The patient is a 54 year old woman who has Type II diabetes, hypertriglyceridemia, and obesity. She
had a mild increase in serum AST level with normal serum alkaline phosphatase and total bilirubin
values. A liver biopsy was performed to assess the putative fatty liver disease, the presence of which
was confirmed. However, hemosiderin was present in the liver, prompting several issues for the
- Would you quantitate the liver iron? If so, how would you do it? Is it necessary to perform
quantitative iron analysis in this case?
- In your daily practice, would you describe the iron and hope the clinician knows what to do to work
it up, or would you actively intervene and join the attempt to determine the cause of the hemosiderosis?
- What additional clinical testing would you advise?
(See end of syllabus for my answers to these.)
A Brief Medical History of Hemochromatosis
In 1889 von Recklinghausen
identified excess tissue iron obtained at autopsy and termed the condition "hamochromastose".1 This
iron was felt to be derived from the blood until 1935 when the British gerontologist Sheldon published
his findings from 311 selected patients, concluding that the iron overload was secondary to increased
iron absorption .2
It was not until 1952 that a therapy became available, when Davis and Arrowsmith reported benefits
from treating these patients with phlebotomy.3 In 1962 the antemortem diagnosis of hemochromatosis
became available with the developing of a grading system for iron in liver samples was developed by
Scheuer and colleagues.4 Easier antemortem diagnosis was made available by the development of
reliable serum iron, iron binding capacity, and ferritin testing in the 1960's and 70's. While an HLA
locus association was identified by Simon et al in 1975,5 it was not until 1996 when Feder and
colleagues identified two missense mutations on chromosome 6p that accounted for the vast majority of
cases of hemochromatosis.6
Terms and Current Definition of Hemochromatosis:
"hemochromatosis" had been defined clinically based on the presence of a combination of an otherwise
unexplained iron overload, frequent presence of iron overload in relatives, and in later stages end-organ
damage (liver, pancreas, etc). Following the discovery of the major gene mutations described below, a
"genetic" definition came into being: ". . . hemochromatosis is now defined as a disorder of iron
metabolism that is inherited as an autosomal recessive trait due to two mutant HFE alleles" 7 This
definition has the advantage of correctly characterizing individuals early in the disease course, it does
not account well for the approximately 10% of individuals who have phenotypic expression of the disease
but lack the major mutations referred to in this definition (C282Y and H63D).
Genetics of Hereditary Hemochromatosis
Population Genetics There is a current general consensus that the
initial hemochromatosis mutation occurred within the last 100-120 generations (@2800) years or so in
Northwestern Europe in a Celtic, Viking, or Germanic population.(Fairbanks) The propagation of this gene
likely occurred because the adverse complications did not become evident until after the reproductive
years and the iron stores were of benefit to a younger adult population living in a region of possibly
limited dietary intake of iron. Affected women were better able to tolerate menstrual or child-birth
related blood loss and all affected individuals could better cope with blood loss associated with trauma
or blood letting (Fairbanks). The worldwide distribution of hemochromatosis reflects the migrations of
the originally affected populations – Caucasians in Northwestern Europe, Canada, USA, Australia, New
Zealand, and South Africa all have roughly similar gene frequencies of 0.06-1.0 and heterozygous carriage
in 10-15% for the major gene mutations.
The HFE Gene Feder et al identified two mutations in an MHC class I
gene on chromosome 6 which is now named the "HFE" gene. The "major" mutation was a G to A substitution
at nucleotide 845 which lead to a cysteine to tyrosine substitution at the amino acid 282, now commonly
referred to a the "C282Y" mutation. The "minor" mutation was a C to G change at nucleotide 187 which
resulted in a histidine to aspartic acid substitution at position 63 ("H63D").
Among patients with marked iron overload ("phenotypic hemochromatosis"), approximately 80-85% (range
59-100%) will demonstrate a C282Y/C282Y genotype, 5% (range 4-8%) a C282Y/H63D phenotype, and the
remainder are not clearly explained currently.(Fairbanks) Hardy-Weinberg analyses indicate that the
C282Y/wild type, H63D/wild type, and H63D/H63D phenotypes are not associated with phenotypic disease and
these patients are regarded as "carriers". There continues to be searching for genes to explain the
5-10% of hemochromatosis patients without the aforementioned mutations, however at the time of this
writing no strong candidates have emerged.
