Study population
Three hundred fifty-four liver transplantations were performed on 326 adults by the Queensland Liver Transplant Service between January 1985 and December 1998. Twenty-eight retransplantations and 44 primary liver transplant recipients without cirrhosis were excluded from the analysis, resulting in a study population of 282 subjects. The etiology of the cirrhosis and indication for OLT were confirmed by review of explant histology and patients' medical records. Records were reviewed for factors known to affect body iron stores, including a history of portosystemic shunt surgery, chronic hemolysis, phlebotomy, gastrointestinal bleeding, and red blood cell transfusions. Written informed consent was obtained from each patient and the study was approved by the Princess Alexandra Hospital Research Ethics Committee.
Hepatic iron assessment
Liver explant iron stores were qualitatively assessed and graded (0-4) following Perls' Prussian blue stain by a single hepatopathologist (A.C.) according to the method described by Searle et al.24 Quantitative hepatic iron measurements were performed on 102 of the 104 explant livers with stainable iron. Paraffin-embedded specimens were deparaffinized by heating to 80°C followed by immersion in xylene (twice for 10 minutes) followed by sequential washes in 100% ethanol (2 minutes) and deionized water (1 minute). Deparaffinized specimens were dried for 72 hours at 65°C, weighed and acid-digested with Aristar grade nitric acid (200 µL) in a sand bath until the digest solution was clear and colorless (approximately 3 to 5 hours). After cooling, specimens were transferred to acid-washed 5-mL volumetric flasks and made up to volume with deionized water. Iron standards 50 to 400 µg Fe/100 mL were prepared by a dilution of iron standard solution (1 g/L; Fluka Chimika/Biochimika, Buchs, Switzerland) in 0.5% hydrochloric acid (Aristar grade). Hepatic iron concentration (HIC) was measured using an atomic absorption spectrophotometer (Varian AAS 4) at a wavelength of 248.3 nm in an air-acetylene flame with deuterium background correction. Linear regression analysis was performed and the concentration of iron in the experimental samples was calculated from the standard curve and expressed as µmol/g dry weight. HIC for each subject was reported as the mean hepatic iron concentration obtained from a minimum of 3 random sites. In view of the previously recognized substantial intraorgan variability of hepatic iron stores in the presence of cirrhosis,25,26 30 patients with marked differences between the initial 3 HIC measurements (defined as HIC standard deviation greater than 20 µmol/g and in whom the initial mean HIC value was near to or above 40 µmol/g) had a further 5 samples analyzed. Iron-loaded rat liver (160 µmol/g dry weight) was included in each assay as a control. A preliminary examination of 25 patients with no stainable hepatic iron showed that all had an HIC less than 40 µmol/g. Patients were separated into 3 groups based on their mean HIC: less than or equal to 40 µmol/g, 41 to 79 µmol/g, or greater than or equal to 80 µmol/g. The separation of patients into these 3 groups was based on published references ranges for HIC.27,28 Normal hepatic iron concentrations are less than 40 µmol/g for men and women.27 Values between 40 and 80 µmol/g are regarded as abnormal, however, patients with hereditary hemochromatosis usually have HIC measurements greater than 80 µmol/g.28 The hepatic iron index (HII) was calculated by dividing the HIC (µmol/g dry weight) by the age (years) of the patient.27
Laboratory investigations
Serum ferritin concentration and transferrin saturation measured on fasting pretransplantation specimens were available in 239 and 241 patients, respectively. Serum 
2 microglobulin levels were determined by immunoassay (DAKO, Glostrup, Denmark) on stored pretransplantation serum in 68 of the 104 patients with stainable hepatic iron. The presence of spur cell anemia was defined as the presence of significant acanthocytosis (
20%) on peripheral blood film and a serum hemoglobin level of less than 10.5 g/dL in the absence of any other identifiable cause of anemia.8,29 The Child-Pugh score calculated at the time of liver transplantation was used as a measure of liver disease severity.30
Genotyping of the HFE mutations and HLA status
HLA typing was performed before OLT using standard microlymphocytoxicity assays on DNA extracted from peripheral blood lymphocytes in 277 patients. HFE mutation status was determined in 79 of the 104 patients with positive hepatic iron staining. Mutational analysis on DNA extracted from buffy coats was performed as previously described,31 in all 79 subjects using polymerase chain reaction amplification of exon 4 (C282Y) and exon 2 (H63D) followed by restriction enzyme digestion using SnaB1 and DpnII, respectively.
