Pathology of Hepatic Iron Overload

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Abbreviations

AD

autosomal dominant

AR

autosomal recessive

DMT1

divalent metal transporter‐1

FPN

ferroportin 1

HAMP

hepatic antimicrobial protein

HCC

hepatocellular carcinoma

HH

hereditary hemochromatosis

HIC

hepatic iron concentration

HJV

hemojuvelin

NAFLD

nonalcoholic fatty liver disease

TFR

transferrin receptor

Overview

Hemochromatosis is the group of disorders caused by systemic iron overload. These can be inherited (hereditary hemochromatosis [HH]) or secondary to a number of conditions, such as multiple blood transfusions, dyserythropoiesis, and chronic liver disease. The liver is commonly affected, and in severe cases, these disorders can lead to cirrhosis and hepatocellular carcinoma (HCC). This review will cover the histological pattern of hepatic iron overload and its diagnostic and therapeutic implications.

HH

HH is one of the most common inherited disorders in individuals of northern European descent and is defined as pathological iron overload caused by hepcidin deficiency. ¹ , ² Because the human body has no physiological mechanism to excrete excess iron, iron homeostasis depends on the regulation of duodenal absorption of dietary iron and cycling of erythrocyte iron by macrophages (Fig. 1). ² In HH, a variety of genetic defects ultimately results in hepcidin deficiency and subsequent increase in iron stores in the body.

FIG 1.

Iron homeostasis. In normal conditions, iron is absorbed by enterocytes and exported to the plasma through the iron transporter FPN, which is negatively regulated by hepcidin. Hepcidin is a hormone synthetized in the liver in response to hepatocellular proteins HFE and TFR2, which detect the iron concentration and stimulate hepcidin expression. Binding of hepcidin to FPN inhibits iron export from enterocytes and macrophages to the plasma, ultimately resulting in decreased plasma iron concentration. HFE is the protein product of HFE gene (H represents high and FE represents iron).

Four major HH categories have been described (Table 1). Type 1 HH is the most frequent genetic iron overload disease and is caused by mutations in the HFE gene. Homozygous C282Y mutation is classified as type 1a, whereas compound C282Y and H63D mutations are classified as type 1b. Type 2 HH or juvenile hemochromatosis is caused by defects in the hemojuvelin (HJV) gene or hepcidin (HAMP [hepatic antimicrobial protein]) gene, and is the most severe form of systemic iron overload, presenting at an earlier age than the other HH types. Type 3 HH is linked to mutations in the transferrin receptor 2 (TFR2). Type 4 HH is caused by defects in the ferroportin 1 (FPN or SLC4A1) gene and is the only HH category with increased hepcidin. ² , ³

TABLE 1.

Major Categories of HH

HH Category	Frequency and inheritance	Mutated Gene	Normal Protein Function	Presentation	Liver Biopsy Findings
Type 1 (classic HH)	Common; AR	HFE C282Y−/− or compound C282Y/H63D	Interacts with TFR1, hepcidin synthesis	4th‐5th decade	Parenchymal hemosiderin
Type 2 (juvenile HH)	Rare; AR	HJV (type 2A) or HAMP (type 2B)	Hepcidin synthesis	2nd‐3rd decade	Parenchymal hemosiderin
Type 3	Very rare; AR	TFR2	Interacts with transferrin, hepcidin synthesis	2nd‐4th decade	Parenchymal hemosiderin
Type 4	Rare; AD	FPN (SLC40A1)	Iron export from enterocyte and macrophages	Mild disease, splenic iron accumulation	Kupffer cell hemosiderin

Most HH patients are asymptomatic because the disease tends to manifest late in its course. Clinical features of iron accumulation are not limited to the liver and include cardiomyopathy, arthropathy, skin hyperpigmentation, diabetes, and hypogonadism. Laboratory studies show increased transferrin saturation and elevated ferritin levels. The liver is most commonly affected in type 1 HH, and progression to cirrhosis can occur in as much as 10% of untreated patients, particularly in individuals with very high serum ferritin levels (>1000 ng/mL). ⁴

