What constitutes a functionally adequate graft? Volume and technical considerations
The main objective of LDLT is to provide adequate functional liver tissue which is estimated to be one third of standard liver mass of the recipient (>0.8% of the recipient's body weight). This volume is either expressed as the ratio of graft weight to recipient body weight (GRBW), or as graft weight as a percentage of standard liver mass. There is excellent linear correlation between these 2 values, and GRBW of 1% corresponds to approximately 50% of standard liver mass. This requirement is easily met in adult left lobe or left lateral segments (segments 2,3,4 and segments 2,3 respectively) intended for children or recipients under 60 kgs, but is a critical issue in recipients over 60 kgs, i.e. most adult recipients. In the initial days of right lobe LDLT, a GRBW of 1% was considered to be the ideal ratio; but current cumulative experience allows for a minimum GRBW of 0.8% for successful transplantation. Successful transplantation has been accomplished with smaller segments (GRBW of 0.6%) [4]; on the other hand, small-for-size syndrome has been reported with GRBW of 0.7% [5]. Host factors that affect regeneration of the liver would expectedly affect the minimum graft volume required for transplantation. These include the medical condition of the recipient, for e.g., a recipient with fulminant hepatic failure has additional stressors, and requires more graft volume than a medically stable recipient.

The graft volume is corrected for the degree of steatosis. In cadaveric grafts, the combination of steatosis and cold ischemia results in graft dysfunction due to microcirculatory disturbances and changes in cell membranes. While cold ischemia is not a significant problem in LDLT, the degree of steatosis reduces the functional mass; every 1% of steatosis reducing GRBW by 1% [6].

For the grafted liver segment to function, it should have adequate portal and arterial blood flow; and proper venous and biliary drainage. Right lobe allografts usually consist of segments 5-8; extended right lobes which consist of segments 4-8 increase the complexity of procedure for both donor and recipient, and are associated with more donor complications [6]. It has emerged that integrity of the middle hepatic vein which drains segments 5 and 8 is very important for regeneration and function of the graft. Many surgical techniques have evolved to preserve this vein using vein grafts and fashioning them on the back table. The integrity of segment 4, on the other hand, is very important for regeneration of the liver remnant in the donor. There is also a concerted effort to refine biliary reconstruction and reduce the almost 30% incidence of biliary complications [1, 6] .

Rarely, other segments of the liver have been used for LDLT: whole left lobe (segments 1-4), right posterior segments (S6, S7) or 2 left lateral segments (S2, S3). The inclusion of segment 1 in whole left lobe grafts (segments 1-4) adds 9% to the graft volume and 3% to GRBW. A right posterior lobes graft (S6 and S7) provides larger volume than the left liver graft (S2, 3, 4) and accomplishes complete venous drainage through the right hepatic vein. However, harvesting of these lobes is technically very difficult [1].

Utility of donor biopsy in LDLT
The role of liver biopsy as a mandatory step in donor evaluation and selection for LDLT is uncertain at the present time, a controversy centered on the relative risks vs. benefits of this invasive procedure [7, 8, 9] . A survey of transplant centers performing LDLT showed that 14% of centers performed a liver biopsy in all donors and 26% not in any [3] (Brown).

The major utility of donor liver biopsy is in the evaluation for steatosis. The accuracy of imaging studies in predicting hepatic steatosis is perhaps a reflection of the specific imaging modalities used [7, 8, 10] . Additionally, BMI does not adequately predict degree of hepatic steatosis [7, 10] . In one study, 15 of 89 potential donors were excluded on the basis of liver biopsy that showed significant steatosis [6]. A significant numbers of donors with a high BMI demonstrate <10% hepatic steatosis [7, 10] . It is however generally agreed that donors with BMI<25, normal liver function tests, normal lipid profile, and no history of diabetes, hypertension or history of excess alcohol consumption can be safely excluded from requiring a biopsy [8, 10] . On the other hand, most centers would perform a liver biopsy on donors with BMI >25 who are otherwise eligible for donation [8, 11] .

