Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients

The aim of the present study was to describe histologic features of the liver in insulin resistance-associated hepatic iron overload (IR-HIO), defined as the association of metabolic disorders and hepatic iron overload. We included 139 patients in the study on the basis of one or more metabolic disorders and liver iron overload unrelated to usual causes. Liver biopsy specimens were reviewed, and histologic data were compared with those of a previously published, well-defined population with genetic hemochromatosis. Iron overload was characterized by a mixed pattern with iron deposits in hepatocytes and sinusoidal cells. Steatosis was present in 59.7% of patients with inflammation in 32.4% of cases. Periportal fibrosis was found in 67.4% of patients. These patients were older, had higher sinusoidal iron scores, and had a higher prevalence of steatosis and inflammation than patients without fibrosis. Iron overload in IR-HIO was histologically different from that in genetic hemochromatosis.     

Iron state and hepatic disease in patients with thalassaemia major, treated with long term subcutaneous desferrioxamine.

Liver biopsies were performed on 51 regularly transfused patients with beta thalassaemia, age range 5-36 (mean 18.6) years, who had received regular subcutaneous desferrioxamine (DFX) treatment for periods between one and eight years (40 for eight years). The biopsy specimens were examined by light microscopy and immunofluorescence for hepatitis B virus surface and core antigens (HBsAg and HBcAg), and the iron content was determined chemically. The results were compared with serum ferritin concentration and aspartate transaminase (AST) activity and with hepatitis B virus serology. Biopsy specimens, in which chemical liver iron had been determined in 12, were also available from 17 patients. Mean serum ferritin (+/- SD) had fallen from 5885 (3245) micrograms/l to 1638 (976) micrograms/l in 36 patients after eight years' chelation, while mean (+/- SD) liver iron concentration had fallen from 2945 (900) micrograms/100 mg dry weight to 857 (435) micrograms/100 mg dry weight in 12 of them. All biopsy specimens examined were negative for HBs and HBc antigens. The presence of histological features of hepatitis was associated with increased liver iron content, increased fibrosis, and with progression of fibrosis between the two biopsies. Procollagen III peptide was assayed in 28 patients but did not correlate with the degree of hepatitis, fibrosis, or with chemical liver iron content. We conclude that with regular subcutaneous DFX, mean concentrations of serum ferritin and liver iron are maintained in these patients at about five and 10 times the normal value, respectively, and that progression of liver damage is more likely to be due to viral hepatitis, presumably related to the parenterally transmitted non-A, non-B agents than to iron overload.     

The changing role of liver biopsy in diagnosis and management of haemochromatosis

Liver biopsy with histological examination of liver tissue was for many years the cornerstone of the diagnosis of haemochromatosis, allowing assessment of the degree of iron overload and examination of liver histology for the acute and chronic effects of iron overload. In the past two decades the role of liver biopsy in haemochromatosis has changed dramatically. Liver biopsy is rarely requested for two main reasons: (1) genetic testing for human haemochromatosis (HFE) mutations has proved to be very reliable in the diagnosis of haemochromatosis in Caucasian populations, and (2) the majority of patients with haemochromatosis are now diagnosed at an early stage well before permanent tissue damage occurs, so the need to assess tissue and organ damage has diminished. Liver biopsy continues to have a very important role in a small number of haemochromatosis patients for whom it has both diagnostic and prognostic implications. Liver biopsy is essential for the accurate assessment of patients with non-HFE haemochromatosis and in patients who have dual pathology. It is also useful where there appears to be a discrepancy between HFE genotypes and iron studies, particularly in HFE heterozygotes. Finally, liver biopsy is currently the 'gold standard' for the diagnosis of fibrosis and cirrhosis, although this is changing as non-invasive methods for assessing fibrosis become more reliable and available. Therefore, it is important that pathologists maintain their knowledge and skills in the use of liver biopsy in haemochromatosis and other iron storage disorders.     

Transfusional iron overload: the relationship between tissue iron concentration and hepatic fibrosis in thalassaemia.