Secondary Iron Overload

Secondary iron overload can occur in a variety of conditions (Table 2), including anemias with ineffective erythropoiesis, blood transfusions, hemodialysis, and anemia of chronic disease. Significant hepatic iron overload can also occur in the setting of chronic liver disease, including alcoholic and nonalcoholic fatty liver disease (NAFLD), chronic viral hepatitis, porphyria cutanea tarda, and cirrhosis. In fact, stainable iron is a fairly common finding in cirrhosis of any cause. ⁵ The mechanisms by which chronic liver disease leads to iron accumulation are only partially elucidated. In the setting of alcohol abuse, for instance, metabolism of excess ethanol results in downregulation of hepcidin expression leading to increased intestinal iron absorption and elevated serum ferritin. ⁶ In patients with NAFLD, insulin resistance appears to play a role in decreased hepcidin levels, causing the so‐called dysmetabolic hepatic iron overload syndrome. ⁷ When untreated, severe cases of secondary iron overload can result in the same complications seen in HH.

TABLE 2.

Causes of Secondary Iron Overload

Iron overload related to systemic disease

Anemias with ineffective erythropoiesis (e.g., thalassemia, sickle cell anemia, hereditary spherocytosis)

Multiple blood transfusions

Long‐standing dialysis

Parenteral iron overload

Anemia of chronic disease

Iron overload related to chronic liver disease

Neonatal hemochromatosis

Porphyria cutanea tarda

Chronic viral hepatitis B and C

Alcoholic liver disease

Cirrhosis

Liver Biopsy

Even though the use of magnetic resonance imaging has reduced the need for liver biopsies to diagnose and grade hepatic iron overload, liver biopsy remains the preferred method to stage hepatic fibrosis and/or evaluate for other liver disease causative factors. ⁴ This is particularly important because advanced fibrosis is associated with higher risk for HCC and increased mortality. In fact, it has been shown that severe liver fibrosis can regress with therapy, and significant fibrosis regression is associated with significant reduction in long‐term risk for HCC. ⁸ Because HH can be occult in the initial phase of disease, liver biopsy sometimes allows for the early identification of patients with HH. Histological evaluation of the liver can also identify underlying or superimposed conditions and help guide clinical management.

The histological pattern of hepatic iron overload is determined according to the cellular compartment where iron accumulates and correlates with the disease etiology. Liver cells (hepatocytes and Kupffer cells) store iron in the form of ferritin, heme, and lysosomal hemosiderin, the latter being the predominant form of stainable iron. ⁹ On routine hematoxylin and eosin stains, hemosiderin deposits are golden‐brown refractile granules. Because small amounts of iron can be difficult to visualize, histochemical stains are typically done. The most commonly used method, the Perls’ Prussian blue stain, highlights hemosiderin granules in blue (Fig. 2). Because normal liver is negative for stainable iron, any positive staining requires mention and interpretation by the pathologist. Two main patterns of iron accumulation have been described: parenchymal iron overload and Kupffer cell hemosiderosis.

FIG 2.

Patterns of hepatic iron accumulation. (A) Normal liver with no iron accumulation. (B) Parenchymal iron overload pattern with pericanalicular hemosiderin present in nearly all hepatocytes. (C) Secondary iron overload with iron accumulation in sinusoidal Kupffer cells (arrows). (D) Mixed pattern of iron accumulation with both Kupffer cell hemosiderosis (arrows) and hepatocellular iron deposition (arrowheads). (E, F) Hepatocellular iron accumulation is zonal and starts in periportal hepatocytes before extending toward zones 2 and 3. (E) An example of grade 1 hepatocellular hemosiderosis; (F) grade 3 hepatocellular iron deposition. Perls’ Prussian blue staining: (A–D) 200× original magnification; (E and F) 100× original magnification.

Parenchymal iron accumulation is the pattern seen in inherited forms of iron overload (also known as primary iron overload) and is characterized by iron accumulation in hepatocytes and bile duct epithelium. In hepatocytes, hemosiderin initially accumulates in a pericanalicular distribution. In the lobules, hepatocellular iron buildup is zonal and first affects periportal (zone 1) hepatocytes, extending toward zones 2 and 3 as iron overload progresses. This results in a gradient of staining from the periphery of the lobules toward the central veins (Fig. 2E,F). Many different grading systems are available for subjective quantification of parenchymal iron. The grading system described by Scheuer, for instance, scores hepatocellular iron on a scale of 1 to 4, ranging from minimal iron accumulation (grade 1) to diffuse accumulation that involves the entire lobule and obliterates the typical gradient (grade 4). ¹⁰ In more severe cases, iron can be found in bile duct epithelial cells and may also extend to the Kupffer cells and portal macrophages (Fig. 3C). In advanced cases, portal fibrosis and eventually cirrhosis develop with an increased risk for HCC (Fig. 3E,F). This pattern of iron accumulation is characteristic of HH and in the absence of underlying liver disease, it should be followed up with serum iron studies and HFE gene testing.