Resource utilization
Resource utilization was defined by assessing the length of stay in the intensive care ward, duration of mechanical ventilation, and total hospital stay. Intensive care ward stay was defined from anesthesia start time for OLT until the patient's departure from the intensive care ward. Subsequent visits to the intensive care ward were not included in the calculation. Ventilator time was defined from anesthesia start time until the time of extubation or spontaneous respiration in the event of a tracheostomy. Length of hospital stay was defined as the total duration of hospitalization in the posttransplantation period. Pretransplantation hospitalization and intensive care ward admissions were not included in the analysis. Data retrieved from medical records were complete in 279 of 282 patients.
Statistical evaluation
Normally distributed variables were expressed as mean ± standard deviation, and the mean HIC between groups was compared using analysis of variance. Nonparametric tests (e.g., Kruskal-Wallis and Mann-Whitney) were used to compare the medians of continuous variables that were not normally distributed between groups. Crude survival outcomes were calculated by Kaplan-Meier survival analysis. The Cox proportional hazard method was applied to evaluate patient survival after adjusting for potentially confounding variables. Comparisons between proportions were made by Pearson's 
2 analysis.
Clinical and laboratory data
The causes of cirrhosis in the 282 patients grouped by the presence or absence of stainable hepatic iron are outlined in Table 1.
Table 1. Comparison of Diagnoses in the 282 Patients With Cirrhosis Undergoing Liver Transplantation Classified by Presence of Stainable Hepatic Iron
 | Positive Iron Staining | Negative Iron Staining |
| Hepatocellular diseases (n = 156; 55%) | | |
| ALD (n = 43) | 28 (65%) | 15 (35%) |
| Hepatitis C cirrhosis (n = 29) | 18 (62%) | 11 (38%) |
| HCV + ALD (n = 12) | 10 (83%) | 2 (17%) |
| Hepatitis B cirrhosis ± HDV (n = 9) | 5 (56%) | 4 (44%) |
| Autoimmune hepatitis (n = 18) | 2 (11%) | 16 (89%) |
| Cryptogenic-hepatocellular (n = 23) | 12 (52%) | 11 (48%) |
| Hereditary hemochromatosis (n = 7) | 7 (100%) | 0 (0%) |
| Wilson's disease (n = 8) | 8 (100%) | 0 (0%) |
 1AT deficiency (n = 4) | 2 (50%) | 2 (50%) |
| Hepatocellular cirrhosis, other (n = 3) | 2 (67%) | 1 (33%) |
| Cholestatic diseases (n = 126; 45%) | | |
| Primary biliary cirrhosis (n = 44) | 7 (16%) | 37 (84%) |
| Primary sclerosing cholangitis (n = 53) | 1 (2%) | 52 (98%) |
| Biliary cirrhosis, other (n = 29) | 2 (7%) | 27 (93%) |
| Total number of patients (n = 282) | n = 104 | n = 178 |
Abbreviations: ALD, alcohol-induced liver disease; HCV, hepatitis C virus; HDV, hepatitis D virus; 1AT, -1-antitrypsin |
Hepatocellular cirrhosis was the principal diagnosis in 156 patients (55%) and cholestatic liver disease was the primary indication for OLT in 126 patients (45%). One hundred four of the 282 subjects (37%) with cirrhosis had evidence of stainable iron in their explant liver. Sixty percent of patients (94 of 156) with hepatocellular cirrhosis had stainable iron in their explant liver, whereas only 8% of patients (10 of 126) with cholestatic liver diseases showed positive hepatic iron staining (P < .001).
Of the 104 patients with stainable hepatic iron, 36 (35%) had grade 1, 41 (39%) had grade 2, 16 (15%) had grade 3, and 11 (11%) had grade 4 hepatic iron stores. Two subjects with grade 2 iron on whom liver tissue was not available for measurement of HIC were excluded from the analysis. Fourteen (14%) subjects had a mean HIC 80 µmol/g, 21 (21%) had an HIC between 41 and 79 µmol/g, and the remaining 67 (65%) had an HIC 
40 µmol/g. The 245 patients categorized with an HIC of 40 µmol/g included 178 subjects with negative Perls' staining and the 67 subjects described above.
