transplantation? Transplant Proc 23:1941, 1991 69. Pohorecki R, Landers DF, Peters RK, et al: Effect of e-aminocaproic acid on blood products usage in orthotopic liver transplantation--a double blind prospective study. Presented at the American Society of Transplant Surgeons Annual Meeting, 1993 70. Horrow JC, Hlavacek J, Strong MD, et al: Prophylactic tranexamic acid decreases bleeding after cardiac operations. J Thorac Cardiovasc Surg 99:70-74, 1990 71. Puigdellivol E, Carral ME, Moreno J, et al: Pharmacokinetics and absolute bioavailability of intramuscular tranexamic acid in man. Int J Clin Pharmacol Ther Toxicol 23: 298-301, 1985 72. Hardy JF, Desroches J: Natural and synthetic antifibrinolytics in cardiac surgery. Can J Anaesth 39:353-365, 1992 73. Roysten D: The serine antiprotease aprotinin (Trasylol): A novel approach to reducing postoperative bleeding. Blood Coagul Fibrinolysis 1:55-69, 1990 74. Lottenberg R, Sjak-Shie N, Fazleabas AT, et al: Aprotinin inhibits urokinase but not tissue-type plasminogen activator. Thromb Res 49:549-556, 1988 75. Himmelreich G, Dooijewaard G, Breinl P, et al: Evolution of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in orthotopic liver transplantation (OLT). Thromb Haemostas 69:56-59, 1993 76. Neuhaus P, Bechstein WO, Lefebre B, et al: Effect of aprotinin on intraoperative bleeding and flbrinolysis in liver transplantation. Lancet 2:924-925, 1989 77. MaUett S, Rolles K, Cox D, et al: Intraoperative use of aprotinin (Trasylol) in orthotopic liver transplantation. Transplant Proc 23:1931-1932, 1991 78. Himmelreieh G, Muser M, Neuhaus P, et al: Different aprotinin applications influencing hemostatic changes in orthotopic liver transplantation. Transplantation 53:132-136, 1992 79. Grosse H, Lobbes W, Frambach M, et al: The use of high dose aprotinin in liver transplantation: The influence on fibrinolysis and blood loss. Thromb Res 63:287-297, 1991 80. Patrassi GM, Viero M, Sartori MT, et al: Aprotinin efficacy on intraoperative bleeding and transfusion requirements in orthotopic liver transplantation. Transfusion 34:507511, 1994 81. Smith O, Hazlehurst G, Brozovic B, et al: Impact of aprotinin on blood transfusion requirements in liver transplantation. Transfusion Med 3:97-102, 1993

CSETE AND DE WOLF 82. Bhargava N, Dhasmana KM, Banerjee AK, et al: Aprotinin reduces hypothermic mortality--an experimental model. Lab Anim 22:223-228, 1988 83. Lie TS, Seger R, Hong GS, et al: Protective effect of aprotinin on ischemic hepatocellular damage. Transplantation 48:396-399, 1989 84. Morgan GR, Harvey PRC, Strasberg SM: Aprotinin for the pretreatment of liver allograft donors. Transplantation 49:1203-1204, 1990 (letter) 85. Baubillier E, Cherqui D, Dominique C, et al: A fatal thrombotic complication during liver transplantation after aprotinin administration. Transplantation 57:1664-1666, 1994 86. Center for Biologic Evaluation and Research. Points to consider in the safety evaluation of hemoglobin-based oxygen carriers. Transfusion 31:369-371, 1991 87. Looker D, Abbott-Brown D, Cozart P, et al: A human recombinant designed for use as a blood substitute. Nature 356:258-260 1992 88. Reiss JG: Overview of progress in the fluorocarbon approach to in vivo oxygen delivery. Biomat Art Ceils Immoh Biotech 20:183-202, 1992 89. Kessler DA, Siegel JP, Noguchi PD, et al: Regulation of somatic-cell therapy and gene therapy by the Food and Drug Administration. N Engl J Med 329:1169-1173, 1993 90. Challita PM, Kohn DB: Lack of expression from a retroviral vector after transduction of murine hematopoietic cells is associated with methylation in vivo. Proc Natl Acad Sci USA 91:2567-2571, 1994 91. Grompe M, Jones SN, Loulseged H, et al: Retroviralmediated gene transfer of human ornithine transcarbamylase into primary hepatocytes of spf and spf-ash mice. Human Gene Ther 3:35-44, 1992 92. Kay MA, Baley P, Rothenberg S, et al: Expression of human a l-antitrypsin in dogs after autologous transplantation ofretroviral transduced hepatocytes. Proc Natl Acad Sci USA 89:89-93, 1992 93. Bilheimer DW, Goldstein JL, Grundy SM, et al: Liver transplantation to provide low density lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia. N Engl J Med 311:1658-1664, 1984 94. Chowdhury JR, Grossman M, Gupta SJ, et al: Long term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR deficient rabbits. Science 254:1802-1805, 1991 95. Simon RH, Engelhardt JF, Yang Y, et al: Adenovirusmediated transfer of the CFTR gene to lung of nonhuman primates: Toxicity study. Human Gene Ther 4:771-780, 1993 96. Csete ME, Drazan KE, van Bree M, et al: Adenovirusmediated gene transfer in the transplant setting. 2. Conditions for expression of transferred genes in cold-preserved hepatocytes. Transplantation 57:1502-1507, 1994 97. Wilson JM, Grossman M, Raper SE, et al: Clinical protocol. Ex vivo gene therapy of familial hypercholesterolemia. Human Gene Ther 3:179-222, 1992 98. Rilo HR, Carroll PB, Shapiro R, et al: Effect of intraportal human islet transplantation on kidney graft survival in simultaneous kidney-islet allografts. Transplant Proc 25:955956, 1993

