Potential conflict of interest: Nothing to report.
Previously we reported the presence of portal fibrosis in 31% (n = 84) of the grafts in protocol biopsies 1 year after pediatric liver transplantation (LTx). To assess the natural history of graft fibrosis after pediatric liver transplantation, we extended the analysis of graft histology in follow-up protocol biopsy specimens obtained 5 and 10 years after transplantation. We correlated histological results with clinical parameters at the time of LTx and during follow-up, to allow identification of risk factors for the development of fibrosis. From 1 year to 5 years after LTx, the prevalence of fibrosis increased from 31% to 65% (n = 66) but remained stable thereafter (at 10 years, 69%, n = 55). At 10 years after LTx, however, the percentage of patients with severe fibrosis had increased from 10% (at 5 years) to 29%. Of the 69% of children without fibrosis at 1 year post-transplantation, 64% (n = 39) had developed some degree of fibrosis at 10 years. Fibrosis was strongly related to transplant-related factors such as prolonged cold ischemia time, young age at the time of transplantation, high donor/recipient age ratio, and the use of partial grafts (P < 0.05). Fibrosis was not significantly related to rejection, chronic hepatitis, or the nature of the immunosuppressive therapy. Conclusion: Biopsies after pediatric LTx show that most grafts developed fibrosis within 5 years. At 10 years after LTx, the graft fibrosis had progressed to severe fibrosis in at least 25% of the patients. Development of fibrosis, starting either before or after the first year post-LTx, was strongly related to transplant-related factors, indicating the importance of these factors to long-term graft prognosis. (HEPATOLOGY 2008.)
Liver transplantation (LTx) has become the standard treatment for children with end-stage liver disease. Survival above 80% has been reported at 10 years after pediatric LTx.1 In contrast to adults,2 most pediatric liver transplant recipients do not have a primary liver disease with a high tendency of recurrence in the graft. Nevertheless, relatively little information is available on the long-term histology and function of the liver graft.
Evans et al.3 recently described a high prevalence of chronic hepatitis, associated with fibrosis, in protocol biopsies: 43% at 5 years and 64% at 10 years after pediatric liver transplantation. The authors speculated that this chronic hepatitis may be a hepatic form of chronic rejection.
Previously, we reported that in 31% of liver grafts at 1 year after pediatric LTx,4 portal fibrosis was present. Chronic hepatitis was not observed in these grafts. Transplant-related factors correlated positively with the presence of fibrosis, including cold ischemia time (CIT) and biliary complications. The long-term prognosis of portal fibrosis at 1 year after transplantation has remained unclear. Also, to what extent fibrosis develops after 1 year post-transplantation (“late” fibrosis) and whether “late” fibrosis is related to specific transplant-related or follow-up–related factors are not known. In the current study, we aimed to elucidate these questions. We evaluated graft histology in protocol biopsy specimens to 10 years after pediatric LTx and investigated possible relationships between transplant-related or post-transplantation parameters and progression of early fibrosis or development of “late” fibrosis.
In our center, 105 children underwent transplantation, with a total of 132 liver grafts from 1982 to 1996. We have performed annual protocol evaluations of all our patients since the start of our liver transplant program. The annual evaluation comprised clinical assessment, biochemical and serological screening, and protocol liver biopsies at 1, 2, 3, 5, and 10 years after transplantation. Previously we reported the histology of the available 1-year biopsy specimens (n = 84).4 Seven of these 84 specimens showed miscellaneous changes (rejection, viral infection, or vascular changes) and were excluded from evaluation. In the remaining 77 grafts, three major histological categories were observed: The first category showed normal histology, the second category showed reactive changes (minimal infiltrates in some portal tracts), and the third category showed a variable degree of portal fibrosis. The primary liver disease of the included patients is shown in Table 1.
Table 1. Primary Liver Disease of the Included Patients
Biopsies performed at 3, 5, and 10 years of the 77 grafts included in our previous study4 were reviewed by the same pathologist (A.S.H.G). Liver biopsies were performed percutaneously with a 16-gauge Menghini biopsy needle. Biopsy specimens were evaluated for the presence and severity of fibrosis. Mild reactive changes, rejection, hepatitis, and other abnormalities were also noted. Severity of fibrosis was evaluated using a four-point scale: 0 = no fibrosis, 1 = mild fibrosis, portal fibrosis without bridging, 2 = moderate fibrosis, portal fibrosis with occasional bridging, 3 = severe fibrosis/cirrhosis, diffuse portal bridging. Blood samples were taken concurrently with the liver biopsies for biochemical liver tests in relation to the severity of fibrosis and serological testing for cytomegalovirus, Epstein-Barr virus, and hepatitis B and C virus.