The interrelationships between liver iron concentration, the duration of iron-loading, and hepatic fibrosis, assessed morphometrically, have been studied in 32 specimens of liver obtained from 19 heavily transfused patients with thalassaemia major whose age ranged from 4 to 19 yr. Similar observations were made in a matched group of thalassaemic patients treated with long-term chelation therapy. The degree of liver damage ranged from very slight increase in fibrous tissue to severe fibrosis and cirrhosis. The severity of the fibrosis was closely correlated both with liver iron concentration and with age. While the relationship between fibrosis and age was linear, both the severity and the rate of fibrosis were exponentially related to liver iron concentration, damage accelerating as liver iron concentration exceeded 3 per cent, dry weight. By producing a modest but significant reduction in liver iron concentration chelation therapy resulted in a disproportionate but predictable retardation in the progression of the fibrosis. The factors affecting the distribution of iron between parenchymal and reticuloendothelial cells were also examined. In general stainable iron was uniformly distributed between parenchymal and reticuloendothelial cells from the early stages of iron-loading. Parenchymal siderosis was relatively heavier in splenectomised patients and in patients with liver iron concentrations above 3 per cent, dry weight than in non-splenectomised patients or patients with liver iron levels of less than 3 per cent, dry weight, but this did not affect the severity of the fibrosis. The relevance of these findings to the traditional concepts of the pathology of transfusional siderosis is discussed.     

Congenital dyserythropoietic anemia--type II (CDA-II) in 3 siblings with long-term follow up and iron overload

The diagnosis of congenital dyserythropioetic anemia-type II (CDA-II) was established in 1974 in three siblings aged 20, 18 and 5 years, respectively. Liver biopsy performed in two elder siblings on admission revealed liver siderosis. Anemia showing haemolytic component with destruction of erythrocytes in the spleen was corrected after splenectomy. Increased number of erythrocytes showing "the double membrane phenomenon" was found in the peripheral blood after splenectomy. All three siblings developed cholecystolithiasis with choledocholithiasis and obstructive jaundice in two of them. Two patients at the age of 49 and 34 years (the third died in an accident at the age of 40 years) developed 29 years after the diagnosis of CDA-II had been established signs of iron overload with transferin saturation 99%, serum ferritin 1450.4 microg/l and 1131.7 microg/l respectively, and hepatic iron concentration (dry weight) 14,843 microg/g and 15,415 microg/g (norm 70-1400 microg/g) respectively. No mutations of HFE gene (C282Y and H63D) were found. Liver biopsy showed heavy accumulation of hemosiderin in hepatocytes and reticuloendothelial cells. The structure of the liver tissue was not changed, only mild fibrosis in portal area was present in the older patient. Because of iron overload therapy with phlebotomy once monthly (400 ml) has been started in both patients. In peripheral blood films excess of Pappenheimer bodies was found.     

Role of liver biopsy in the diagnosis of hepatic iron overload in the era of genetic testing

We studied hepatic iron overload (HIOL) patterns in 32 patients who underwent liver biopsies and testing for HFE mutations (C282Y, H63D). Iron-stained biopsy specimens were examined for patterns of iron deposits: hereditary hemochromatosis (HH) pattern or non-HH pattern. Visual iron grade based on amount of cellular and lobular iron was evaluated. We found the HH pattern in 17 biopsy specimens (53%) and the non-HH pattern in 6 specimens (19%). HH with superimposed non-HH was noted in 9 cases (28%). In 25 patients with HFE mutations, HH alone and combined with non-HH patterns was noted in 22 specimens (88%). Visual iron grade correlated approximately with the hepatic iron index. Heavy HIOL was noted in C282Y homozygotes and 1 patient with cirrhosis without either HFE mutation. Mild steatohepatitis was found in 21 specimens (66%); it was associated with the non-HH pattern in 80% (12/15) and the HH pattern in 62% (16/26) of cases. Liver biopsy can identify pattern and grade of HIOL and associated pathology for diagnosis and management of patients with abnormal iron studies and elevated liver function test results. Genetic tests for HFE mutations and liver biopsies are complementary in the workup of these patients.     

