Medical Liver Disease: Problem Diagnoses for Practicing Pathologists
Dr. Grace Kim
Dr. Linda D. Ferrell
Dr. Sanjay Kakar
Section 4 -
Linda D. Ferrell
University of California
San Francisco, CA
Iron metabolism is a complex process that is currently becoming more understood, but many aspects of
these processes still remain unknown. The details of iron metabolism is beyond what can be discussed,
but D Trinder, et al have written a good review of the current status of
these investigations (see Ref 2).
Iron balance is essentially controlled by the regulation of iron absorption by the duodenum and
proximal jejunum, but no good mechanism is present for the excretion of excess iron stores. Iron is a
potentially highly toxic chemical, so it must be sequestered in non-toxic forms while still available for
synthesis of such iron-containing proteins as hemoglobin, myoglobin, and catalase. Iron stores in the
liver are present primarily as ferritin; hemosiderin is also present but to a much lesser degree. The
ferritin is present both as free ferritin within the cytosol and within siderosomes. The free cytosolic
ferritin is seen by light microscopy as the blue blush of liver cells by Perls' stain, while the granules
are the siderosomes, a form of secondary lysosome filled with predominantly ferritin. With increased
iron stores, the granules become more prominent, forming first near the bile canalicular membrane in
hepatocytes nearest to the portal zone (zone 1). As iron stores become more pronounced within
hepatocytes, this storage extends to zone 2 and then zone 3 (central zone). The free ferritin within the
cytosol reaches a plateau beyond which the ferritin is than transferred to siderosome. The amount of
iron/ferritin in the siderosomes, however, can increase indefinitely.
Mechanisms of Iron-Induced Fibrosis and Injury
The iron-loaded hepatocytes may release profibrogenic substances, which activate hepatic stellate
cells (which in turn, are thought to be the principal sources of collagen and other matrix proteins in
chronic liver disease), or they may release substances which stimulate Kupffer cells to produce
profibrogenic substances which in turn activate the stellate cells. Iron in toxic amounts can also
induce lipid peroxidation of organic membranes, leading to cell injury and death. These lipid
peroxidation products can also stimulate stellate cells to produce collagen and can stimulate Kupffer
cells to stimulate the stellate cells as well. In addition, the increased iron stores in hepatocytes
leads to catalyzed oxidative destabilization of lysosomes, which then leak digestive enzymes and cause
Assessment of Iron
Light microscopic evaluation of the iron stores in the liver can provide rapid and valuable
information about the levels of iron stores. Three main elements need to be assessed: the grade (or
amount) of stainable iron, its distribution in the various cell types of the parenchyma or portal zones,
and the degree of fibrosis. We commonly use a methodology of 0-4+ for grading of iron stores, by
assessing the overall amount of iron in all cell types together. One practical method of how to
determine the various levels in a relatively reproducible manner is given in the Table 1 below.
Table 1. Histologic Grading of Iron Stores
|Grade ||Magnification (eyepiece and objective) required for observation|
|0 ||Granules absent or barely seen x 400|
|1+ ||Barely seen x250, easily seen x400|
|2+ ||Discrete granules seen x100|
|3+ ||Discrete granules seen x25|
|4+ ||Masses visible x10, or naked eye|
Adapted from MacSween: Pathology of the Liver, Chap 5., Ref 1
Hepatic iron concentration can be obtained by measuring the dry weight of iron in the liver tissue.
Tissue that has been previously embedded in paraffin will still work well for this determination. Using
the iron concentration, the hepatic iron index (HII) can be determined by dividing the concentration (in
umol/g dry weight) by the age of the patient (in years). Values more than or equal to about 1.9 to 2 can
be used to separate patients who are homozygous for the hemochromatosis gene from the heterozygotes, and
can be helpful to identify the homozygous patients from other causes of iron overload in many instances
as well. However, genetic testing is now the preferred methodology to determine hereditary
Classification of Iron Overload
Iron overload can be divided into familial or hereditary forms versus acquired forms. The following
table lists many of the currently known iron overload states (adapted from ref 2 and 5).
