00019606-200903000-00007ReportDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2009 Lippincott Williams & Wilkins, Inc.18March 2009
p 53-60Diagnosis of Hepatic Iron OverloadA Family Study Illustrating Pitfalls in Diagnosing HemochromatosisCase ReportSchranz, Melanie MD*; Talasz, Heribert MD†; Graziadei, Ivo MD*; Winder, Thomas MD*; Sergi, Consolato MD, PhD‡; Bogner, Klaus MD§; Vogel, Wolfgang MD*; Zoller, Heinz MD**Department of Medicine, Clinical Division of Gastroenterology and Hepatology†Biocenter, Clinical Biochemistry‡Department of Pathology, Innsbruck Medical University, Innsbruck§Department of Internal Medicine, Krankenhaus Vöcklabruck, Vöcklabruck, AustriaSupported by a grant from the Austrian Science Funds FWF P19579.Conflict of Interest Statement: We declare that we have no conflict of interest.Reprints: Heinz Zoller, MD, Clinical Department of Gastroenterology and Hepatology, Anichstrasse 35, A-6020 Innsbruck, Austria (e-mail:
[email protected]).AbstractRecent identification of genetic variants in iron storage disease has changed the classification system and diagnostic algorithms for hemochromatosis. Clinical diagnosis of the disease requires phenotypic evidence of iron overload because the commonly disease-associated HFE genotypes have an incomplete penetrance. Furthermore, ∼20% of patients with a clinical diagnosis of hemochromatosis have no disease-associated genotype, which underlines the importance of clear phenotypic criteria of hemochromatosis. A diagnosis of hemochromatosis cannot be made even in patients with liver cirrhosis simply on the basis of genetic testing that indicates that iron overload is the cause of the disease and not its consequence. Proper diagnosis requires integration of clinical presentation, family history, and the results of biochemical and histopathologic tests. Here we propose a rational diagnostic algorithm for hepatic iron overload syndromes and illustrate potential pitfalls by presenting a family study in a pedigree with rare HFE variants (H63D and E168Q), in cis on the same chromosome. Although the clinical suspicion of hemochromatosis was confirmed by histology, chemical analysis of liver tissue revealed a normal hepatic iron concentration, which is compatible with the genetic finding of 1 normal and 1 doubly mutated allele. In conclusion, clinical suspicion of hemochromatosis and elevated serum iron parameters should prompt HFE genotyping for C282Y and H63D. Should they be uninformative, further genetic tests should be recommended only if iron overload in liver tissue has been confirmed chemically.Hemochromatosis is a systemic disorder in which tissue iron overload causes damage to organs, mainly the liver, heart, pancreas, and pituitary. The most frequent clinical manifestations of hemochromatosis include fatigue, liver cirrhosis, and hepatocellular carcinoma, and, in rare cases, heart failure, hypogonadism, and diabetes.1–3A characteristic feature of hemochromatosis is the presence of hepatic iron concentrations (HICs) of >25 μmol/g liver tissue (dry weight) detected by chemical analysis. The hepatic iron index (HII) is calculated by dividing the HIC by the patient's age and makes it possible to distinguish hereditary hemochromatosis from iron overload secondary to alcoholic liver disease, porphyria cutanea tarda, and chronic viral hepatitis.4 Histologic assessment of the distribution of stainable iron and histologic grading of iron overload correlate well with HII and HIC.5 In hereditary hemochromatosis, most stainable iron is found in hepatocytes, but also biliary epithelia may contain siderotic granules. In contrast, secondary iron overload primarily affects Kupffer cells. Furthermore, histologic investigation of liver specimens enables determining the degree of tissue damage, which can range form mild fibrosis to frank cirrhosis.6Disease-associated polymorphisms of the HFE gene were identified in 1996, and since then it has become possible to identify patients at risk for developing hemochromatosis at presymptomatic stages of the disease. The 2 HFE genotypes that are commonly associated with hemochromatosis are homozygosity for C282Y and compound heterozygosity for C282Y and H63D.7,8 However, ∼20% of patients with the clinical and pathologic features of hemochromatosis and hepatic iron overload are neither homozygous for C282Y nor do they demonstrate compound heterozygosity for C282Y/H63D.9A search for other genetic causes of hemochromatosis in such patients resulted in the discovery of several rare variants in the HFE gene. These variants include S65C, V59M,10 R66C,11 G93R, I105T,10,12 