Hepatitis C virus in sickle cell disease

PURPOSE: To determine the prevalence of hepatitis C virus antibodies (anti-HCV) in patients with sickle cell disease. PATIENTS AND METHODS: Between 1983 and 2001, 150 patients from the Howard University Hospital Center for Sickle Cell Disease were screened for HCV antibody (52% women, 48% men, mean age 34 years). Frozen serum samples from 56 adult sickle cell patients who had participated in previous surveys (1983-92) of HIV and HTLV-1 serology and who were tested in 1992 for anti-HCV antibody--when commercial ELISA test (Ortho) became available--were included in this paper. Of the 150 patients in the study, 132 had sickle cell anemia genotype (SS), 15 had sickle cell hemoglobin-C disease (SC) and three had sickle beta thalassemia. Clinical charts were reviewed for history of blood transfusion, IV drug abuse, homosexuality, tattooing, iron overload, and alcohol abuse. RESULTS: Antibodies to HCV were detected in 53 patients (35.3%). Of the 55 patients who had frozen serum samples tested in 1992, 32 (58%) were reactive for anti-HCV, while only 21 of the 95 patients (22%) tested after 1992 were positive for HCV antibodies (P<0.001). Thirty-nine of 77 patients (51%) who received more than 10 units of packed red blood cells were positive for HCV antibody, and only 14 of 61 patients (23%) who received less than 10 units of packed red blood cells transfusion were positive for HCV antibodies (P<0.001). None of the 12 patients who never received transfusion were positive for HCV antibody. In the 53 anti-HCV positive patients, the mean alanine amino-transferase (ALT) value was 98- and 81 U/L, respectively, for males and females. These values were normal for the HCV-antibody negative patients. The aspartate amino-transferase (AST) and the total bilirubin were also higher in the anti-HCV positive patients compared to patients in the anti-HCV negative group. Forty-four patients (57.1%) who were transfused more than 10 units developed iron overload defined by a serum ferritin level higher than 1,000 ng/ml. A total of 20 of the patients with iron overload underwent liver biopsies. Seven of these 20 patients (35%) were HCV positive. These patients often had more severe liver disease and higher degree of iron deposition. CONCLUSION: The prevalence of HCV antibody and iron overload is directly related to the number of blood transfusions in patients with sickle cell disease. The prevalence of HCV infection has decreased significantly, since blood donor screening for HCV became available. Chronic HCV infection and iron overload place sickle cell patients at risk for significant liver disease.     

Histological picture of liver disease in thalassemia intermedia

Thalassemia Intermedia (TI) is a clinical definition in use for a spectrum of clinical conditions ranging in severity from the asymptomatic carrier status to the transfusion-dependent status. The histological lesions of the liver in patients affected by TI has not seen well characterized yet. The aim of this study was to define the histological picture of liver disease in TI. To this end we studied our pool of 22 Thalassemia Intermedia patients who underwent liver biopsy; none of them had blood transfusion. We took into consideration fibrosis according to Ishak's staging and iron overload according to Sciot's grading. Moreover, we determined the hepatic iron content by atomic absorption spectroscopy. Our results have shown that, in all patients studied, the histological picture was characterized by the increase in liver iron stores. By Perls's stain haemosiderin appeared to be stored mainly in hepatocytes in the majority of cases. Inspite of the large amounts of liver iron in these patients, fibrosis was absent or mild. We also noted the presence of iron-free-foci, in part of the patients. Our data show that the histological and histochemical picture of liver disease in Thalassemia Intermedia is very similar to that found in Thalassemia Major. The finding of iron-free-foci could induce to consider a follow up of these patients, even if asymptomatic.     

Role of non-transferrin bound iron in iron overload and liver dysfunction in long term survivors of acute leukaemia and bone marrow transplantation.

AIMS: To determine whether nontransferrin bound iron is present in the serum of long term survivors of acute leukaemia and bone marrow transplantation who have liver dysfunction as indicated by consistently raised serum aspartate aminotransferase (AST) activities. METHODS: Thirty eight patients, who were at least three years from the end of treatment, were studied. Serum samples were analysed for hepatitis C, hepatitis B, AST, ferritin, and non-transferrin bound iron. A bleomycin based assay was used to detect non-transferrin bound iron. Patient and blood bank records were examined to determine the number of units of transfused blood received by each patient. RESULTS: Ten patients had consistently raised serum AST activities. Of these, two had evidence of hepatitis C infection, one had chronic hepatitis B infection and one had chronic graft versus host disease affecting the liver. None of these four patients had detectable non-transferrin bound iron. The remaining six patients had no obvious reason for raised AST activities, but four had non-transferrin bound iron detectable in their serum as compared with only two out of 28 patients with normal AST activities. Patients with abnormal AST activities had higher serum ferritin concentrations than those with normal AST, though serum ferritin was raised in 21 of 28 patients without liver dysfunction. CONCLUSION: Non-transferrin bound iron may be found in this group of patients, suggesting that iron overload is the cause of the observed liver dysfunction. Non-transferrin bound iron may also be a more specific indicator of iron overload than the serum ferritin concentrations.     