Demographic and laboratory characteristics of the 280 patients with cirrhosis in relation to their HIC are shown in Table 2.
Table 2. Clinical and Laboratory Characteristics of Patients With Cirrhosis in Relation to Hepatic Iron Concentration
| Hepatic Iron Concentration (µmol/g) |
| 40 | 41-79 | 80 | P Value |
| Number of patients (n = 280)* | 245 | 21 | 14 | |
| Gender (M:F) | 121:124 | 15:6 | 10:4 | .05 |
| Age (yr)† | 43.49 ± 12.72 | 46.67 ± 8.78 | 47.07 ± 11.34 | .33 |
| Child-Pugh score† | 8.81 ± 2.48 | 10.75 ± 2.22 | 10.86 ± 1.96 | <.001 |
| Serum creatinine (mmol/L) | 0.10 ± 0.07 | 0.11 ± 0.08 | 0.09 ± 0.03 | .412 |
| Cirrhosis type (cholestatic:hepatocellular) | 125:120 | 0:21 | 1:13 | <.0001 |
| Transferrin saturation (%)† | 37.3 ± 25.82 | 59.4 ± 24.49 | 81.5 ± 15.27 | <.0001 |
| Serum ferritin concentration (ng/mL)‡ | 110(8-2600) | 480(185-2980) | 1600(455-2440) | <.0001 |
| Spur cell anemia | 5/245 (2.2%) | 1/21 (4.8%) | 8/14 (57%) | <.0001 |
NOTE. Laboratory reference ranges for transferrin saturation, serum ferritin concentration, and serum creatinine are 15% to 45%, 10 to 200 ng/mL, and 0.07 to 0.12 mmol/L, respectively.
*Data on 2 patients with grade 2 hepatic iron on whom HIC was not performed are not included in the table.
† Values are expressed as mean ± standard deviation.
‡ Values are expressed as median and range. |
The mean age at OLT of patients with increased hepatic iron (i.e., 41-79 µmol/g and 80 µmol/g) was not significantly different from the 245 patients with an HIC 40 µmol/g (P = .33). Sixty-six percent of the 104 patients with stainable hepatic iron were men and 81% were Caucasian with the Japanese patients being the second largest ethnic group (13%) reflecting the referral pattern of the Queensland Liver Transplant Service. There were significant differences in the mean transferrin saturation and median serum ferritin concentration between the 3 HIC groups (P < .0001 and P < .0001, respectively).
Increased hepatic iron concentration was associated with a higher Child-Pugh score (P < .001), male gender (P = .05), and hepatocellular liver disease (P < .0001) on univariate analysis. Thirty-four of the 35 patients (97%) with an HIC greater than 40 µmol/g had an underlying hepatocellular etiology for their cirrhosis. Hepatocellular cirrhosis remained significantly associated with HIC after adjustment for Child-Pugh score at the time of OLT (P = .0018). Of the 282 patients, 58 (21%), 99 (35%), and 124 (44%) patients had Child-Pugh class A, B, and C cirrhosis, respectively, at the time of OLT. There was a significant difference in hepatic iron stores (nil = 0; mild = grade 1 or 2; Severe = grade 3 or 4) between the 282 patients when grouped by Child-Pugh class (Table 3).
Table 3. Relationship of Severity of Liver Disease and Hepatic Iron Stores in the 282 Patients With Cirrhosis
| Child-Pugh Class |
| A (n = 58) | B (n = 99) | C (n = 124) |
| Nil iron (grade 0) | 47 (81%) | 83 (84%) | 48 (38%) |
| Mild iron loading (grade 1 or 2) | 9 (16%) | 13 (13%) | 54 (44%) |
| Severe iron loading (grade 3 or 4) | 2 (3%) | 3 (3%) | 22 (18%) |
NOTE. Child-Pugh class was not calculated in one patient because of missing data. |
Eighteen percent of subjects with class C cirrhosis had severe iron loading compared with 3% of subjects with class A or B cirrhosis (P = .008) whereas 44% of patients with class C cirrhosis had mild iron loading compared with 13% and 16% of subjects with class A and B cirrhosis, respectively (P < .0001).