Histopathology of Hereditary Hemochromatosis
The hallmark of genetic hemochromatosis is the deposition of hemosiderin in hepatocytes and biliary
epithelium. Hemosiderin is insoluble and particulate in nature and will appear granular with iron
stains.9 Alternatively, iron may be deposited in the form of ferritin which is soluble and
characterized by diffuse, non-granular light blue staining of the hepatocyte or macrophage cytoplasm and
is very non-specific but not characteristic of genetic hemochromatosis.
In early phases of disease, hemosiderin is deposited in periportal (zone 1) hepatocytes .As
progressive iron accumulation takes place, midzonal (zone 2) and centrilobular (zone 3) hepatocytes will
progressively accumulate iron as will biliary epithelium. Neither inflammation nor fatty infiltration is
a feature of uncomplicated hemochromatosis and their presence suggests alcoholic liver injury or chronic
As iron accumulates, eventually individual hepatocytes will accumulate lethal levels of iron and
undergo "sideronecrosis". Generally sideronecrosis will become evident when liver iron is in the
12-15,000 microgm/gm dry weight liver range although there seems to be considerable variation in this
threshhold. The locally released iron is then taken up into macrophages, however hepatocellular iron
will continue to dominate. By contrast, iron deposition in hematologic disorders occurs primarily in the
reticuloendothelial system (Kupffer cells) and, when present in large quantities, "spills over" into the
hepatocytes to a lesser degree. Thus, the finding of iron deposited primarily in Kupffer cells is not in
keeping with hemochromatosis and does not require measurement of hepatic iron concentration for
diagnostic purposes. Quantification of hepatic iron may, however, be useful in assessing the need for
treatment in exogenous iron overload.
With continuing iron accumulation and sideronecrosis, progressive fibrosis and eventually cirrhosis
may occur. Iron related fibrosis tends to begin when approximately 15,000 microgm Fe/gm dry weight liver
is present although this is just a generalization. Precirrhotic portal and periportal fibrosis takes on
a "holly leaf" configuration. When cirrhosis is present, it is bland in nature with fine fibrous tissue
septa surrounding regenerative nodules.
Another histologic feature described in the hemochromatotic liver is the iron free focus
characterized by a relative reduction of hepatocytic iron and dysplastic changes.10 This lesion is
purported to represent an early step in the development of hepatocellular carcinoma. Both the clinician
and the pathologist should maintain a high index of suspicion for hepatocellular carcinoma as it may
occur in up to one-third of patients with cirrhotic stage hemochromatosis.11
Differential Diagnosis of Hemosiderosis
It should be recognized that hepatic hemosiderin deposition can occur in a wide variety of disease
states other than genetic hemochromatosis. In general, thorough clinical evaluation and careful
histologic examination can distinguish these conditions from genetic hemochromatosis although biochemical
analysis of liver tissue iron (see below) or genotypic analysis may be necessary in ambiguous cases.
One of the most common non-hemochromatotic cause of hemosiderosis is alcoholic steatohepatitis.
Histological features include fatty infiltration and varied degrees of inflammatory reaction. Generally,
stainable hepatic iron is only mild in such instances, but at times it is sufficient to cause confusion
with regard to the question of genetic hemochromatosis. Measurement of hepatic iron concentration
usually allows one to distinguish these conditions. The mechanism whereby mild hemosiderosis occurs in
conditions other than hemochromatosis is unknown;12 increased iron absorption has been postulated in
alcoholic liver disease,13 but has not been supported by measurement of iron absorption following
administration of alcohol.14 The demonstration of increased iron absorption in a small number of
chronic alcoholic men may be pertinent, however.15 In some other instances, mild hepatic iron
deposition likely reflects the heterozygous state of hemochromatosis, given the fact that approximately
10-15% of the Caucasian North American population is so affected and mild iron overload is manifested in
Transfusions and chronic hemolytic disorders commonly lead to hepatic hemosiderin deposition.
Transfusional iron tends to accumulate in Kupffer cells and is thus easily distinguished from the
hepatocellular iron of genetic hemochromatosis. Iron following hemolysis tends to be deposited in both
hepatocytes and Kupffer cells and thus demonstration of hemolysis by laboratory means is very helpful in
distinguishing hemolysis-related hemosiderosis from genetic hemochromatosis.