FIG 3.

Primary iron overload. (A) In this example of HH type 1, hemosiderin is easily visualized in periportal hepatocytes as golden‐brown refractile granules (arrow). (B) In the late stages of disease, iron accumulation (arrows) involves hepatocytes and bile duct epithelium (bd). (C) In severe cases, iron diffusely accumulates within hepatocytes, bile duct epithelium, and even Kupffer cells. (D) When untreated, chronic iron overload leads to fibrosis and ultimately cirrhosis. (E) HCC in a patient with HH‐related cirrhosis. (F) Although the background cirrhotic liver (left) shows marked hemosiderin accumulation (arrows), the tumor (right) contains no noticeable iron. (A and B) Hematoxylin and eosin staining, 400× and 200× original magnification, respectively. (C and D) Perls’ Prussian blue staining, 400× and 20× original magnification, respectively. (E and F) Hematoxylin and eosin staining, 20× and 200× original magnification, respectively.

Kupffer cell hemosiderosis, or secondary iron overload pattern, refers to hemosiderin accumulation in Kupffer cells, and sometimes portal macrophages and endothelial cells (Fig. 2C,D). ¹¹ This pattern is seen in a number of conditions summarized in Table 2. It can also be seen after hepatocellular injury (e.g., acute hepatitis) because of the increased turnover of injured hepatocytes leading to transient Kupffer cell hemosiderosis. This pattern is also described in HH type 4 (FPN disease). Even though no formal grading system exists for Kupffer cell hemosiderosis, it can be generally categorized as focal (when it involves sparse Kupffer cells) and diffuse (when most Kupffer cells show stainable iron) (Fig. 4).

FIG 4.

Secondary iron overload. (A) Hemosiderin accumulation occurs predominantly in Kupffer cells, endothelial cells (v), and portal macrophages (not shown). A cluster of hemosiderin‐laden Kupffer cells named siderotic body is seen at the center (arrow). (B) Minimal hepatocellular hemosiderin is not an uncommon finding in severe cases and should not be misinterpreted as primary iron overload. (A and B) Perls’ Prussian blue staining, original magnification 200× and 400×, respectively.

Mixed patterns of iron accumulation are not uncommon and can be challenging to interpret. They typically occur in cases of severe iron overload, chronic liver disease, and multifactorial conditions (Figs. 2D and 4B).

In addition to stainable iron and fibrosis, liver tissue can be used to determine the hepatic iron concentration (HIC). HIC results can help confirm genetic iron overload in patients who lack the most common gene mutations. ⁴ In addition, it is used to assess the need for iron chelation therapy in patients with secondary overload, in whom serum ferritin levels may not correlate with the degree of hepatic iron deposition. ⁴ , ¹² , ¹³ HIC can be determined by colorimetry or atomic absorption. Accurate HIC measurements require adequate liver samples with >1 mg dry weight. This test can be accomplished using fresh or formalin‐fixed tissue. However, since iron accumulation can be heterogeneous in the liver, fixed tissue is preferred because it allows the pathologist to assure the specimen quality (i.e., mostly parenchyma, avoiding scar or capsular areas). ¹⁴ , ¹⁵

The hepatic iron index (HII) is calculated from the HIC and can be useful in early/mild cases of iron overload. It accounts for the fact that iron stores accumulate progressively over time and is calculated by dividing the HIC in μmol/g dry weight by the patient’s age in years. The normal range for HII is less than 1.0. ¹⁵

In summary, hepatic iron overload can be primary, as a result of genetic defects in iron regulatory proteins, or secondary, as a result of increased erythrocyte turnover/hemolysis, systemic diseases, and chronic liver disease. Histological evaluation of the liver is used to establish the predominant pattern of iron accumulation, the degree of iron overload, and the fibrosis stage. This information can ultimately inform the causative factor of iron overload and guide the need for phlebotomy, chelation therapy, and cancer surveillance in patients with cirrhosis.