To identify predictive factors for the evolution of fibrosis, potential contributing factors in the donor and the recipient and parameters of the transplant procedure and follow-up were analyzed. In our previous study,4 the following independent risk factors were correlated with fibrosis at 1 year after LTx: CIT, biliary complications, cytomegalovirus status of the recipient, and the occurrence of an acute rejection episode. To these factors we added the following parameters, which we hypothesized could be related to fibrosis: recipient's age at the time of LTx, donor/recipient (D/R) age ratio, donor age, graft type, type of biliary reconstruction, and immunosuppressive scheme. Table 2 shows the demographic data of the included patients.
Table 2. Demographic Data
77 Liver Grafts
Abbreviations: ITBL, ischemic type of biliary lesions.
ABO Blood group incompatibility
Cold ischemia time (h)
Anhepatic phase (min)
Operation time (h)
University of Wisconsin
Cyclosporine azathioprine, prednisolone
Switched to tacrolimus
Acute rejection < 1 year
Acute rejection > 1 year
The major immunosuppression regimen for these patients was based on a triple therapy with cyclosporine (trough levels of 100-150 μg/L after the first month), prednisolone (after 3 months tapered to 0.25-0.4 mg/kg/2 days, alternate-day dosing), and azathioprine (2 mg/kg/day). Before 1988, an immunosuppression regimen was used in five children consisting of prednisolone (after 3 months tapered to 0.4-0.6 mg/kg/2 days, alternate-day dosing) and azathioprine (2.5 mg/kg/day). Cyclosporine was withdrawn in 44 patients after the evaluation at 2 years. These 44 patients continued with the immunosuppression regimen consisting of azathioprine and prednisolone in the dosages mentioned above.5, 6 Immunosuppression was switched to tacrolimus (trough levels of 4-6 μg/L) in cases of severe side effect(s) of cyclosporine, severe acute rejections, or chronic rejection. In this group, prednisolone was tapered to 0.1 mg/kg/day-1 Rejection in the first 4 weeks after transplantation was treated by intravenous bolus injections of methylprednisolone (20 mg/kg/day) on 3 successive days. Rejections after this 4-week period were treated with a temporary increase of the oral prednisolone dosage (4 mg/kg/day).
Data were analyzed using SPSS version 14. Continuous variables are presented as median with the range and categorical variables as number with percentages. Continuous variables were compared using nonparametric tests, or with Student t test if data were normally distributed. Categorical variables were compared using the Pearson's chi-squared test or the Fisher's exact test if suspected cell frequency was less than 5. For analysis of the association between the ordered fibrosis categories and the ranks of laboratory test, we used the Jonckheere-Terpstra test, a test for ordered alternatives, correcting for unequal numbers in groups. P < 0.05 was considered statistically significant.
In the original study of 77 included biopsy specimens at 1 year after LTx, 26 showed fibrosis of varying severity (Fig. 1). At 3 years after LTx, 64 of 77 (83%) of the biopsy specimens were available, and fibrosis was present in 31 (48%). At 5 years after LTx, 66 of 77 (85%) biopsy specimens were available, and fibrosis was present in 43 (65%). At 10 years after LTx, 55 of 77 (71%) biopsy specimens were available, and fibrosis was present in 38 (69%). At 10 years after LTx, 16 patients (29%) had developed severe fibrosis (grade 3). Thus, the prevalence of fibrosis increased from 31% to 65% of the biopsies between 1 year and 5 years after LTx but remained stable thereafter (at 10 years, 69%, n = 55). The severity of fibrosis progressed during follow-up (Fig. 1). At 10 years after LTx, the percentage of patients with severe fibrosis had increased from 10% (at 5 years) to 29%. The incidence of severe fibrosis after 10 years was significantly higher in the patients with fibrosis at 1 year after LTx (P = 0.004) compared with patients without fibrosis.