Table 2. Classification of Iron Overload
|Familial or hereditary forms of hemochromatosis|
| ||Hereditary hemochromatosis (HHC, HFE1, HFE gene and Hfe protein defect) C282Y homozygosity, C282/H63D heterozygosity, other HFE gene mutations|
| ||Juvenile hemochromatosis (HFE2, Type 2A: HJV gene and hemojuvelin protein;Type 2B: HAMP gene and hepcidin protein defects)|
| ||Transferrin receptor 2 mutation (HFE3, TFR2 gene and transferring receptor 2 protein defect)|
| ||Ferroportin mutation (HFE4, SCL40A1 gene and ferroportin 1 protein defect)|
| ||Autosomal dominant hemochromatosis (from Soloman Islands)|
|Acquired iron overload|
| ||Iron loading anemias, such as Thalassemia major, sideroblastic anemias, chronic hemolytic anemias|
| ||Dietary iron overload (African iron overload)|
| ||Iron via parenteral route, with blood transfusions, parenteral iron medications|
| ||Chronic liver disease such as end-stage cirrhosis, hepatitis C, alcoholic liver disease, non-alcoholic steatohepatitis|
| ||Neonatal hemochromatosis|
| ||Porphyria cutanea tarda|
Hereditary Hemochromatosis (HHC, HFE1)
Hereditary Hemochromatosis, commonly known in the past as primary hemochromatosis, is the most common
form of iron overload, and is an autosomal recessive disorder seen in Anglo-Celtic/Northern European
populations due to mutations in the HFE gene found on chomosome 6p. The HFE gene product is a
transmembrane glycoprotein homologous to Class I major histocompatibility complex. Mutation of the gene
prevents the formation of a disulfide bond that disrupts an important association with a B2microglobulin,
which in turn alters its normal cell surface expression. HFE also associates with transferrin receptors,
and so mutations may decrease the affinity for iron-bound transferrin, altering intestinal absorption.
Two major gene defects have been specifically identified. The most common defect is a single G to A
mutation at position 282, resulting in a cysteine to tyrosine substitution, designated as the C282Y
mutation. The second most common defect is the H63D mutation, which results in a histadine to aspartic
acid substitution, and causes a reduced function of the HFE gene product rather than the more severe
complete loss of function seen with the C282Y mutation.
Pathology - Homozygotes for C282Y
The patients with both gene copies of the C282Y mutation may have the full-blown syndrome of
hereditary hemochromatosis, but the actual penetrance of disease is very low (ref 6), and additional
factors such as alcohol exposure may be factors that enhance the development of disease. In patients
with liver disease, iron deposits can be seen in teenage years in periportal hepatocytes, but no Kupffer
cell iron or fibrosis is typically present. Young males typically have more iron stores than young
females of the same age. With increasing age, iron deposits become heavier in parenchymal cells, with
extension from the periportal zone to the central zone (Zone 1-3 gradient), and Kupffer cells as well as
other cell types such as portal tract macrophages show increased iron stores as well. Fibrosis then is
likely to begin, which probably corresponds to the development of mesenchymal iron deposits. The
fibrosis begins in the periportal areas, causing portal-to-portal septa and a portal-based pattern of
fibrosis. The cirrhosis is typically micronodular in type, with only mild to moderate inflammatory
changes mostly associated with foci of hepatocyte damage and necrosis, and no significant bile ductular
proliferation. The hepatocyte damage (sideronecrosis) is a late stage finding seen in the highest levels
of iron overload.