Chronic viral hepatitis is not uncommonly accompanied by hepatic hemosiderin. The hemosiderin
deposits are largely in Kupffer cells and often contain diffuse, ferritin-type iron, both of which are in
contrast to genetic hemochromatosis.
Accumulation of hemosiderin is fairly common in cirrhosis of any type. Ludwig et al noted stainable
hemosiderin in 145/449 (32.4%) of cirrhotic livers and increased chemically-determined iron concentration
in 91/449 (20.3%), including 38 cases(8.5%) in which the level was in the hemochromatosis range (hepatic
iron index > 1.9).16 In this study, it was felt that hemozygous hemochromatosis only accounted for
5 instances of iron overload. While many of the other cases may have reflected incidental heterozygous
genetic hemochromatosis, it seems clear that iron deposition can occur in cirrhosis as an secondary
phenomenon although the etiology remains unclear. Patients with biliary cirrhosis seem to be less prone
to accumulate iron (7-20%) than do patients with non-biliary cirrhosis(22-67%). In cases of cirrhosis
with iron deposition, one is usually able to determine whether homozygous genetic hemochromatosis is
present using traditional means of assessment, however in ambiguous cases genetic analysis may play a
Quantitative Analysis of Liver Tissue Iron
Introduction In the past 25 years, measurement of iron concentration in
liver biopsy specimens has proven to be a valuable diagnostic tool which has also served to broaden our
concept of hemochromatosis.17 Tissue iron analysis must be performed in a highly qualified laboratory
where rigorous control of processing and analytical procedures are followed to assure accurate results.
The actual analysis is performed in a graphite atomic absorption spectrophotometer. The results are
reported in μmol/g dry weight or in μg/g dry weight. Normal values in the Mayo Metals
Laboratory are 7.2-39.4 μmol (400-2,200 μg) per gram dry weight in men and 1.8-28.6 μmol
(100-1,600 μg) per gram dry weight in women. The variation of repeated determinations on the same
test sample is approximately 5%.
Interpretation of Hepatic Iron Concentration Despite the somewhat
subjective nature of histochemical assessment of deposited iron, it correlates reasonably well with the
hepatic iron concentration as measured chemically. This is especially true at higher concentrations,
which almost always show grade 3 or 4 stainable iron. Nonetheless, occasional instances of disparity are
encountered. Also, the significance of moderate degrees of iron overload (2-3+) may be difficult to
interpret, particularly in adults in the 30-60 years of age range. In such cases, measurement of hepatic
iron concentration is of particular value (see below regarding histologic hepatic iron index). Although
the hepatic iron concentration provides a very useful piece of diagnostic information, in routine
clinical practice this determination is not necessary in every biopsy specimen obtained for suspected
hemochromatosis. For instance, if the pathologist finds little in the way of stainable iron, measurement
of hepatic iron concentration is unnecessary. Conversely, if there is histological evidence of heavy
iron deposition, the hepatic iron concentration may be superfluous.
Heavy iron overload (defined arbitrarily as hepatic iron concentration >10,000 μg/g dry
weight or iron stores as estimated by quantitative phlebotomy exceeding 10 g) is the hallmark of genetic
hemochromatosis. As mentioned above, iron accumulation to this degree is almost never seen in other
conditions. With the advent of HLA typing, however, it has become apparent that homozygous
hemochromatosis can be associated with only modest degrees of iron overload, especially in young
individuals and in premenopausal women. Furthermore, only a portion of those who are genotypically
homozygous manifest disease-related mobidity with advancing age.
The concept of the hepatic iron index was introduced by Bassett, et al. in 1986,17 and it has
proven useful, especially in the interpretation of mild to moderate degrees of hepatic iron overload.
The index is simply calculated by dividing the hepatic iron concentration in μmoles/g by the
patient's age and is expressed in micromoles/g/year. Since the hepatic iron concentration is often
reported in μg/g, the value must be divided by 55.8 to convert it to μmoles/g. The utility of
this determination is based on the observation that hepatic iron concentration rises progressively with
age only in homozygous hemochromatosis. Thus, an hepatic iron index of 1.9 or greater (in normals, the
index is usually <1.0.) generally indicates homozygous hemochromatosis, whereas values <1.9 are
compatible with the heterozygous state, alcoholic liver disease, or other conditions not accompanied by
significant iron overload. Borderline values may be difficult to interpret and a number of caveats exist about the use of the hepatic iron index (below). In ambiguous
cases, genetic analysis is often helpful.