Of the 11 missing biopsy specimens at 5 years, six patients had died (four with cirrhosis), and two patients had a retransplantation (both with cirrhosis). In three patients, a biopsy was not performed at 5 years; however, biopsy specimens from each of the three patients were available at 10 years. Twenty-two samples were missing at 10 years after LTx: eight patients had died, five had undergone retransplantation, and nine patients did not undergo a liver biopsy, in most cases because they were referred to the adult hepatology department. Of the patients who died or had a retransplantation, 10 patients had cirrhosis and three patients had normal histology. Of the nine patients who did not undergo a liver biopsy at 10 years after LTx, all had a biopsy at 5 years after LTx, with the following results: no fibrosis in four, mild fibrosis in one, and moderate fibrosis in four. Analysis of the biopsy results at 5 years did not show a difference in the amount of fibrosis and the severity of fibrosis between the group with a biopsy at 10 years after LTx and the nine patients who did not undergo a biopsy at 10 years. If we combine the last biopsy results at 5 years of these nine patients with the 10-year biopsy results, we see fibrosis in 63% and severe fibrosis in 21% of the patients who are still alive and did not have a retransplantation at 10 years.
Correlation Between Biochemical Liver Tests and Progression of Fibrosis.
As stated, the results were obtained from protocol biopsies at indicated time points after LTx. The high incidence of (severe) fibrosis prompted the investigation of whether the presence and/or severity of fibrosis correlated with biochemical liver function indices obtained at the time of the biopsies. Table 3 shows the biochemical liver tests in relation to the presence and severity (grade) of fibrosis at 5 years and 10 years after LTx. Jonckheere-Terpstra analysis showed a significant upward trend for higher laboratory values with a more severe grade of fibrosis in most of the laboratory values [aspartate aminotransferase, alanine aminotransferase, and gamma glutamyl transferase (GGT) at 5 years, alkaline phosphatase and GGT at 10 years). This observation was most pronounced for GGT at 5 and 10 years. However, the results also indicated a considerable variation in each biochemical liver test among the three different categories. Thus, it was not possible to use the laboratory values to predict the severity of fibrosis.
Table 3. Biochemical Liver Tests and Histological Findings in the Liver Biopsies 5 Years and 10 Years After Transplantation
Fibrosis Grade 1 and 2
Fibrosis Grade 3
Abbreviations: AP: alkaline phosphatase, GGT: γ-glutamyl transferase, AST: aspartate aminotransferase, ALT: alanine amino transferase, Dir Bilir: direct bilirubin
Laboratory values median (range)
Significant upward trend (Jonckheere-Terpstra test, P < 0.05)
5 yrs after LTx Total group: n = 66, no fibrosis: n = 23, fibrosis grade 1 and 2: n = 36, Fibrosis grade 3: n = 7
10 yrs after LTx, total group: n = 55, no fibrosis: n = 17, fibrosis grade 1 and 2: n = 22, fibrosis grade 3 n = 16.
To evaluate the clinical significance of severe fibrosis in the biopsy specimens, we evaluated these patients for portal hypertension. As parameters for portal hypertension we used the number of thrombocytes and the spleen size by the abdominal ultrasound performed during the clinical review 10 years after transplantation. Nine of the patients with severe fibrosis (9/16, 56%) had an enlarged spleen, whereas in 13 of 38 of the patients with no or mild fibrosis an enlarged spleen was seen (34%, not significant). Median number of thrombocytes in the patients with severe fibrosis was 176 (44-323) × 109/L and 232 (44-562) × 109/L in the patients with no or mild fibrosis (not significant).
Predictive Factors of Fibrosis at 10 Years After Liver Transplantation.
We analyzed whether pre-transplantation–related, transplantation-related, and post-transplantation–related variables correlated with graft fibrosis at 10 years after pediatric LTx (Table 4). Post-transplantation–related (follow-up) factors such as rejection, biliary complications, and immunosuppression (with or without calcineurin inhibitors) were not significantly related to fibrosis. Further analysis of the three different immunosuppressive regimens showed that distribution of fibrosis in these three groups was almost the same (not significant).
Table 4. Results of Univariate Analysis of Risk Factors for Fibrosis in the 10-Year Biopsies After Pediatric LTx
All variables that significantly correlated with fibrosis at 10 years after LTx were transplant-related factors: young age at the time of LTx, high D/R age ratio, a long cold ischemia time, and the use of partial grafts (Table 4). In our previous study, biliary complications were related to the development of fibrosis, and an episode of acute rejection seemed to prevent against fibrosis at 1 year after LTx.4 However, these factors were not related to the presence of fibrosis at 10 years after LTx.