E168Q,13 R224G,11 and V295A,14 which have all been identified in patients with hemochromatosis and heterozygosity for the C282Y mutation on the other parental allele. Among these mutations, E168Q has been exclusively found in association with the H63D mutation, both of which occur in cis on the same chromosome.15However, in a considerable proportion of hemochromatosis patients, no HFE gene variants can be identified. This type of iron storage disease is referred to as “non-HFE hemochromatosis.”16 The search for genetic causes and modifiers of iron storage disease in the latter group has led to the identification of mutations in the genes encoding transferrin receptor 2 (TFR2),17 hepcidin (HAMP),18 hemojuvelin (HFE2),19 and ferroportin (FPN1).20The penetrance of disease-associated HFE genotypes, which is <20% for patients homozygous for C282Y and <10% for C282Y/H63D heterozygotes,21–23 must be kept in mind while interpreting HFE gene test results. Hemochromatosis is a polygenic disease with a strong sex dependence24; furthermore, environmental factors such as alcohol consumption and dietary iron content can lead to increased iron deposition in the liver (reviewed in Ref. 25).Nevertheless, genetic testing for hemochromatosis has changed the diagnostic algorithm of iron overload. In patients with increased serum iron parameters and disease-associated genotypes of the HFE gene, therapeutic phlebotomy can be started without patients having to undergo liver biopsy.DIAGNOSTIC WORKUP OF PATIENTS WITH SUSPECTED IRON OVERLOADAccording to the American Association for the Study of Liver Diseases (AASLD) guidelines for the diagnosis and management of hemochromatosis, symptomatic patients in whom iron overload is suspected should be tested for hemochromatosis. In addition, the following groups of asymptomatic individuals should be evaluated for the presence of iron overload: (1) first-degree relatives of a confirmed case of hemochromatosis; (2) individuals with abnormal serum iron markers discovered during routine testing or in cases of unexplained elevation of liver enzymes; and (3) individuals with asymptomatic hepatomegaly or radiologic detection of enhanced computed tomography attenuation of the liver.Among patients with unexplained liver disease or with a presumably known cause of liver disease, testing for the common HFE gene polymorphisms C282Y and H63D is indicated if elevated fasting transferrin saturation is found. The algorithm proposed by the AASLD suggests to “exclude other liver or hematologic diseases with or without liver biopsy in cases with compound heterozygosity, heterozygosity for C282Y, and patients with non-C282Y genotypes of the HFE gene.”26The availability of a facile treatment for hemochromatosis renders early diagnosis of iron overload syndromes mandatory and many patients with pathologic serum iron parameters are referred for hemochromatosis testing for indications not included in the list of the AASLD algorithm. Besides liver diseases and hematologic disorders, dysmetabolic siderosis is a frequent cause of pathologic serum iron parameters and may be suspected in obese patients with metabolic syndrome. Acute or chronic inflammation is also a common cause of abnormal serum iron parameters and we propose exclusion of these conditions before further investigations for suspected hemochromatosis are initiated (Fig. 1). If other liver diseases are also excluded as suggested in the AASLD guidelines, genetic testing for rare HFE variants may confirm hemochromatosis in patients heterozygous for C282Y or H63D and hepatic iron overload. In such patients, HFE genotyping by reversed hybridization assays or direct gene sequencing has been suggested as the next diagnostic step.3 Interpretation of gene sequencing results should include family studies to avoid pitfalls such as occurrence of heterozygous variants in cis.JOURNAL/dimp/04.03/00019606-200903000-00007/figure1-7/v/2021-02-17T200000Z/r/image-jpeg