Although the validity of the hepatic iron index has been confirmed in several studies, there are
occasional instances where available evidence and clinical judgment support another interpretation.
Firstly, iron quantitation and hepatic iron index determination for diagnostic purposes should be
restricted to cases in which the histologic distribution of suggests genetic hemochromatosis, i.e.
hepatocellular iron predominates over Kupffer cell iron, and a reasonable clinical explanation for iron
overload does not exist. Transfusional hemosiderosis, particularly in younger individuals, can easily
lead to hepatic iron indices in excess of 1.9. Secondly, the utility of the hepatic iron index in the
pediatric population has not been demonstrated and thus should be used in this group with utmost caution
if at all. Thirdly, as discussed above, it should be kept in mind that the index was originally applied
to cases of early hemochromatosis and must be interpreted cautiously in patients with advanced
chronic liver disease where exceptions appear to occur quite frequently.16 Lastly, sampling
variations may occur (see below).
Because measurement of hepatic iron concentration is not always available, Deugnier, et al. ,18
have investigated the utility of a histologic hepatic iron index based on detailed and systematic grading
of iron deposits in various locations of the liver lobule. In their hands, this provided satisfactory
differentiation of heterozygous and homozygous patients, but other conditions were not evaluated. This
method or the computerized assessment of hepatic iron described by Olynyk, et al. ,19 can be performed
on a standard histologic slide stained for iron. The findings from both methods correlate reasonably
well with the chemical measurement of hepatic iron and support the opinion that determination of hepatic
iron concentration may not be essential in every instance. A pathologist, experienced in assessing
hepatic iron deposition, should assist the clinician in this decision.
Potential Difficulties in Interpretation of Liver Biopsy and Hepatic Iron
Concentration With regard to measurement of hepatic iron concentration, certain potential problems
can give rise to spurious results. The distribution of iron, although generally uniform in the
non-cirrhotic liver, might be quite irregular when cirrhosis is present. Irregular distribution is not
likely to affect the final interpretation if hepatic iron concentration is very high, but it might cause
confusion when iron overload is moderate in degree. As mentioned above, a sample that consists largely
of scar tissue might lead to underestimation of the degree of iron deposition.
The finding of modest hepatic siderosis in a patient with histological evidence of established
cirrhosis should be interpreted with caution as mild iron overload is unlikely to be the cause of
cirrhosis. It has been observed that a threshold level of hepatic iron concentration of approximately 12
times the upper limit of normal is required for the production of significant fibrosis.17 This set of
circumstances was encountered when hepatic iron concentration was measured in livers explanted in the
course of orthotopic liver transplantation.16 In several cases, the hepatic iron index was mildly
elevated above the level of 1.9 and the amount of iron present was considered insufficient to have caused
cirrhosis. The explanation for the mild siderosis observed was uncertain but earlier liver biopsies,
which were available in some cases, clearly showed cirrhosis to be present before excess iron deposition
occurred, thus providing strong evidence against the diagnosis of hemochromatosis.
The occurrence of siderosis in conjunction with alcoholic liver disease presents a common and often
vexing problem. As indicated above, serum iron parameters are often elevated in this setting and
stainable hepatic iron may appear greater than the hepatic iron concentration would suggest. The hepatic
iron concentration, however, rarely exceeds two to three times the upper limit of normal in alcoholic
liver disease.20 On the other hand, alcoholic patients with heavy iron overload (for example,
>5 times normal hepatic iron concentration) have been shown to have genetic hemochromatosis of the
homozygous form.12 Alcohol likely works in synergy with iron to aggravate tissue injury and may give
rise to cirrhosis in the hemochromatotic earlier than would be expected with iron overload alone.17,21,22
The rest of the story . . . .
- The iron was reported semi-quantitatively as mild zone 1 hepatocellular hemosiderosis, with a comment
that formal quantitation was not pursued but that the quantity would likely have been in the 3000-5000
micrograms of iron/g dry weight liver range.
- Active participation in the evaluation of iron overload is usually appreciated by the clinician and
can help minimize costs of the work-up. A phone call was made and there was a discussion about optimum
work-up with the clinician.