Helicobacter spp. and liver diseases

To test whether Helicobacter species play a role in the enhancement of liver necro-inflammation and fibrosis and in the development of hepatocellular carcinoma (HCC), we sought DNA sequences of Helicobacter species in liver specimens from patients with viral-related chronic hepatitis, HCC or metastatic liver carcinoma. We enrolled 28 consecutive patients with ultrasound evidence of hepatic nodule(s) on their first liver biopsy: 21 had histological evidence of HCC (Group I) and 7 of metastatic liver carcinoma (Group II). In the same period we observed 27 consecutive patients with chronic hepatitis on their first liver biopsy (Group III). Helicobacter sequences were sought by PCR using primers for the 16S rDNA of Helicobacter spp, designed to amplify a 400 base-pair fragment, and detected by 2% agarose gel and hybridization with a specific biotinylated probe. We used, as positive controls for the DNA extraction from liver tissue, hepatic biopsy sections in which HBV infection was confirmed by HBcAg positivity and in which we amplified HBV-DNA by specific primers; positive controls for the amplification of Helicobacter spp were obtained from gastric biopsy sections in which Helicobacter pylori infection was confirmed by biochemical and histochemical tests. HBV-DNA was found in all five HBcAg positive liver biopsies. Helicobacter spp 16S rDNA was detected in all five biopsy specimens of gastric mucosa and in none of liver specimens from patients in any group. Our data suggest that Helicobacter species were not involved in the pathogenesis of virus-related HCC, chronic hepatitis or liver carcinoma metastasis.     

HIV replication is associated with increased severity of liver biopsy changes in HIV-HBV and HIV-HCV co-infection

Histological parameters were assessed in liver biopsies (n = 48) performed in patients co‐infected with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and/or hepatitis C virus (HCV) in order to evaluate factors which were associated with significant liver disease. Necroinflammation and fibrosis was scored by the Ishak classification system, and binary logistic regression analysis was used to assess HIV and antiretroviral‐related determinants of necroinflammation and fibrosis. A total of 46 biopsies were included; 33 were from HIV‐positive patients co‐infected with HCV and 15 biopsies were from HIV‐positive patients co‐infected with HBV. One HIV‐positive patient was co‐infected with HBV and HCV. Median biopsy inflammatory grade for the cohort was 8.5 (IQR 6–10), the median fibrosis Stage 2 (IQR 1.8–4), and the median steatosis score was 1 (IQR 0–2). At the univariate level, HIV‐related variables that were significantly associated with more severe biopsy changes were higher HIV RNA at the time of biopsy (associated with inflammatory Grade 10+; P = 0.018) and any exposure to didanasine (ddI) or stavudine (D4T; associated with fibrosis Stage 3+; P = 0.022). HIV RNA at the time of biopsy remained significant at the multivariate level. Patients with HIV hepatitis co‐infection in this cohort had surprisingly mild changes in liver histology, and there were no statistically significant differences between biopsy results in HBV compared to HCV co‐infection. The association between HIV RNA and necroinflammation supports current recommendations for earlier initiation of HAART in patients with HIV‐hepatitis co‐infection. J. Med. Virol. 84: 993–1001, 2012. © 2012 Wiley Periodicals, Inc.     

Special stains in diagnostic liver pathology

The comprehensive histopathologic evaluation of liver tissue, including biopsy, explant, and postmortem specimens, utilizes a standard panel of special histochemical stains as well as selective immunohistochemistry. These methods provide increased accuracy in addressing common diagnostic problems such as determining the stage of fibrosis in chronic hepatitis, documenting the presence of cirrhosis or other causes of portal hypertension, iron, and copper overload, disorders of the biliary tract, and tumor histogenesis. This review discusses the indications for various staining methods and the specific uses of trichrome and reticulin connective tissue stains, periodic acid-Schiff (PAS) and diastase-pretreated PAS (DPAS), iron, and Victoria blue methods. Diagnostic applications of immunohistochemical stains are also described.     