Algorithm for the differential diagnostic workup of patients with suspected iron overload. Symptoms and signs indicating iron overload syndromes include fatigue, arthropathy, diabetes, abnormal skin pigmentation, impotence, or signs of chronic liver or heart failure. In patients with these symptoms, serum iron parameters should be determined. If serum transferrin saturation and/or serum ferritin is increased, basic laboratory tests should include the indicated parameters to exclude hematologic, inflammatory metabolic, and liver disease other than hemochromatosis. HFE genotyping is the first step in differential diagnostic workup of “primary” iron overload and C282Y homozygosity or compound heterozygosity for C282Y/H63D is sufficient for the diagnosis of HFE-associated hemochromatosis. In patients without a disease-associated HFE genotype, a magnetic resonance imaging may help estimating the degree of hepatic overload. In patients with high suspicion of hepatic iron storage, a liver biopsy should be performed to determine the degree of tissue damage and reveal the histologic and cellular distribution of iron. Patients with Kupffer cell iron overload are candidates for ferroportin gene sequencing, where heterozygous mutations are sufficient to cause “ferroportin disease.”27 In patients with predominantly hepatocellular iron overload, rare genetic hemochromatosis syndromes should be considered and genetic tests carried out according to clinical presentation. In isolated cases with severe disease manifestation, occurrence of bigenic hemochromatosis has been reported.28 Aceruloplasminemia or Wilson disease may be a rare genetic cause in patients with, non-HFE hemochromatosis and genetic tests should be requested if iron overload is associated with neurologic impairment.29,30Magnetic resonance imaging offers a noninvasive alternative to liver biopsy for quantification of tissue iron. It has only recently received Food and Drug Administration approval and has therefore not yet been included in the current AASLD guidelines. If histology of liver tissue sample reveals that stainable iron is confined to Kupffer cells and secondary causes of iron overload such as repeated transfusions have been excluded, sequencing of the ferroportin gene may be considered in patients with isolated hyperferritinemia. Dysmetabolic siderosis is a differential diagnosis in patients with isolated hypoferritinemia and is further characterized by hypercholesterolemia, increased HbA1c, impaired oral glucose tolerance, and obesity.31In patients presenting with familial and histologically confirmed hepatic iron overload, and who are negative for HFE and ferroportin gene mutations, genetic testing may be extended to other “iron genes.” A rational diagnostic algorithm in such patients is proposed in Figure 1 and is based on the age on clinical presentation. If clinically overt iron overload occurs before 20 years of age, the clinical diagnosis of juvenile hemochromatosis can be made and the hemojuvelin gene (HJV) should be sequenced. In patients negative for HJV mutations, genetic variants of the HAMP have been reported.18 Clinically apparent iron overload in patients above 20 years of age has been described in association with mutations in the gene encoding transferrin receptor 2, which spans 18 exons and a coding region of ∼3 kb, limiting its analysis to dedicated laboratories.28Despite the availability of genetic tests, the diagnosis of hemochromatosis still represents a challenge, requiring careful clinical, histopathologic, and chemical assessment.3CASE PRESENTATIONThe index patient presented at the age of 53 years with icterus and progressive asthenia to a primary care hospital. His past medical history was unremarkable, and he consumed moderate amounts of alcohol (60 to 90 g/d). Clinically, the patient showed signs of liver cirrhosis with portal hypertension including ascites and an enlarged spleen. Diagnostic laparoscopy and a computed tomography scan revealed a dark liver with micronodular cirrhosis and an exophytic tumor of the left liver lobe (Fig. 2). At the time of presentation, the serum concentration of α-1-fetoprotein was normal (5.5 μg/L).JOURNAL/dimp/04.03/00019606-200903000-00007/figure2-7/v/2021-02-17T200000Z/r/image-jpeg
Computed tomography scan of the abdomen showing a cirrhotic liver, ascites, and a protruding hepatocellular carcinoma.A blood test showed hyperbilirubinemia (5.78 mg/dL), thrombocytopenia (77 g/L), and mild anemia (113 g/L). Increased serum aminotransferase activities (aspartate aminotransferase 29 U/L and γ-glutamyl transferase 43 U/L) and alkaline phosphatase activity (356 U/L) were also noted. Impaired liver function was further indicated by coagulation abnormalities (International Normalized Ratio 1.8) and hypoalbuminemia (3.87 g/L).Differential diagnosis revealed marked hyperferritinemia of 843 μg/L and increased transferrin saturation of 70%. Chronic viral hepatitis and autoimmune hepatitis, as well as primary biliary cirrhosis and α-1-antitrypsin deficiency were excluded by appropriate tests. The patient was then referred to our center for liver