- The following tests (with results) were performed:
- Serum Ferritin 1031
- Serum iron 122, iron binding capacity 256, 48% saturation
- HFE genotyping – C282Y/H63D compound heterozygote
The final impression is that this patient has hereditary hemochromatosis with a potentially
clinically significant genotype but relatively mild penetrance. While she may never have suffered
morbidity from the iron, a phlebotomy program was undertaken. Screening of first degree relatives with
serum iron saturation assay was advised.
- von Recklinhausen FD, Uber hemochromatose. Tagelb Versamml Natur Artze Heidelberg 1889;62:324-325.
- Sheldon JH. Haemochromatosis. London, Osford University Press, 1935.
- Davis WD, Arrowsmith WR. The effect of repeated phlebotomomies in hemochromatosis; report of three
cases. J Lab Clin Med 1952;39:526-532.
- Scheuer PJ, Williams R, Muir AR. Hepatic pathology in relatives of patients with hemochromatosis. J
Pathol Bacteriol 1962;84:53-64.
- Simon M, Pawlotsky Y, Bourel M, Fauchet R, Genetet B. Hemochromatose idiopathique: maladie associee
a l'antigene tissulaire HLA 3. Nouv Presse Med 1975;19:1432.
- Feder JN, Gnirke A, Thomas W, et al. A novel MHC class-1-like gene is mutated in patients with
hereditary hemochromatosis. Nat Genet 1996;14:399-408.
- Edwards CQ, Ajioka RS, Kushner JP. Hemochromatosis: a genetic definition. In, Hemochromatosis.
Genetics, pathophysiology, diagnosis, and treatment. Barton JC, Edwards CQ, eds. Cambridge University
Press, 2000; 8-11.
- Fairbanks VF. Population Genetics. In, Hemochromatosis. Genetics, pathophysiology, diagnosis, and
treatment. Barton JC, Edwards CQ, eds. Cambridge University Press, 2000; 42-50.
- Deugnier YM, Loreal O, Turlin B, et al. Liver pathology in genetic hemochromatosis: A review of 135
homozygous cases and their biochemical correlations. Gastroenterology 1992; 102:2050-9
- Deugnier YM, Guyader D, Crantock L, et al. Primary liver cancer in genetic hemochromatosis: A
clinical, pathogenetic study of 54 cases. Gastroenterology 1993; 104:228-34.
- Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer G. Long-term survival in
patients with hereditary hemochromatosis. Gastroenterology 1996; 110:1107-19.
- Powell LW, Fletcher LM, Halliday JW. Distinction between haemochromatosis and alcoholic siderosis.
In: Hall P, ed. Alcoholic Liver Disease. 2nd ed. 1995:199-216.
- Charlton RW, Jacobs P, Seftel H, Bothwell TH. Effect of alcohol on iron absorption. British Med J
- Celda A, Rudolf H. Donath A. Effect of a single ingestion of alcohol on iron absorption. Am J
Hematol 1978; 5:225-37.
- Duane P, Raja KB, Simpson RJ, Peters TJ. Intestinal iron absorption in chronic alcoholics. Alcohol
and Alcoholism 1992; 27:539-44.
- Ludwig JL, Hashimoto E, Porayko MK, Moyer TP, Baldus WP. Hemosiderosis in cirrhosis: A study of 447
native livers. Gastroenterology 1997; 112:882-8.
- Bassett ML, Halliday JW, Powell LW. Value of hepatic iron measurements in early hemochromatosis and
determination of the critical iron level associated with fibrosis. Hepatology 1986; 6:24-9.
- Deugnier YM, Turlin B, Powell LW, et al. Differentiation between heterozygotes and homozygotes in
genetic hemochromatosis by means of a histological hepatic iron index: A study of 192 cases. Hepatology
- Olynyk J, Hall P, Sallie R, Reed W, Shilkin K, Mackinnon M. Computerized measurement of iron in liver
biopsies: A comparision with biochemical iron measurement. Hepatology 1990;12:26-30.
- LeSage GD, Baldus WP, Fairbanks VF, et al. Hemochromatosis: Genetic or alcohol-induced?
Gastroenterology 1983; 84:1471-7.
- Irving MG, Halliday JW, Powell LW. Association between alcoholism and increased hepatic iron stores.
Alcoholism: Clinical and Experimental Research 1988; 12:7-13.
- Adams PC, Agnew S. Alcoholism in hereditary hemochromatosis revisited; prevalence and clinical
consequences among homozygous siblings. Hepatology 1996; 23:724-7.