Binding of serum ferritin to concanavalin A in patients with iron overload and with chronic liver disease.

Total serum ferritin and the proportion of serum ferritin binding to concanavalin A (glycosylated ferritin) was measured in 18 healthy volunteers and in 84 patients, eight with primary haemochromatosis, 43 with beta-thalassaemia major and secondary iron overload and 33 with chronic liver diseases without iron overload. The total serum ferritin was either equally or even more closely related than either the non-binding or the concanavalin A binding ferritin, to the liver iron concentration in all patients with iron overload, and with the units of blood transfused in non-chelated thalassaemic patients. The total serum ferritin showed a significant correlation with serum aminotransferase for the group of 84 patients. In the thalassaemic patients the ferritin binding to concanavalin A also correlated with aminotransferase. However, in the other groups it was the ferritin not binding to concanavalin A which showed a significant correlation with aminotransferase activity. These results suggest that measuring the fraction of serum ferritin which binds to concanavalin A does not offer any advantage over estimation of the total serum ferritin concentration in the assessment of iron stores in patients wit iron overload and liver damage.     

Iron-induced mucosal injury to the upper gastrointestinal tract

AIMS: To define the causes and associations of mucosal iron deposition in upper gastrointestinal biopsy specimens and to describe the morphological features of iron-related injury. METHODS: The histological pattern, intensity and distribution of iron in biopsies obtained from 1991 to 2005 were recorded and correlated with endoscopic and clinical findings. RESULTS: Twenty-five biopsies (16 gastric, four duodenal, five oesophageal) were accrued. Iron deposition was seen in two groups: 10 cases showed erosive injury, with brown-black crystalline material overlying eroded epithelium. These patients were taking oral iron tablets. The remaining 15 cases showed variable iron deposition in the surface epithelium, lamina propria and glands. In nine patients, there was a history of oral iron intake and at least eight had had blood transfusions. The most intense iron deposition was noted in patients with end-stage liver disease. The mean age of patients with erosive injury was 43% higher than in the iron overload group (76 versus 53 years). Iron stains were also performed on 15 normal gastric biopsies and five biopsies with chronic, non-specific gastritis; all were negative for haemosiderin deposition. CONCLUSIONS: Iron-related erosive injury is related to oral iron pill ingestion and occurs in older patients. Mucosal iron deposition is also associated with iron overload disorders.     

Glomus tumour of the trachea: report of a case with microscopic, ultrastructural and immunohistochemical examination and review of the literature

In the 6-year period 1984-1989, 101 liver biopsies or 'needle necropsies' from human immunodeficiency virus positive patients were examined histologically. Of these, only nine showed no abnormality whatsoever. The commonest histological findings were either fatty change or changes related to co-existent chronic viral hepatitis. Granulomas were seen in 15 cases, four of which were positive for acid-fast bacilli. A range of organisms were recorded: cytomegalovirus (4); Histoplasma capsulatum (1); Pneumocystis carinii (2); Cryptococcus neoformans (1); and Leishmania donovani (1). There were two cases of non-Hodgkin's lymphoma, but no cases of Kaposi's sarcoma. Marked iron deposition, which correlated with multiple blood transfusions was seen in nine biopsies. We were unable to identify any histological feature in the liver as being specific for HIV infection. The high incidence of liver abnormalities reflects: (i) the coincident exposure to hepatotropic viruses; (ii) the presence of opportunistic infections and neoplasms, usually part of a disseminated multi-organ process arising in the setting of profound immune depression; (iii) iatrogenic causes, in particular iron overload related to multiple blood transfusions received for treatment of zidovudine-induced anaemia; and (iv) non-specific changes associated with chronic debilitating disease.     

Stainable bone iron in undecalcified,plastic-embedded sections. Occurrence in man related to the presence of "free" iron?

Iron demonstrable with the Prussian blue reaction at the osteoid/mineralized tissue interphase (osteoid seam) of trabecular bone was observed in only 2.3% of a total of 1536 conventionally fixed and processed, undecalcified, plastic-embedded biopsy specimens taken from the iliac crest of patients for various diagnostic purposes. In marked contrast, clearly stainable bone iron was noticed in all 4 biopsy specimens from the iliac crest and in 11 of 15 vertebral bone fragments obtained at autopsy from individuals with verified primary or secondary hemochromatosis. Findings, including results obtained in vitro, suggest that a positive Prussian blue reaction at the surface of trabecular bone signals the presence of low-molecular-weight ("free") iron, which can bind to the osteoid matrix directly, ie, without the help of osteoblasts. Stainable bone iron may thus be a useful criterion for early detection of hemochromatosis and other types of potentially toxic iron overload.     

