Review

Pathology of Hepatic Iron

Overload
Marcela A. Salomao, M.D.

OVERVIEW (Fig. 1).2 In HH, a variety of genetic defects ultimately re-

Hemochromatosis is the group of disorders caused by
systemic iron overload. These can be inherited (hereditary
hemochromatosis [HH]) or secondary to a number of con-
ditions, such as multiple blood transfusions, dyserythro-
poiesis, 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 defi-
ciency.1,2 Because the human body has no physiological
mechanism to excrete excess iron, iron homeostasis de-
pends on the regulation of duodenal absorption of die-
tary iron and cycling of erythrocyte iron by macrophages
sults in hepcidin deficiency and subsequent increase in iron
stores in the body.
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 hemochroma-
tosis 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, present-
ing 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
Most HH patients are asymptomatic because the dis-
ease tends to manifest late in its course. Clinical features

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.
From the Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, AZ.
Potential conflict of interest: Nothing to report.
Received July 17, 2020; accepted October 11, 2020.

View this article online at wileyonlinelibrary.com

© 2021 by the American Association for the Study of Liver Diseases

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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).

TABLE 1. MAJOR CATEGORIES OF HH

Frequency and
HH Category inheritance Mutated Gene Normal Protein Function Presentation Liver Biopsy Findings
−/−
Type 1 (classic HH) Common; AR HFE C282Y or compound Interacts with TFR1, hepcidin 4th-5th decade Parenchymal
C282Y/H63D synthesis 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, 2nd-4th decade Parenchymal
hepcidin synthesis hemosiderin
Type 4 Rare; AD FPN (SLC40A1) Iron export from enterocyte Mild disease, splenic Kupffer cell hemosiderin
and macrophages iron accumulation

of iron accumulation are not limited to the liver and in- TABLE 2. CAUSES OF SECONDARY IRON OVERLOAD
clude cardiomyopathy, arthropathy, skin hyperpigmen- Iron overload related to systemic disease
tation, diabetes, and hypogonadism. Laboratory studies Anemias with ineffective erythropoiesis (e.g., thalassemia, sickle cell
anemia, hereditary spherocytosis)
show increased transferrin saturation and elevated ferritin Multiple blood transfusions
levels. The liver is most commonly affected in type 1 HH, Long-standing dialysis
and progression to cirrhosis can occur in as much as 10% Parenteral iron overload
Anemia of chronic disease
of untreated patients, particularly in individuals with very Iron overload related to chronic liver disease
high serum ferritin levels (>1000 ng/mL).4 Neonatal hemochromatosis
Porphyria cutanea tarda
Chronic viral hepatitis B and C
Alcoholic liver disease
SECONDARY IRON OVERLOAD Cirrhosis

Secondary iron overload can occur in a variety of condi-

tions (Table 2), including anemias with ineffective erythro- occur in the setting of chronic liver disease, including alco-
poiesis, blood transfusions, hemodialysis, and anemia of holic and nonalcoholic fatty liver disease (NAFLD), chronic
chronic disease. Significant hepatic iron overload can also viral hepatitis, porphyria cutanea tarda, and cirrhosis. In

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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 ex-
pression leading to increased intestinal iron absorption and
elevated serum ferritin.6 In patients with NAFLD, insulin re-
sistance 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.
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 evalua-
tion of the liver can also identify underlying or superimposed
conditions and help guide clinical management.
The histological pattern of hepatic iron overload is de-
termined according to the cellular compartment where
iron accumulates and correlates with the disease etiol-
ogy. 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 de-
posits are golden-brown refractile granules. Because small
amounts of iron can be difficult to visualize, histochem-
ical stains are typically done. The most commonly used
method, the Perls’ Prussian blue stain, highlights hemo-
siderin 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: paren-
chymal iron overload and Kupffer cell hemosiderosis.
Parenchymal iron accumulation is the pattern seen in
inherited forms of iron overload (also known as primary
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iron overload) and is characterized by iron accumulation
in hepatocytes and bile duct epithelium. In hepatocytes,
hemosiderin initially accumulates in a pericanalicular distri-
bution. 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 mac-
rophages (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 character-
istic of HH and in the absence of underlying liver disease,
it should be followed up with serum iron studies and HFE
gene testing.
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 con-
ditions 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 tran-
sient Kupffer cell hemosiderosis. This pattern is also de-
scribed 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).
Mixed patterns of iron accumulation are not uncom-
mon 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 pa-
tients who lack the most common gene mutations.4 In ad-
dition, it is used to assess the need for iron chelation therapy
in patients with secondary overload, in whom serum ferri-
tin levels may not correlate with the degree of hepatic iron
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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.