Hepatic iron analysis
The median dry weight of deparaffinized liver specimens for HIC determination was 2.7 mg (range, 0.8-7.6 mg) and the median HIC coefficient of variation was 28% (range, 1%-76%). There was no significant difference in the median HIC coefficient of variation in each of the 3 HIC groups with values of 29%, 26%, and 26% for 40 µmol/g, 41 to 79 µmol/g, and 80 µmol/g, respectively (P = .29). The intraorgan variability of HIC measurements quantified by standard deviations of the HICs varied widely across livers, ranging from 7 to 99 µmol/g (median, 31 µmol/g) for livers with a mean HIC value of greater than 80 µmol/g and from 1 to 37 µmol/g (median, 15 µmol/g) for livers with a mean HIC value between 41 and 79 µmol/g. The difficulty of calculating accurate HICs in cirrhosis is emphasized by the fact that in 22 patients, the HIC was 80 µmol/g in at least one liver section; however, only 14 patients were included in the 80 µmol/g HIC group. Similarly, 15 patients had a mean HII greater than 1.9; however, an additional 4 patients had at least one liver section where the HII exceeded 1.9. The extra HIC measurements in the 30 patients with marked variability of hepatic iron resulted in only minor changes in any individual's mean HIC measurement and did not change the grouping of any subject.
In the 27 patients with grade 3 or 4 stainable iron, deposition was greatest in hepatocytes; however, significant amounts were also seen in Kupffer cells. There was variable iron deposition in septal macrophages, biliary epithelium, and vessels. In 7 cases, there was heavy staining (3+) in proliferating bile ductules, which was not seen in the anatomical bile ducts, suggesting differences in uptake mechanisms between these 2 structures. Iron distribution was uniform throughout the nodules in 63% (17 of 27) of the livers and the remainder showed variation within and between the parenchymal nodules.
Pathogenesis of hepatic iron accumulation
Spur cell anemia was significantly associated with increased hepatic iron concentration (P < .0001). Eight of the 14 (57%) patients with an HIC 80 µmol/g had spur cell anemia compared with 5 of 245 (2.2%) patients and 1 of 21 (4.8%) patients with an HIC 40 µmol/g and between 41 and 79 µmol/g, respectively (Table 2
). Ten of the 14 (71%) patients with spur cell anemia had alcohol-induced cirrhosis and the remaining 4 patients had primary biliary cirrhosis, extrahepatic biliary atresia, hepatitis C cirrhosis, and cryptogenic cirrhosis. Many patients had acanthocytes identified in their peripheral blood on at least one occasion but failed to fulfill the requirements for a diagnosis of spur cell anemia.
Serum 2 microglobulin levels were normal or slightly increased in all 68 patients on whom pretransplantation serum was available for analysis. There was no significant difference in the serum 2 microglobulin concentration between HIC groups with mean values of 4.5 mg/L, 2.8 mg/L, and 4.11 mg/L (reference range, 1-3 mg/L) for 40 µmol/g, 41 to 79 µmol/g, and 80 µmol/g, respectively (P = .1). Similarly, the frequency of the HLA-A3 allele was not significantly different between HIC groups (22%, 14.3%, and 35.7%, P = .324).
Of the 79 subjects on whom HFE mutational analysis was performed, 7 had a pretransplantation diagnosis of hereditary hemochromatosis on the basis of an elevated serum transferrin saturation and serum ferritin concentration associated with an HII greater than 1.9 on pretransplantation liver biopsy. Clinical and genotypic characteristics of all 7 patients are shown in Table 4.