FUTURE DIRECTIONS IN LIVER TRANSPLANTATION 99. Evans MI, Adzick NS, Johnson MP, et al: Fetal thera p y - 1994. Curt Opin Obstet Gynecol 6:58-64, 1994 100. Starzl TE: Liver allo- and xenotransplantation. Transplant Proc 25:15-17, 1993 101. Starzl TE, Fung J, Tzakis A, et al: Baboon-to-human liver transplantation. Lancet 341:65-71, 1993 102. Starzl TE, Tzakis A, Fung JJ, et al: Prospects of clinical xenotransplantation. Transplant Proc 26:1082-1088, 1994 103. Makowka L, Wu GD, Hoffman A, et al: Immunohistopathologic lesions associated with the rejection of a pigto-human liver xenografl. Transplant Proc 26:1074-1075, 1994 104. Bach FH. Xenotransplantation: A view to the future. Transplant Proc 25:25-29, 1993 105. Calmus Y, Ayani E, Cardoso J, et al: Target antigens of hyperacute xenogeneic rejection in the rat to guinea pig and guinea pig to rat discordant combinations. Transplantation 56:778-785, 1993 106. Mafiez R, Kelly RH, Marino IR, et al: Complement activation correlates with graft damage in baboon-to-human liver xenotransplantation. Transplant Proc 26:1249-1250, 1994 107. Bailey LL, Nehlson-Cannarella SL, Concepcion W, et al: Baboon-to-human cardiac xenotransplantation in a neonate. JAMA 254:3321-3329, 1985 108. Porter KA: Pathological changes in transplanted kidneys, in Starzl TE (ed): Experience in renal transplantation. Philadelphia, PA, Saunders, 1964, pp 284-298

109

109. Murase N, Starzl TE, Demetris AJ, et al: Hamsterto-rat heart and liver xenotransplantation with FK506 plus antiproliferative drugs. Transplantation 55:701-708, 1993 1 t0. Tuso PJ, Cramer DV, Yasunaga C, el al: Removal of natural human xenoantibodies to pig vascular endothelium by perfusion of blood through pig kidneys and livers. Transplantation 55:1375-1378, 1993 111. Ohta K, Tanaka N, Orita K: Discordant liver xenograff transplantation. Transplant Proc 26:2435-2438, 1994 112. Valdivia LA, Fung JJ, Demetris AJ, et al: Donor species complement after liver xenotransplantafion. The mechanism of protection from hyperacute rejection. Transplantation 57:918-922, 1994 113. Stalzl TE, Demetfis AJ, Murase N, et al: Cell migration, chimerism, and graft acceptance. Lancet 339:1579-1582, 1992 114. Nardo B, Valdivia LA, Pan F, et al: Pretransplant xenogeneic blood transfusion combined with FK 506 prolongs hamster-to-rat liver xenograff survival. Transplant Proc 26: 1208, 1994 115. Valdivia LA, Demetris A J, Langer AM, et al: Dendritic cell replacement in long-surviving liver and cardiac xenografts. Transplantation 56:482-484, 1993 116. Chiche L, Adam R, Caillat-Zucman S, et al: Xenotransplantation: Baboons as potential liver donors? Scientific and ethical issues. Transplantation 55:1418-1421, 1993 117. Post SG: Baboon livers and the human good. Arch Surg 128:131-133, 1993