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 pa-
renchyma, 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 ac-
counts 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

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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.

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.

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guide the need for phlebotomy, chelation therapy, and
cancer surveillance in patients with cirrhosis.
CORRESPONDENCE
Marcela A. Salomao, M.D., Department of Laboratory Medicine and
Pathology, Mayo Clinic, 13400 E. Shea Blvd., Scottsdale, AZ 85259.
E-mail: salomao.marcela@mayo.edu
REFERENCES
1) Edwards CQ, Griffen LM, Goldgar D, et al. Prevalence of hemochro-
matosis among 11,065 presumably healthy blood donors. N Engl J
Med 1988;318:1355-1362.
2) Brissot P, Pietrangelo A, Adams PC, et al. Haemochromatosis. Nat
Rev Dis Primers 2018;4:18016.
3) Pietrangelo A, Caleffi A, Corradini E. Non-HFE hepatic iron overload.
Semin Liver Dis 2011;31:302-318.
4) Kowdley KV, Brown KE, Ahn J, et al. ACG clinical guideline: heredi-
tary hemochromatosis. Am J Gastroenterol 2019;114:1202-1218.
5) Ludwig J, Hashimoto E, Porayko MK, et al. Hemosiderosis in cirrhosis:
a study of 447 native livers. Gastroenterology 1997;112:882-888.
6) Duane P, Raja KB, Simpson RJ, et al. Intestinal iron absorption in
chronic alcoholics. Alcohol Alcohol 1992;27:539-544.
7) Barisani D, Pelucchi S, Mariani R, et al. Hepcidin and iron-
related gene expression in subjects with Dysmetabolic Hepatic Iron
Overload. J Hepatol 2008;49:123-133.
237  
| Clinical Liver Disease, VOL 17, NO 4, APRIL 2021
20462484, 2021, 4, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/cld.1051 by HACETTEPE UNIVERSITY, Wiley Online Library on [30/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SalomaoPathology of Hepatic Iron Overloa Salomao
8) Bardou-Jacquet E, Morandeau E, Anderson GJ, et al. Regression of
fibrosis stage with treatment reduces long-term risk of liver cancer
in patients with hemochromatosis caused by mutation in HFE. Clin
Gastroenterol Hepatol 2020;18:1851-1857.
9) Turlin B, Deugnier Y. Evaluation and interpretation of iron in the liver.
Semin Diagn Pathol 1998;15:237-245.
10) Scheuer PJ, Williams R, Muir AR. Hepatic pathology in relatives
of patients with haemochromatosis. J Pathol Bacteriol 1962;84:
53-64.
11) Bottomley SS. Secondary iron overload disorders. Semin Hematol
1998;35:77-86.
12) Pakbaz Z, Fischer R, Fung E, et al. Serum ferritin underesti-
mates liver iron concentration in transfusion independent
thalassemia patients as compared to regularly transfused thal-
assemia and sickle cell patients. Pediatr Blood Cancer 2007;49:
329-332.
13) Allali S, de Montalembert M, Brousse V, et al. Management of
iron overload in hemoglobinopathies. Transfus Clin Biol 2017;24:
223-226.
14) Imbert-Bismut F, Charlotte F, Turlin B, et al. Low hepatic iron con-
centration: evaluation of two complementary methods, colori-
metric assay and iron histological scoring. J Clin Pathol 1999;52:
430-434.
15) Ludwig J, Batts KP, Moyer TP, et al. Liver biopsy diagnosis of homo-
zygous hemochromatosis: a diagnostic algorithm. Mayo Clin Proc
1993;68:263-267.
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