Table 4. Clinical and Genotypic Characteristics of Seven Patients With a Pretransplantation Diagnosis of Hereditary Hemochromatosis
| Patient | Age/Sex | 2nd Cause | HIC (µmol/g) | C282Y | H63D | Vx | Tf | Comments |
| 1 | 52/M | — | 2 | +/+ | –/– | Yes | 4 | HCC |
| 2 | 57/M | — | 39 | +/+ | –/– | Yes | 0 | HCC |
| 3 | 53/M | — | 60 | +/+ | –/– | Yes | 0 | HCC |
| 4 | 55/M | — | 440 | +/+ | –/– | Yes | 0 | — |
| 5 | 51/M | MZ-1AT | 25 | –/+ | –/– | Yes | 0 | — |
| 6 | 43/F | ALD | 166 | –/– | –/– | Yes | 4 | Spur cell anemia |
| | SZ-1AT | | | | | | Congenital dyserythropoesis |
| 7 | 53/M | ALD | 107 | –/– | –/– | No | 10 | Spur cell anemia |
Abbreviations: Vx, history of venesections; Tf, number of packed cell transfusions; 2nd cause, second etiology of cirrhosis; +/+, homozygosity for mutation; +/–, heterozygosity for mutation; HCC, hepatocellular carcinoma; 1AT, -1-antitrypsin deficiency; ALD, alcohol-induced liver disease. |
Only 4 homozygotes and 7 heterozygotes for the C282Y mutation were identified. Three of the latter were compound heterozygotes. The 4 patients homozygous for the C282Y mutation were middle-aged Caucasian men and 3 had hepatocellular cancer complicating their liver disease. These 4 patients had a mean follow-up period of 47 months (range, 11-111 months) after OLT. No patient has evidence of hepatic iron accumulation on posttransplantation liver biopsies. Patient 4 (see Table 4
) had multiple liver biopsies over the nine years following his liver transplantation and the hepatic iron concentration measurements have always been less than 40 µmol/g. All 3 patients deironed before OLT (patients 1, 2, and 3 in Table 4
) presently have normal iron studies (transferrin saturation: 22%-44%; serum ferritin concentration: 43-300 ng/mL) after OLT. Of interest, patient 4 (only diagnosed 2 months prior to OLT) had abnormal iron indices during the first 2 years after OLT with elevations of transferrin saturation (45%-57%) and serum ferritin concentration (414-670 ng/mL); however, these indices then normalized for the remaining period of follow-up. The HFE status of the donor for this patient is not known. With the exception of one patient with transfusion-dependent spur cell anemia, all patients with a pretransplantation diagnosis of hereditary hemochromatosis had commenced venesection before liver transplantation. Alternative diagnoses were present in 2 of the 3 patients with non-HFE hepatic iron overload. Two patients had a history of significant alcohol intake (>80 g/d for more than 10 years) and heterozygosity for 1 antitrypsin deficiency was present in 2 patients.
Resource utilization and survival data
Table 5 shows the relation between the resource variables measured and hepatic iron concentration.
Table 5. Relationship of Hepatic Iron Concentration and Resource Utilization
| Hepatic Iron Concentration (µmol/g) |
| 40 (n = 245) | 41-79 (n = 21) | 80 (n = 14) | P Value |
| Ventilator time (hours) | 61 (8-3174) | 88 (12-1560) | 81 (27-380) | .422 |
| Intensive care ward (hours) | 91 (8-3174) | 127.5 (37-1800) | 145 (32-935) | .145 |
| Hospital stay (days) | 21 (0-132) | 27.5 (7-225) | 24.5 (11-67) | .464 |
NOTE. Values are expressed as median and range. |
There were no significant differences between patients with increased hepatic iron stores and those with normal hepatic iron stores (i.e., 40 µmol/g) for each of the 3 resource variables (ventilator time, duration of intensive care ward stay, and total hospital stay). The posttransplantation unadjusted survival of the 178 patients with an HIC of less than 40 µmol/g was not significantly different from that of the 35 subjects with hepatic iron overload (i.e., HIC 41-79 µmol/g and 80 µmol/g) with 1-year and 5-year survival rates of 80% versus 74% and 72% versus 63%, respectively (P = .27). The adjusted Kaplan-Meier posttransplantation survival curves for the 282 patients when separated into the 3 HIC groups are shown in Fig. 1.
Fig. 1. Actuarial patient survival after liver transplantation by hepatic iron concentration. Patient survival is adjusted for age, gender, Child-Pugh score, and serum creatinine at time of OLT (P = .81).