Predictive Factors of Late Fibrosis.
To evaluate whether different risk factors correlated with the development of fibrosis after the first year after LTx, we performed a subgroup analysis of the patients who developed fibrosis after the first year (“late” fibrosis). Fifty-one grafts did not have fibrosis in the first-year biopsies after LTx. Liver biopsy specimens from 39 of these patients were available at 10 years after LTx (71%). Most patients who did not have fibrosis at 1 year had developed fibrosis at 10 years [late fibrosis, 25/39 patients (64%)]. We evaluated whether predictive factors could be identified for the development of late fibrosis (Table 5). Late fibrosis was associated with the same transplant-related risk factors as were observed for the total group of patients in relation to the development of fibrosis in the 10-year biopsy specimens.
Table 5. Results of Univariate Analysis of Risk Factors for the Development of “Late” Fibrosis After Pediatric Liver Transplantation
Although all of these factors were significantly associated with fibrosis in univariate analysis, we realize that they may mutually be associated and interrelated. We tested these variables for mutual association in a correlation matrix. Most of the identified variables indeed proved to be interrelated. Young age was associated with a long CIT (P = 0.039) and a high D/R age ratio (P = 0.014) A high D/R age ratio was associated with the use of partial grafts (P = 0.04).
Graft Loss and Its Predictive Factors.
Graft loss in the first 10 years occurred in 13 of the original 77 patients. Eight patients had died, two of complications of cirrhosis, and two of complications of a retransplantation. Four died of other causes: two patients died of post-transplantation lymphoproliferative disease, one of bacterial sepsis and one of toxic epidermal necrolysis. Five patients successfully underwent retransplantation.
Fibrosis in the first-year biopsy correlated with retransplantation in the first 10 years after transplantation: five of the seven retransplantations (71%) had fibrosis in the first-year biopsies, compared with 21 of 70 (30%) of the patients without retransplantation (P = 0.04). Fibrosis in the first-year biopsy did not significantly correlate with mortality. The presence of biliary complications was associated with retransplantation. Six of the seven (85%) patients with a retransplantation had biliary complications, compared with 15 of 70 (21%) of the patients who did not have a retransplantation (P = 0.03). Predictive factors that were related to fibrosis after 10 years (CIT, age at the time of LTx, and the use of partial grafts) were not associated with retransplantation.
In this study we have shown a profound increase in the prevalence of fibrosis in pediatric grafts from 31% at 1 year after transplantation to almost 70% after 10 years, and an increase in the severity of fibrosis with a progression to severe fibrosis in more than 25% of the grafts after 10 years. Transplant-associated factors are related to the development of fibrosis, and even to the development of “late” fibrosis (that is, starting after the first year). Identified transplant-associated factors that have shown to be important include long cold ischemia time (CIT), young age at the time of transplantation, high D/R age ratio, and the use of partial grafts.
A high frequency of histological abnormalities (70%-90%) has been reported previously in long-term follow-up biopsies of adult recipients of liver grafts.7–9 However, these abnormalities were mostly related to the recurrent primary disease. Only limited data are available on long-term graft histopathology after pediatric liver transplantation. Rosenthal et al.10 investigated biopsies up to 3 years after transplantation and found only minor pathological changes: mild fibrosis in 8% of the biopsy specimens. Fouquet et al.11 described results of 10-year liver biopsies as part of a clinical follow-up study of patients with biliary atresia after LTx. These authors found normal histology in 27% of the biopsy specimens, which is comparable to our study. The major abnormalities found by Fouquet et al. were mild to moderate chronic rejection (41%) and mild centrolobular fibrosis (22%), which is different from our findings. However, no histopathological details are given in this study.