transplantation evaluation.The differential diagnosis of hemochromatosis was made on the basis of the observed hyperferritinemia. After recompensation of chronic liver disease, quantitative phlebotomy was carried out biweekly over a period of 2 months, removing ∼1.2 g of iron. After 4 phlebotomies, the hemoglobin concentration dropped to 10.8 g/dL and phlebotomy was stopped after decompensation of the liver disease. At this stage, the serum ferritin concentration was 467 μg/L and transferrin saturation 37%.The patient was referred for genetic testing for hemochromatosis, which revealed heterozygosity for H63D. On a subsequent reverse hybridization multiplex polymerase chain reaction (PCR) assay, the patient tested positive for the E168Q mutation in exon 3 of the HFE gene, which was confirmed by direct gene sequencing.Family studies indicated that E168Q and H63D were both located on the same allele. The pedigree is depicted in Figure 3 where the patient and his sister have identical HFE genotypes with heterozygosity for E168Q and H63Q. The observation that H63D and E168Q are inherited from only 1 parent demonstrates that both mutations are located on one and the same allele. Serum iron parameters of the pedigree show (Table 1) normal iron status in the female relatives (sister II.1 and daughter III.1 of the index case), but high normal serum ferritin in the male relative (son of the index case III.2) suggests a potential risk for the development of iron overload.JOURNAL/dimp/04.03/00019606-200903000-00007/table1-7/v/2021-02-17T200000Z/r/image-tiff Demographic and Clinical Biochemical Parameters of Study SubjectsJOURNAL/dimp/04.03/00019606-200903000-00007/figure3-7/v/2021-02-17T200000Z/r/image-jpeg
Pedigree—the index case is marked by an arrow.In view of the advanced stage of liver disease (Child B, Model of End Stage Liver Disease 40) and the (3.2×2 cm) hepatocellular carcinoma stage Union Internationale Contre le Cancer stage II (United Network for Organ Sharing modified TNM stage T2N0M0), the patient was listed for liver
transplantation and underwent successful orthotropic liver
transplantation at the age of 55 years.Histologic examination of the explanted liver specimen revealed micronodular cirrhosis, and Perl's stain revealed increased stainable iron confined to hepatocytes with a decrease in intensity from periportal to centrilobular zones (total iron score 42 according to Turlin and Deugnier32). Despite advanced cirrhosis, where some stainable iron is frequently found, severity of pathology (Figs. 4A, B) with mainly hepatocellular iron storage (Fig. 4D) and the pattern of hepatic iron deposit within the liver lobule were typical for hemochromatosis (Fig. 4C).JOURNAL/dimp/04.03/00019606-200903000-00007/figure4-7/v/2021-02-17T200000Z/r/image-jpeg
A, CAB stain of the explanted liver showing micronodular cirrhosis. B to D, Perl's stain of explanted liver showing markedly stainable iron confined to hepatocytes with a decrease in staining intensity from periportal to centrilobular zones.Chemical quantification of iron in the explanted liver revealed a concentration of 11.7 μmol/g liver dry weight (normal <25 μmol/g) in a random sample of the paraffin-embedded tissue block, which is known to correlate well with the hepatic iron content determined in fresh tissue.33,34 However, iron quantification in liver samples where hepatocytes and fibrous tissue were separated by microdissection, hepatic iron contents of 53.7 μmol/g hepatocyte dry weight and 8.2 μmol/g fibrous tissue dry weight were measured.Five years after successful liver
transplantation, the patient is well and his serum iron parameters are all within the normal range (serum ferritin 253 μg/L, transferrin saturation 23%). A recent liver biopsy showed minimal periportal inflammation and mild steatosis of 15% with minimal fibrosis, but no stainable iron was found (Fig. 5). Biochemical iron quantification revealed a tissue iron content of 7.75 μmol/g liver dry weight.JOURNAL/dimp/04.03/00019606-200903000-00007/figure5-7/v/2021-02-17T200000Z/r/image-jpeg
Perl's stain of a section of a liver biopsy taken from the graft liver 2 years after liver