Confusing the role of liver biopsies as a standard modality of evaluation is the presence of histological changes in the majority of donors, some of which are of uncertain significance. At least 2 studies have shown the presence of histological findings in 38% and 73% of biopsies from donors with normal biochemistry; these findings included granulomas, minimal portal inflammation ("triaditis"), chronic hepatitis, fibrosis, variable degree of steatosis, steatohepatitis, sinusoidal dilatation and iron deposition [7, 9] . Obviously, not every histological deviation merits disqualification of the donor. However, in the study by Ryan et al, 3 donors were excluded on the basis of their histological findings; 2 patients had chronic hepatitis with fibrosis and 1 had an abnormal vascular pattern [7]. In the series by Tran, 3 biopsies showed >30% steatosis, 1 showed steatohepatitis and 4, chronic hepatitis; all had normal liver function tests [9].

A definite role for liver biopsy exists in evaluation of living donors for a recipient with Alagille's syndrome, an autosomal dominant condition with variable penetrability, in which close relatives may express limited manifestations of the syndrome. In one series of transplantation for Alagille's syndrome, 4 of 11 donors showed bile duct hypoplasia by magnetic resonance cholangiopancreatography in the form of decreased diameter of bile ducts. Bile duct paucity was confirmed by subsequent biopsy [12]. There are reports of aborted operations in asymptomatic donors with normal liver function tests and unsuspected hepatic manifestations of Alagille's syndrome [13]. Another report describes implantation of a segmental graft inspite of intraoperative diagnosis of bile duct paucity. A porto-enterostomy was performed but the recipient developed cholestasis and died of sepsis [12]. Living donor transplantation for Alagille's syndrome may thus constitute an absolute indication for donor liver biopsy. Another indication for donor liver biopsy in LDLT includes elevated serum ferritin levels [14].

Post-transplantation biopsy pathology in LDLT
Biopsies of right lobe grafts performed to rule out rejection due to elevated liver enzymes in the early post-operative period often show a combination of findings that include centrilobular hepatocyte ballooning, cholestasis and biliary proliferation, in the absence of rejection or mechanical obstruction to the bile duct. In one study, bile duct proliferation with cholestasis and cholate injury, and without mechanical obstruction of the biliary tree was found in 29% of patients with right lobe LDLT vs. 2% of those who received cadaveric organs [15]. Similarly, centrilobular hepatocyte ballooning and cholestasis were found in 13 of 25 biopsies from reduced grafts in another study [16] . In a separate series of 9 patients who received right lobe grafts, 5 biopsies performed to rule out acute cellular rejection showed centrilobular hepatocyte ballooning, canalicular cholestasis and biliary proliferation. There was no inflammation, necrosis or acute cellular rejection. Two of these patients had bile leaks, 1 had a stricture and the remaining 2 responded to antibiotics for cholangitis [17].

Evidence of venous outflow obstruction in the form of sinusoidal dilatation, centrilobular spotty necrosis and central venous fibrosis (1 case) was found in 18% of patients who received right lobe LDLT; this finding was not present in patients who received cadaveric organs [15]. This finding might suggest insufficient venous drainage in the regenerating liver.

Graft loss due to massive vascular engorgement and hemorrhage in zones 1 and 2 has been described in a patient who had severe portal hypertension before transplantation, and that probably resulted from massive portal flow in a relatively small graft. The patient subsequently received a cadaveric organ [17].

There appears to be decreased incidence of rejection in recipients of right lobe LDLT [5, 15, 18, 19] .A study of 48 pairs of patients matched for timing of transplantation, age, gender and cause of chronic liver disease found a statistically significant difference in the rate of acute cellular rejection between LDLT from related donors (sibling, parent, child) when compared to cadaveric donors. This difference was not present when the living donor was not genetically related i.e. related by marriage, friends. In the group of related LDLT, rejection tended to occur within the first 3 months and/ or 1 year following transplantation [18]. In the series from Marcos, none of the biopsies performed for elevation of liver function tests showed rejection, clinically significant rejection not being found either in the earlier phase of rapid proliferation of the graft or the later slower phase of regeneration [5].