In later stages of iron overload, small areas of liver parenchyma with very little to no iron can be
found. The Kupffer cells in these foci will contain iron. This change can also be seen in severe
non-hereditary forms of iron overload (secondary hemochromatosis) as well. Some consider these to be
Effects of Iron removal
Therapeutic phlebotomy is the treatment of choice for these patients. The removal of iron occurs in
the reverse order of the accumulation pattern, beginning in the central zone, with the last cells to lose
iron in the periportal area (zone 1). However, the largest clusters of iron deposits may still remain
after the smaller granules are removed, so this should probably be taken into account in the grading
The reversal of fibrosis has been reported. There is well-accepted evidence that there are changes in
the pattern of fibrosis and in the parenchymal architecture. The nodules transform to macronodules,
which contain increased numbers of central veins and tiny portal tract-like structures. These
macronodules are partially separated by thin septa, as seen in the incomplete septal type of cirrhosis.
It is suggested that the iron removal allows the liver to undergo gradual repair and regrowth of normal
structures that expand the size of the original nodules and lead to thinning of the fibrous septa.
However, only a very rare case of restoration of a cirrhotic liver to near normal structure has been
Predisposition for HCC
Homozygotes may have a 2-4x increased risk for
hepatocellular carcinoma (HCC) over those patients with other forms of cirrhosis, and there is definitely
a >200x increase incidence over patients without cirrhosis. The HCC essentially always occurs in
patients with cirrhosis (but rare cases with fibrosis short of cirrhosis have been described). The
removal of iron apparently does not reverse this tendency.
Pathology – Heterozygotes for C282Y
5-10% of C282Y heterozygotes show increased iron stores sufficient to meet the criteria for hereditary
hemochromatosis. These patients will typically have the H63D mutation as the other copy of the gene.
True C282Y heterozygotes with a normal second copy of the gene may show some mild increase in iron
stores, with varying amounts of iron seen from 0-2+, but have no evidence of clinical disease.
Other Familial Forms of Hemochromatosis
The juvenile pattern (HFE2) is an autosomal recessive disorder with the same pattern of iron overload
as seen in the homozygous C282Y form, but instead, the iron overload occurs much sooner, in the second
and third decades. Two gene defects have been identified: HJV on chromosome 1 that codes for
hemojuvelin and HAMP on chromosome 19 that codes for hepcidin. The patients have hypogonadism, other
endocrine manifestations, and cardiac failure as well as cirrhosis.
A defect in the gene coding for transferrin receptor 2 (TfR2) has been labelled as HFE3, a defect in
ferroportin gene as HFE4. The mechanisms underlying the iron overload in these three entities are
unknown. However, it is thought that ferroportin may be key in transporting iron out of the cell, so
lack of this protein may be the cause of the overload in these patients.
A severe defect in ceruloplasmin function (aceruloplasminemia) reduces the rate of iron oxidation
(Iron is oxidased by the ceruloplasmin so that it can be bound to plasma transferrin) and the subsequent
cellular release of iron causes progressive accumulation in the liver, pancreas and brain, resulting in
diabetes and neurodegenerative disease.
A defect resulting in lack of plasma transferrin (atransferrinemia) causes increased iron absorption,
and subsequent iron deposits in liver, pancreas, and heart as well.
An autosomal dominant pattern of inheritance with disease effect similar to HHC has been noted in
Melanesians from the Soloman Islands. The genetic defect remains unknown.
Acquired Iron Overload
The acquired iron overload syndromes are typically those associated with chronic anemias, dietary
overload, and transfusional or parenteral overload.
For the hematologic diseases (such as thalassemia major and most sideroblastic anemias) that are
characterized by severely ineffective erythropoiesis, with markedly hyperplastic marrow and high plasma
iron turnover, the increased iron stores result from both increased absorption and increased
transfusional iron in combination. In the chronic anemias that do not require transfusion therapy, such
as beta thalassemia minor or hereditary spherocytosis, occasional patients develop severe iron overload
that is not apparently routinely associated with HFE gene defect; thus, it is though iron absorption may
be increased in these cases. In the aplastic anemias or hypoplastic anemias, the intestinal absorption
does not appear to be increased to any significant degree, so iron overload in these patients is
Transfusional overload as well as parenteral overload will show heavy preferential early loading of
the Kupffer and other mesenchymal cells such as portal tract macrophages and endothelial sinusoidal cells
as well as iron in hepatocytes. Fibrosis/cirrhosis is extremely rare in these patients.