The role of liver biopsy in the management of chronic hepatitis C in patients infected with the human immunodeficiency virus

We evaluated the role of liver biopsy in the management of chronic hepatitis C in HIV-infected persons. Patients included had abnormal alanine transaminase (ALT) levels, detectable HCV RNA, and an interpretable liver biopsy. Demographic, epidemiologic, clinical, laboratory, histological, and therapeutic data were recorded in all patients. We also registered the clinical diagnosis of cirrhosis (previous to biopsy) and whether the biopsy result deferred the decision of initiating therapy. During the 33-month duration of the study, 112 patients were included. The degree of fibrosis in liver biopsies was none or mild (F0 or F1) in 47 patients (42%) and was significant or severe in 65 (58%). Seventeen patients (15%) had histological cirrhosis. By logistic regression analysis, only portal hypertension (odds ratio [95% confidence interval], 5.3 [1.05-25.9], P = 0.04) was independently associated with significant fibrosis. Overall, cirrhosis was predicted before biopsy in 29 patients (26%) by the caregiving physician, but only 8 of these were confirmed histologically. The clinical prediction of cirrhosis before biopsy had a sensitivity of 47%, a specificity of 78%, a positive predictive value of 28%, and a negative predictive value of 89%. Histological findings changed the decision to initiate HCV therapy in 19 patients (17%) because of little or no fibrosis. Liver biopsy is a useful tool in the management of HCV-HIV-coinfected persons. In addition to allowing grading and staging of the disease far better than any other method or combination of methods, it is important for making management decisions for patients coinfected with HCV and HIV.     

Hepatic steatosis: an emerging cause of cirrhosis in HIV patients

Hepatic steatosis is an emerging cause of morbidity in antiretroviral therapy (ART)-experienced HIV patients. The influence of steatosis on fibrosis is poorly understood. We report two cases of rapid evolution of disseminated macrovacuolar steatofibrosis to cirrhosis. Both patients had no history of alcohol abuse, nor intravenous drug use and were tested negative for HCV (PCR RNA) and had no HBS antigen. Patient 1 had a past history of hypertrygliceridemia, but controlled with dietetic measures for 4 years prior to biopsy. The first hepatic biopsy showed a disseminated macrovacuolar steatosis (>80%). The patient had then cytolysis and an uncontrolled HIV viral load. The second biopsy was performed two years later, and HIV was controlled by a new line of ART. It showed a regression of the steatosis (10%) and a progression of the fibrosis with signs of cirrhosis. Patient 2 had a long history of HIV infection. He also had an uncontrolled dyslipidemia. The first biopsy was realised during a period of uncontrolled HIV infection and elevated liver enzymes. The biopsy showed a major macrovacuolar steatosis (>80%). The second biopsy was realised 6 years after and showed the same steatosis and signs of cirrhosis. The HIV infection was then under control. Observations reported here show a rapid evolution of liver steatosis to cirrhosis in HIV positive / HCV negative patients, despite the control of HIV infection. The implication of HAART remains unclear. In the era of HAART, liver steatosis can rapidly evoluate to cirrhosis without any risk factor except ART.     

Iron in the era of molecular biology

Identification of the HFE gene and its C282Y and H63D mutations has improved the classification of iron overload disorders. Inherited hemochromatosis is due mainly, or perhaps only, to C282Y homozygosity, whereas nonhemochromatosis forms of iron overload are due to other HFE mutations and are usually responsible for mild overload precipitated by another factor such as cirrhosis or insulin-resistance. In practice, the diagnosis of inherited hemochromatosis rests on demonstration of homozygosity for the C282Y mutation; in this setting, the role of liver biopsy is to evaluate the prognosis by looking for fibrosis. In patients who are not homozygous for the C282Y mutation but have severe iron overload, causes other than hemochromatosis should be looked for before the extremely remote possibility of nonC282Y-related hemochromatosis is considered; here, liver biopsy remains of considerable diagnostic usefulness.