Hepatitis C
End-stage liver disease due to hepatitis C (HCV) infection is the commonest indication not only for cadaveric liver transplantation but for right lobe LDLT as well. While it is known that hepatitis C infection recurs universally in the allograft, several studies indicate that the infection recurs earlier and is more severe in right lobe grafts than cadaveric allografts [20, 21] . A separate study identified a risk for developing cholestatic hepatitis in patients who had received right lobe grafts. None of the patients who received cadaveric organs developed this syndrome which was defined as serum total bilirubin greater than 10 mg/dL, with ductular proliferation and bile stasis on liver biopsy and without evidence of extraheptic biliary obstruction [19]. This syndrome is characterized by intense viral replication in serum and liver. It is believed that the actively proliferating hepatocytes offer replicative advantages to the virus. Upregulation of IRES (internal ribosome entry site) and LDL receptor in proliferating hepatocytes probably plays an important role, as the former is involved in viral translation and the latter in viral entry into hepatocytes through endocytosis of the LDL receptor [20].

On the other hand, there are factors specific to right lobe LDLT that are expected to slow down recurrence of hepatitis C infection. These include (1) lower requirement for immunosuppression in this group of patients (2) easier weaning from immunosuppression [22] (3) younger age of donors and (4) better pre-transplantation control of hepatitis C in the recipient [20].

Cadaveric grafts from donors with hepatitis C have been used for recipients with end-stage liver disease without significant adverse effects on outcome. However, positive serology for HCV is a contradiction to LDLT as it can be expected to complicate any future surgical intervention that the donor may require. Additionally, regeneration in the donor remnant may accelerate the disease process [6].

Hepatitis B
After the initial disastrous results of liver transplantation for hepatitis B induced end-stage liver disease, adequate control of the disease has been achieved through lamuvidine and HBIg, making liver transplantation a viable option for this condition. The use of partial grafts from living donors is tricky since the proliferative activity of hepatocytes can be expected to offer replicative advantage to the virus as well leading to its reactivation. However, it has been reported that the use of lamuvidine and HBIg in right lobe LDLT, as in cadaveric transplantation, can effectively control recurrence of disease. The fact that these patients require less immunosuppression may additionally abate recurrence. Being an elective procedure, LDLT can be to be put off until adequate control of viral DNA titres has been achieved as this strategy offers a distinct advantage towards preventing recurrence in the graft [23].

An important issue in LDLT for hepatitis B is the use of anti-HBc positive donors. The anxiety results not only from the risk of recurrence in the recipient, but also a similar risk to the donor with an actively replicating liver. While evidence of prior exposure to hepatitis B may be considered a contraindication to donation in the United States [6, 24] , this would significantly affect the donor pool in other regions where hepatitis B is endemic. A study addressing this issue comes from Hong Kong where prevalence of HBV carriers is 10% and 33.3% of the population has had past exposure as evidenced by positivity for anti-HBc in the absence of HBsAg positivity. Since hepatitis B infection tends to cluster in families, 54% of living donors were positive for anti-HBc so that elimination of these donors would have significantly undermined the rationale of the program. Luckily, use of grafts from these anti-HBc positive donors did not demonstrate any difference in post-operative AST levels and prothrombin time between donors who were positive for anti-HBc and those who were not; however bilirubin levels at days 6 and 7 were significantly higher in donors who were anti-HBc positive, but this difference did not persist at 1 month. The anti-HBc positive donors were older and the impact of this factor on the presence of cholestasis is not clear. More importantly however, these donors did not experience clinical episodes of hepatitis or liver disease at a mean follow-up of 32 months (range 21-76 months) [25].

Graft function in right lobe living donor allografts
Recipients who receive partial grafts experience persistent hyperbilirubinemia following transplantation, even when the graft size represents 60% of expected liver volume. The biopsy findings are those of ballooning change and parenchymal cholestasis [5, 26] . The bilirubin levels however, normalize within 1 week and are subsequently no different from that seen in cadaveric grafts. Donors have normal bilirubin levels at 1 week post-transplantation. No significant differences are observed in levels of ALT and AST or prothrombin time between donors, recipients of partial grafts and recipients of cadaveric grafts. Levels of factor VII were significantly different between recipients of cadaveric grafts vs. those of partial grafts and donors for the first 3 days post-transplantation but normalized thereafter [5].

Proliferation in right lobe living donor allografts
LDLT takes advantage of the legendary regenerative capacity of the liver, a truly unique biological phenomenon in which a mature, terminally differentiated organ not only increases its volume exponentially, but also miraculously stops once optimal volume is achieved. Donors for LDLT are the first group of completely healthy individuals to undergo major hepatic resection and recipients, especially of right lobes, the first group of patients who receive organs that are significantly smaller than normal. In essence, LDLT is an elective technically complicated surgical procedure carried out in a perfectly healthy individual to benefit another that is sick. So, what secrets has this experiment in normal human regeneration, that tests the very limit of "primum non nocere", revealed so far?