Excess dietary overload
Iron overload in patients without chronic anemia but who are self-medicating with iron compounds has
been described. Whether these patients are heterozygotes or not for the HFE gene defects is unknown.
The most studied group is the African population whose source of excess iron was found within the
containers used for brewing their traditional beers. The excess iron storage is predominantly in bone
marrow, spleen, and liver. In the liver, the storage is mainly within macrophages in portal areas and
Kupffer cells, with less storage in hepatocytes. Fibrosis occurs in these patients related to the dense
deposits in portal tracts.
Miscellaneous Iron Storage Conditions
Chronic Liver Disease
It is known that end-stage cirrhosis due to a variety of causes (including alcohol, nonalcoholic
steatohepatitis, HBV, and HCV) can show increased iron stores even without genetic evidence of HFE gene
defects. In these patients, the hepatic iron index (HII) can be >1.9. These cases typically show
prominent hepatocyte iron, lack vascular or biliary epithelial iron deposits, and only have minimal
mesenchymal iron within portal zones, macrophages, or Kupffer cells (unlike hereditary hemochromatosis
which involves all these sites by the cirrhotic stage). The deposits of iron are also more scattered,
with considerable variation in the amount of iron seen from nodule to nodule.
It is also thought that the presence of one copy of the HFE gene mutation (C282Y) can cause mild iron
overload in patients with other forms of liver disease (HBV, HCV, steatohepatitis), but most note that
this degree of iron overload does not enhance the progression of the primary disease.
A current debate still exists as to whether this entity is hereditary or acquired as a secondary
effect to severe antenatal liver disease resulting in feto-placental iron processing, with subsequent
iron deposition in a variety of tissues, including liver, heart, gastric and bronchial mucosa, and
endocrine tissues such as pancreas and thyroid. These infants present with hypoglycemia,
hyperbilirubinemia, and other signs of liver failure. The condition may recur in siblings, suggesting a
hereditary factor. Histologically, the liver shows extensive fibrosis and loss of hepatocytes, with
siderosis. Some giant hepatocytes as well as nodular formation can be seen.
Porphyria Cutanea Tarda (PCT)
A common feature of PCT is the deposition of iron in periportal hepatocytes and Kupffer cells. The
degree of deposition is usually only mild, but more severe storage has been noted in patients with
alcoholic liver disease. Some have suggested that heterozygotes for the HFE mutation at C282Y leads to
the iron overload.
- Iron Storage Diseases, Chap 5, In MacSween (Eds), et al. Pathology of the Liver, 2002.
- Trinder D, Fox C, Vautier G, Olynyk JK. Molecular pathogenesis of iron overload. Gut 2002;51:290-295.
- Olynyk JK. Hereditary Haemochromatosis: diagnosis and management in the gene era. Liver 1999;19:73-80.
- Britton RS, et al. Pathogenesis of Hereditary Hemochromatosis: genetics and beyond. Sem Gastrointeint Dis 2002;13:68-79.
- Roetto A. New insights into iron homeostasis through the study of non-HFE hereditary haemochromatosis. Best Practice & Research Clinical Haematology 2005;18(2):235-50.
- Waalen J, Nordestgaard BG, Beutler E. The penetrance of hereditary hemochromatosis. Best Practice & Research Clinical Haematology 2005;18(2):203-20.
- Deugnier YM, et al. Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations. Gastroenterol 1992;102:2050-2059.
- Nash S, et al. Role of liver biopsy in the diagnosis of hepatic iron overload in the era of genetic testing. Amer J Clin Pathol 2002;118:73-81.
- Hohler T, et al. Heterozygosity of the hemochromatosis gene in liver disease- prevalence and effects on liver histology. Loiver 2000;20:482-486.