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There were no significant differences in survival between patient groups in relation to HIC after adjustment for age, gender, Child-Pugh score and serum creatinine (P = .81). Patients with an HIC 80 µmol/g had an 86% 1-year and an 80% 5-year survival as compared with patients with lower hepatic iron concentrations who had 1-year and 5-year survival rates of 80% to 82% and 71% to 75%, respectively. Child-Pugh score was the only variable significantly associated with patient survival after OLT on multivariate analysis (P = .0008). The results from the survival analyses examining the effect of HIC were conducted in several different ways to examine the effect of different cut-points and group sample size. Cox regression analyses were repeated using only 2 study groups with cut-points at 80 µmol/g, 40 µmol/g, the mean (17.7 µmol/g), and using HIC as a continuous variable. The results were essentially identical for all analyses with corresponding P values of .892, .952, .591, and .963. Survival after OLT was not significantly different between patient groups in relation to hepatic iron staining (i.e., no iron, mild [grade 1 or 2], or severe [grade 3 or 4]; P = .28). Similarly, when patient survival after OLT was examined in relation to HII (i.e., >1.9 or 1.9) there was no significant difference between patient groups (P = .263). Of the 4 patients with HFE- related hemochromatosis, 1 died during follow-up from recurrent hepatocellular cancer at 11 months and the remaining 3 patients are alive at 14 months, 52 months, and 111 months after OLT.
Contrary to previous reports, this study showed that increased hepatic iron stores were not associated with reduced survival after liver transplantation, irrespective of the method of hepatic iron classification (i.e., HIC, HII, or hepatic iron grade). Increased HIC was associated with a higher Child-Pugh score, but the severity of the underlying liver disease, rather than HIC, was the only significant determinant of patient survival.
Recent studies have advanced our understanding of the relationship between end-stage liver disease and hepatic iron overload.11,16-18,32 Ludwig et al. reported increased hepatic iron stores in 20% of subjects undergoing OLT for cirrhosis with 8.5% of subjects having an HII >1.9.32 The majority of these patients had alternative causes for their chronic liver disease such as hepatitis B, hepatitis C, or alcohol-induced cirrhosis and the results of the current study support these observations. Since the cloning of HFE, a number of groups have shown that the observed hepatic iron excess in cirrhosis is not associated with known common HFE mutations.16-18 The current study provides further insight into other potential mechanism(s) of iron overload in cirrhosis. Increased hepatic iron concentration was associated with spur cell anemia, more severe liver disease, male gender, and hepatocellular liver disease.
Spur cell anemia is an uncommon hemolytic disorder caused by an alteration in the erythrocyte cell membrane lecithin-phospholipid ratio resulting in acanthocytosis.20,33,34 The membrane rigidity of spur cell results in splenic entrapment leading to a progressive loss of surface area, spiculation, and finally erythrocyte death.29 Most reported cases of spur cell anemia have been in the setting of alcohol-induced cirrhosis.21,29,35 We identified two patients with cholestatic liver disease, mild hemosiderosis, and spur cell anemia; however, the majority of patients with spur cell anemia had underlying hepatocellular liver diseases, predominantly alcohol-induced cirrhosis. The onset of spur cell anemia is a marker of advanced liver disease and is associated with a poor prognosis of usually less than 6 months.34,36 It is interesting to speculate that spur cell hemolytic anemia may increase intestinal iron absorption resulting in increased hepatic iron deposition; however, destruction of red blood cells in the periphery of the body does not usually stimulate intestinal iron absorption.37 Whether spur cell anemia is an underlying cause of the iron loading or merely a surrogate marker of liver disease, severity is currently under investigation. It is of interest to note that iron was deposited predominantly in hepatocytes and proliferating bile ducts, not reticuloendothelial cells, suggesting that blood transfusions do not play a dominant role in cirrhosis-associated iron overload associated with spur cell anemia. Mild hemolysis has been shown in many forms of liver disease but is most severe in patients with spur cell anemia.35,38
Consistent with reported literature, mutations in HFE were not common in iron-loaded patients with end-stage cirrhosis in the current study.16-18 HFE mutational analysis of 79 of the 104 patients with positive hepatic iron staining identified only 4 patients homozygous for the C282Y mutation and 7 heterozygotes, 3 of whom were compound heterozygotes for the C282Y and H63D mutations. 2 microglobulin is important for the intracellular transport and cell surface expression of HFE,12,15 and this is highlighted by the fact that 2 microglobulin gene knockout mice develop hepatic iron overload, which histologically resembles that of HFE-related hereditary hemochromatosis in humans.39 We did not show any significant reduction in serum 2 microglobulin concentration in patients with stainable hepatic iron. This is consistent with the results of Walker et al., who found no mutations of the 2 microglobulin gene sequence in 6 subjects with non-C282Y hemochromatosis19 and suggests that a defect in 2 microglobulin is not primarily responsible for cirrhosis-associated hepatic iron overload.