Several studies show that liver regeneration begins almost immediately after transplantation and near-normal volumes are reached within 7 -14 days [5, 27, 28] . In a 16 year old Japanese male who received an extended left lobe (361 gm, 0.59% of body weight, 30.3% of standard liver volume) for fulminant hepatic failure, the PCNA labeling index was 50% on post-operative day 3. HGF was expressed in non-parenchymal cells; c-met in the cytoplasm and EGFR on the cell membranes of regenerating hepatocytes in this liver biopsy obtained on the third post-operative day. TGF-a was negative. At 2 weeks, regeneration was 195% in the recipient and 110% in the donor [27]. A separate study showed rapid increases in serum HGF and IL-6 in recipients of right lobe grafts, reaching peak levels 3 hours after reperfusion and then declining rapidly at 6 hours to reach pre-operative levels on the second post-operative day. Levels of TGF-b1 did not show any significant change for the 5 days of post-operative follow-up [29].

A study using imaging techniques to observe hepatic regeneration found a rapid increase in the first week following transplantation (101% increase in donors and 87% in recipients); the peak regeneration in donors was 144% at 60 days, and 119% in recipients at 30 days [5]. In a study of 47 follow-up CT scans of 10 donors and 8 recipients taken for a period of 8 months – 14 months, it was observed that the liver regenerated in the immediate post-operative period. The process was more rapid and reached a higher peak in the recipient than the donor; the liver volume peaking in donors at 6 months (74% increase) and at 2 weeks in recipients at 120%. The difference in volume was statistically significant between these 2 groups at 10 days, 1 month and 2 months post-transplant but not after 2 months [28]. Similarly, a study on left lobe adult donors showed more rapid regeneration in pediatric recipients than their donors [30]. This observed difference in regeneration rates between donors and recipients has not been reported by all investigators. Furthermore, the underlying mechanisms are uncertain but may involve pro-proliferative action of immunosuppressive drugs, or increased portal flow in the recipient due to a persistent hyperdynamic state or denervation of the graft. Needless to say, factors affecting regeneration are multifactorial and include medical condition of the recipient, warm ischemia time, immunosuppression, steatosis and donor age [31].

In a histopathological study of liver biopsies from recipients of right lobe allografts, it was observed that the restoration of liver volume observed by imaging studies occurs by proliferation of mature hepatocytes as seen by an increase in the labeling index of MIB-1. The difference in the labeling index between right lobe allografts and that of whole organ allografts was found to be statistically significant within the first 2 months but not after that period, confirming that most of the regeneration occurs in the immediate post-operative period. The proliferation of hepatocytes is accompanied by a statistically significant decrease in the number of portal tracts in biopsies obtained from patients who have received right lobe grafts when compared with those who have received cadaveric organs [32]. This observation raises questions about the integrity of the hepatic microarchitecture and function over the long-term. These answers will undoubtedly be provided by LDLT donors and recipients in the decades to come.

References

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General reviews on LDLT:

33. Ito T, Kiuchi T, Egawa H et al. Surgery-related morbidity in living donors of right-lobe liver graft: lessons from the first 200 cases. Transplantation 2003; 76: 158-63.
34. Ghobrial RM, Amersi F, Busuttil RW. Surgical advances in liver transplantation. Living related and split donors. Clin Liver Dis 2000; 4: 553-65.
35. Shiffman ML, Brown RS Jr., Olthoff KM et al. Living donor liver transplantation: Summary of a conference at the National Institutes of Health. Liver Transpl 2002; 8: 174-88.
36. Miller C. Living donor liver transplantation – overview after 178 cases. Transplant Proc 2003; 35: 964-5.
37. Chen C, Fan S, Lee S et al. Living-donor liver transplantation: 12 years of experience in Asia. Transplantation 2003; 75 (S): S6-11.
38. Lo C. Complications and long-term outcome of living liver donors: A survey of 1,508 cases in five Asian centers. Transplantation 2003; 75 (S): S12-5.