transplantation, showing no indication of iron storage.MATERIALS AND METHODSClinical biochemical parameters (aminotransferase activities, α-1-fetoprotein, albumin serum iron, and coagulation parameters) were determined by routine clinical chemistry methods (Modular platform, Roche, Basel, Switzerland). Blood count and hemoglobin concentration was determined using routine clinical hematologic protocols on a Coulter Counter (Beckman, Krefeld, Germany).After informed consent was obtained from the index case and all first-degree relatives, DNA was prepared from peripheral white blood cells using the nucleon BACC3 DNA extraction kit (Amersham Biosciences, Vienna, Austria) according to the manufacturer's instructions. Genotyping of the C282Y and H63D polymorphism was carried out by allelic discrimination on the ABI platform according to the method published by Tafe et al.35 Further analysis for common HFE gene variants was carried out by the commercially available reversed hybridization Hemochromatosis StripAssay (ViennaLabs, Vienna, Austria) following the manufacturer's directions.15 The HFE genotype was confirmed by direct DNA sequencing of all exons and exon-intron boundaries of the HFE gene using the following primers exon 1 forward 5′-CAA CAA CAC CCC TTC AGG AT-3′, exon 1 reverse 5′-TGA GCA GGT CCT CCA AAG TT ACA-3′; exon 2 forward 5′-ACA TGG TAA AGG CCT GTT GC-3′, exon 2 reverse 5′-GCT CCC ACA AGA CCT CAG AC-3′; exon 3 forward 5′-CCT GGG GAT GGT GGA AAT AG-3′, exon 3 reverse 5′-CTG CAA CCT CCT CAA CTC TG-3′; exon 4 forward 5′-GTT CCA GTC TTC CTG GCA AG-3′, exon 4 reverse 5′-CTC CTC TCA ACC CCC AAT AG-3′; exon 5 forward 5′-GAT GAG AGC CAG GAG CTG AG-3′, exon 5 reverse 5′-CCC TGG GGC AGA GGT ACT-3′; exon 6 forward 5′-GGT TTG TGA TGA TGC CTC TTT CC-3′, exon 6 reverse 5′-AAT GGG GAA ATC TTT TTG AGG A-3′.Briefly, about 30 μg of genomic DNA was added to a mixture of 1×PCR buffer (Applied Biosystems, Vienna, Austria) 100 μM PCR primers, 2.35 mM MgCl2, and 0.25×Q-solution (Qiagen, Vienna, Austria), which was brought to a final volume of 24 μL with DNase-free water before addition of 1.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Vienna, Austria). After initial denaturation and enzyme activation at 95°C for 5 minutes, PCR was carried out by stepwise heating and cooling to 95°C for 45 seconds, 55°C for 45 seconds, and 72°C for 90 seconds for a total of 36 cycles followed by a final extension phase of 72°C for 7 minutes. Sequencing reaction was carried out using the BigDye Sequencing Kit v1.1 (Applied Biosystems, Vienna), using the reverse PCR primer as sequencing primer. Sequencing was carried out on an ABI prism 3100 capillary sequencer (Applied Biosystems, Vienna) using standard conditions and software for sequence analysis.A block of tissue from the explanted liver was fixed in formalin and embedded in paraffin. For the quantification of liver tissue iron, a random sample from the tissue block was used after deparaffinization with xylene. For iron quantification in liver parenchyma and fibrous tissue, 30-μm sections were cut and the respective tissues separated under the microscope using a needle and forceps. Tissue (1 mg) was collected for each fraction and iron content quantified as follows. Graphite furnace atomic absorption spectrometry was carried out according to the method published by van Deursen et al36 using a Unicam Model Solaar, 939 QZ, atomic absorption spectrometer, equipped with a Zeeman-effect background corrector, an FS90 furnace autosampler, and a longitudinally heated atomizer with extended lifetime graphite tubes (Thermo Electron Corporation, Dreieich, Germany).DISCUSSIONHere we report on a patient who presented with signs of iron storage disease and liver cirrhosis. Increased serum transferrin saturation and hyperferritinemia suggested iron overload and therefore phlebotomy was initiated. However, only 1.2 g of iron was removed over a period of 2 months before anemia developed. The diagnosis of hemochromatosis usually requires that more than 3 g of iron can be mobilized in females and >5 g in males.37 In patients heterozygous for 1 disease-associated HFE polymorphism, liver biopsy is required to differentiate between primary and secondary iron overload syndromes. A diagnosis of hemochromatosis was made based on the amount and distribution of stainable iron in the explanted liver.The HII in this patient was 0.21, which is well below the threshold of 1.9.4 Although the HIC in microdissected liver parenchyma was increased, calculation of HII on the basis of microdissected liver parenchyma resulted in an HII of 1.0, which still makes the diagnosis of hemochromatosis improbable.38 Although it is reported that histopathologic grading of the severity of iron overload correlates with HIC/HII,5 our index case shows that this does not always hold true.The presence of 2 HFE gene variants was indicative for genetic hemochromatosis, but pedigree studies allowed assessment of the relationship between the patient's HFE genotype and iron storage disease. Family studies indicated that both HFE gene variants were in cis on the same chromosome.This case illustrates the difficulties and pitfalls that can arise during the differential diagnostic