Thirty-four (97%) of 35 patients with increased hepatic iron concentration had underlying primary hepatocellular diseases (e.g. alcohol-induced cirrhosis, hepatitis C cirrhosis, Wilson's disease, or 1 antitrypsin deficiency) whereas only 1 patient had underlying cholestatic liver disease, namely secondary biliary cirrhosis. This is virtually identical with the findings of Ludwig et al. who reported that only 2 (5%) of their 38 patients with severe hemosiderosis (i.e., HII >1.9) had underlying biliary cirrhosis.32 In the current study, hepatocellular liver disease remained significantly associated with increased hepatic iron concentration even after adjustment for severity of liver disease. The mechanisms resulting in increased hepatic iron deposition in hepatocellular liver disease compared with cholestatic liver disease are unknown. A potential mechanism may relate to differences in whole body iron absorption as bile duct ligated rodents (i.e., an animal model of cholestatic liver disease) have reduced intestinal iron absorption.40 Other mechanisms such as surgical porto-systemic41,42 or spontaneous intrahepatic shunts43 may also contribute to the increased hepatic iron stores observed in cirrhosis-associated iron overload. However, hemosiderosis secondary to shunting fails to explain the increased frequency of hepatic iron overload in hepatocellular liver disease compared with cholestatic liver disease.
Our study highlights 2 common causes of hepatic iron overload in the setting of cirrhosis. The first cause is true HFE- related hereditary hemochromatosis of which homozygosity for the C282Y mutation is the most common form in Caucasian populations. However, the vast majority of patients with cirrhosis and excess hepatic iron do not have the common HFE mutations, rather their hepatic iron appears to be secondary to their advanced liver disease. This observation has been reported by previous investigators16-18 and the documented absence of HFE mutations in such patients is confirmed in the present study. The observations of the present study suggest fundamental clinical characteristics of cirrhosis-associated iron overload and these include advanced liver disease (usually hepatocellular in nature), modest increases in hepatic iron stores, and the absence of the common HFE mutations or a family history of hemochromatosis. Spur cell anemia may be present, but is not a universal feature.
Exact quantitation of hepatic iron stores in cirrhosis remains a difficult problem. Consistent with previous studies,11,25,26,32 we found substantial variability in hepatic iron measurements within the same cirrhotic liver, with the greatest variability in patients with the highest HIC measurements. Iron distribution is often patchy in cirrhotic livers because of an uneven distribution within the hepatic parenchyma, variable degrees of steatosis, and large bands of fibrous tissue containing little or no iron.11,25,26 Furthermore, small sample size has been reported to be another significant factor in the imprecision of HIC measurements from a single needle biopsy.26,44 In the current study, hepatic iron concentration for each subject was reported as the mean value of tissue iron concentration from 3 randomly selected sites. In those subjects in whom the standard deviation between these measures exceeded 20 µmol/g and the mean hepatic iron concentration was close to, or above, 40 µmol/g, a further 5 measurements were performed. Thus, the reported mean hepatic iron concentration reflects sampling from at least 3, and in 30 subjects, 8 different sites within the liver.
There is conflicting evidence in the literature regarding the outcome of liver transplantation in patients with hepatic iron overload. Pillay et al. reported a 1-year survival of 83% in 6 patients, all of whom had undergone venesection prior to OLT.45 The HFE status of these patients is not known, but their early diagnosis and HLA haplotype (A3B7) suggest they were homozygous for the C282Y mutation. However, 2 larger studies have shown a 1-year survival of 53% to 58%3,7 in iron overloaded patients undergoing OLT. Neither of these studies corrected for other variables, which may influence patient survival after OLT. Kilpe et al.3 reviewed the 1-year and 5-year survival rates of over 5,000 OLT recipients and found significantly reduced short- and long-term survival for 56 patients with a diagnosis of hemochromatosis. Included among these 56 subjects were the 37 patients reported by Farrell et al. of which at least 17 (46%) had additional causes for their liver disease. It is unclear how many of the 56 patients in the study by Kilpe et al. were diagnosed as having hereditary hemochromatosis before OLT and the criteria used to diagnose hemochromatosis. In the current study, 3 of the 7 subjects with a pretransplantation diagnosis of hereditary hemochromatosis had an additional cause for their liver disease.