In the most comprehensive study of graft histology up to 10 years after pediatric LTx, Evans et al.3 reported (near) normal graft histology in 77% at 1 year, 61% at 5 years, and 31% at 10 years. At 10 years, Evans et al. observed fibrosis in 69%, of which 14% had already developed severe fibrosis. In some aspects, our data are in accordance with these: (near) normal graft histology in 66% at 1 year, 35% at 5 years, and 31% at 10 years. Also, our data on the incidence of fibrosis are similar, although in our patients we found more progression to severe fibrosis. We observed fibrosis in 69%, of which 40% had progressed to severe fibrosis at 10 years. Fibrosis in the study of Evans et al.3 was associated with an entity described as a “chronic hepatitis of unknown cause.” It was speculated that this chronic hepatitis could be a new presentation of chronic rejection in the grafted liver. We observed hepatitis in the graft histology of only four patients. One patient had hepatitis C. One had a recurrence of autoimmune hepatitis, as confirmed by positive auto-antibodies and elevated immunoglobulin levels. In the other two patients, no abnormalities could be found in the laboratory values. Thus, our results clearly differ from those obtained by Evans et al. An explanation for this observed difference could be that our different immunosuppression regimen, with a continued use of low-dose steroids in our cohort, prevented chronic hepatitis.
We attempted to identify the (severity) of fibrosis using biochemical liver functions. Laboratory values at 5 and 10 years indeed showed a significant relation between biochemical liver tests and grade of fibrosis. However, most of the liver tests were only mildly elevated, and even normal in most individual cases. Elevated GGT was the most consistent finding, which is compatible with biliary damage. However, we could not find clinical signs of biliary complications, such as abnormalities of the biliary tree on ultrasound or elevated bilirubin levels.
Earlier we had shown that fibrosis at 1 year was related to biliary complications, long CIT, positive cytomegalovirus status of the recipient, and a negative correlation with acute rejection.4 At 10 years after LTx, the presence of fibrosis correlated with a long CIT, young age at the time of LTx, high D/R age ratio, and the use of partial grafts. A subgroup analysis of the grafts without fibrosis at 1 year after LTx showed that fibrosis developed in 64% of these grafts. Even the development of “late” fibrosis was related to the same risk factors associated with the transplantation procedure as was seen for fibrosis at 10 years.
We have shown in this study that a long CIT contributes to the development of fibrosis in pediatric liver grafts. The biliary tree seems to be more vulnerable for a prolonged CIT than the liver parenchyma, which subsequently could lead to more ischemic damage and fibrosis in the portal tracts. A CIT above 11.5 hours is associated with the development of ischemic type of biliary lesions.12 Conversely, the contribution of a long CIT to fibrosis could be caused by confounding factors. For example, a long CIT could represent the cut-down procedure for preparation of partial grafts. Young age is related to the use of partial grafts and high ratio of D/R age. Partial grafts have to adjust to different blood flow and space after LTx. Partial grafts have to expand during growth, especially in small children. We cannot exclude that one or more of these factors is involved in the development of fibrosis, even after the first year after transplantation.
Our study also provides information on the clinical significance of the development and progression of fibrosis. Early fibrosis (developed in the first year) was associated with retransplantation. However, these retransplantations were mainly related to biliary complications, which was one of the risk factors in the development of fibrosis in the first year. Although long CIT, young age, and the use of partial graft are associated with the development of fibrosis in our study, the function of these grafts still seems adequate at 10 years. We do realize, however, that the progression of fibrosis toward cirrhosis could endanger graft function and graft survival in the longer term (>10 years after liver transplantation).
Theoretically, the results of our study might be biased by the missing histology results of nine patients who did not undergo a biopsy at 10 years after LTx. As we have shown, however, analysis of the 5-year biopsy specimens of these patients shows that the amount and severity of fibrosis at 5 years after LTx is similar to the patients who did have a biopsy at 10 years after LTx. If we would add the results of the 5-year biopsy of these nine patients to the available 10-year biopsy results (n = 55), the prevalence of fibrosis would be 63% and of severe fibrosis 21%. This slightly better outcome is still very similar to our original outcome.
We evaluated the possibility that the current results are influenced by the fact that many patients used an immunosuppressive scheme without calcineurin inhibitor. However, in our study, 1 year after LTx,4 we found portal fibrosis already present in 33% of the patients, who at that time all used cyclosporine. Furthermore, we did not find any significant difference in the prevalence of fibrosis in the patients with or without calcineurin inhibitor. And, finally, other studies,3, 11 have shown fibrosis in long-term biopsy specimens after pediatric liver transplantation.
In conclusion we have shown that transplant-related factors are associated with the development of fibrosis and cirrhosis of pediatric liver grafts in most patients. Liver function does not seem to be influenced by this development of fibrosis at 10 years after LTx. Our data do not show a strong impact of the development of fibrosis on graft survival up to 10 years after LTx. It is unclear whether severe fibrosis leads to graft failure and subsequent graft loss after this period.