workup of patients with liver cirrhosis and increased serum iron parameters. Testing for hemochromatosis-associated polymorphisms of the HFE gene is standard clinical practice.39 However, hemochromatosis-associated HFE gene variants have an incomplete penetrance and must therefore be considered as risk factors for the development of hemochromatosis.40 Furthermore, HFE gene mutations are only one of the many known causes of hemochromatosis. The observed frequency of non-HFE hemochromatosis is variable across Europe. More than 90% of all hemochromatosis patients are homozygous for C282Y or compound heterozygous for C282Y and H63D in Northern Europe,41 whereas only ∼75% of all Italian hemochromatosis patients have one of these HFE genotypes.42 The reported frequency of non-HFE hemochromatosis is even higher in the cohort of hemochromatosis patients who require liver
transplantation for end-stage liver disease.43 This suggests that non-HFE hemochromatosis has more severe manifestations and worse prognosis than classic HFE-associated iron storage disease.In summary, hemochromatosis is a complex disease and no consensus has been reached on diagnostic criteria in patients with advanced stage liver disease and iron overload where the clinical, biochemical, and histopathologic presentation is blurred by hepatic dysfunction, which could be a cause or a consequence of disturbed iron metabolism.ACKNOWLEDGMENTThe authors are indebted to the patient and his family for participation in the study. Gisela Egg, Veronika Lacher, Barabara Wimmer, and Anna Schloegl are acknowledged for excellent technical assistance. The authors thank Rajam Csordas for critical discussions.REFERENCES1. Pietrangelo A. Hereditary hemochromatosis—a new look at an old disease. N Engl J Med. 2004;350:2383–2397.[Context Link][Full Text][CrossRef][Medline Link]2. Andrews NC. 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Algorithm for the differential diagnostic workup of patients with suspected iron overload. Symptoms and signs indicating iron overload syndromes include fatigue, arthropathy, diabetes, abnormal skin pigmentation, impotence, or signs of chronic liver or heart failure. In patients with these symptoms, serum iron parameters should be determined. If serum transferrin saturation and/or serum ferritin is increased, basic laboratory tests should include the indicated parameters to exclude hematologic, inflammatory metabolic, and liver disease other than hemochromatosis. HFE genotyping is the first step in differential diagnostic workup of “primary” iron overload and C282Y homozygosity or compound heterozygosity for C282Y/H63D is sufficient for the diagnosis of HFE-associated hemochromatosis. In patients without a disease-associated HFE genotype, a magnetic resonance imaging may help estimating the degree of hepatic overload. In patients with high suspicion of hepatic iron storage, a liver biopsy should be performed to determine the degree of tissue damage and reveal the histologic and cellular distribution of iron. Patients with Kupffer cell iron overload are candidates for ferroportin gene sequencing, where heterozygous mutations are sufficient to cause “ferroportin disease.”27 In patients with predominantly hepatocellular iron overload, rare genetic hemochromatosis syndromes should be considered and genetic tests carried out according to clinical presentation. In isolated cases with severe disease manifestation, occurrence of bigenic hemochromatosis has been reported.28 Aceruloplasminemia or Wilson disease may be a rare genetic cause in patients with, non-HFE hemochromatosis and genetic tests should be requested if iron overload is associated with neurologic impairment.29,30
Computed tomography scan of the abdomen showing a cirrhotic liver, ascites, and a protruding hepatocellular carcinoma. Demographic and Clinical Biochemical Parameters of Study Subjects
Pedigree—the index case is marked by an arrow.
A, CAB stain of the explanted liver showing micronodular cirrhosis. B to D, Perl's stain of explanted liver showing markedly stainable iron confined to hepatocytes with a decrease in staining intensity from periportal to centrilobular zones.
Perl's stain of a section of a liver biopsy taken from the graft liver 2 years after liver
transplantation, showing no indication of iron storage.Diagnosis of Hepatic Iron Overload: A Family Study Illustrating Pitfalls in Diagnosing HemochromatosisSchranz Melanie MD; Talasz, Heribert MD; Graziadei, Ivo MD; Winder, Thomas MD; Sergi, Consolato MD, PhD; Bogner, Klaus MD; Vogel, Wolfgang MD; Zoller, Heinz MDCase ReportCase Report118p 53-60