Recently Brandhagen et al.,16 in a case-matched study found the 5-year survival of patients with significant iron overload was significantly less than controls (48% vs.77%; P = .045). Reduced survival was largely attributed to serious infections (especially fungal), which were more common in patients with severe hemosiderosis (24% vs. 7%; P = .03).16,46 The development of bacterial and fungal sepsis in the setting of OLT is most likely to be related to the level of immunosuppression, and the immunosuppression required to maintain a functioning graft in the individual iron-loaded patients who died was not reported. Furthermore, attributing sepsis-related deaths beyond the immediate postoperative period to increased pretransplantation iron stores is speculative, because there is no evidence that iron stores reaccumulate in these patients. Our data do not support the conclusions of Brandhagen et al. or those of a recently published document on liver transplantation practice guidelines.47 In the current study, when survival after OLT was adjusted for age, sex, Child-Pugh score, and serum creatinine, the survival of patients with an HIC 80 µmol/g was similar to the group of patients with an HIC of 40 µmol/g and 41 to 79 µmol/g (P = .81). In all analyses, the direction of association was that survival was better for higher HIC measurements regardless of HIC cutoff point and patient numbers within groups. Similarly, resource utilization for any of the 3 variables measured did not differ significantly between patient groups in relation to HIC.
Of the 4 patients with HFE-related hereditary hemochromatosis 1 died during follow-up from recurrent hepatocellular carcinoma. There were no differences in the survival rates between these 4 patients and non-HFE homozygous patients with hepatic iron overload (i.e. 40 µmol/g) or the entire adult recipient population transplanted with cirrhosis. In contrast, Brandhagen et al. found that C282Y homozygous patients had reduced survival similar to non-HFE patients with severe iron overload, but worse than patients without hepatic iron overload.16 However, in both studies patient numbers are small. Data on reaccumulation of hepatic iron after OLT in subjects with HFE-related hereditary hemochromatosis is extremely limited. We found no evidence of reaccumulation of iron in the new liver in any of the 4 C282Y homozygous subjects over a mean follow-up period of 47 months. Others (Fagiuoli S, et al., unpublished data),46 have shown recurrent iron deposition in many of their patients with hepatic iron overload dying after OLT; however, peritransplant blood transfusions may have been responsible, because the iron was predominantly observed in Kupffer cells. Significantly, these studies were performed before the cloning of HFE and included patients with alternative causes for their liver disease.
In summary, our data suggest that the severity of liver disease rather than HIC is the most important determinant of outcome after OLT and that, in general, increasing HIC in cirrhosis reflects the severity of the underlying liver disease.
Acknowledgment
The authors express their thanks to Anne Pink for technical assistance in HFE mutational analysis, Glynn Rees for collecting liver explant tissue from pathology archives for the measurement of HIC, and Jill Tate for performing serum 2 microglobulin immunoassays.
1. Weisner RH. A long-term comparison of tacrolimus (FK506) versus cyclosporine in liver transplantation. A report of the United States FK506 group. Transplantation 1998;66:493-499.
2. United Network for Organ Sharing (UNOS) http://www.ew3.att.net/unos.
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- From the Departments of 1Gastroenterology and Hepatology, 2Anatomical Pathology, and 3Surgery, Princess Alexandra Hospital, Brisbane, Australia; and the 4Epidemiology and Population Health Unit, Queensland Institute of Medical Research, Brisbane, Australia.
- Received May 18, 2000.
- Accepted September 25, 2000.
- Address reprint requests to: Darrell H. G. Crawford, M.D., Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, QLD, 4102, Australia. E-mail: crawford@health.qld.gov.au
; fax: (61) 7 3240 5111.
Copyright © 2000 by the American Association for the Study of Liver Diseases.
- 0270-9139/00/3206-0004$3.00/0
- doi:10.1053/jhep.2000.20348
)
