Pediatric and Developmental Pathology 1, 179–199, 1998
Pediatric and Developmental Pathology
r1998 Society for Pediatric Pathology
PERSPECTIVES IN PEDIATRIC PATHOLOGY
Histopathologic Approach to Metabolic
Liver Disease: Part 1
GARETH P. JEVON
AND
JAMES E. DIMMICK*
Department of Pathology and Laboratory Medicine, Children’s & Women’s Health Centre of British Columbia,
University of British Columbia, 4480 Oak Street, Vancouver, BC, Canada V6H 3V4
Received July 28, 1997; accepted January 22, 1998.
Key words: liver, liver pathology, metabolic diseases,
metabolism, inborn errors
INTRODUCTION
Traditional approaches to the diagnosis of genetic
metabolic diseases are often based on descriptions of
metabolic pathways, for example, urea cycle disorders, or by moiety classes, such as carbohydrates,
amino acids, lipids, or structural proteins. The address
might be by clinical presentation, a method of value to
the pediatrician and of assistance to the pathologist.
Some disorders are expressed acutely and severely in
the fetus and neonate, others in a more chronic
manner in older infants and children. Ultrastructural
morphologists, using an organelle-based approach,
discuss peroxisomopathies, mitochondrial and lysosomal disorders. These viewpoints broaden the approach to differential diagnoses, but the analysis of
diagnostic possibilities often begins with the histologic
picture seen by the pathologist examining a liver
biopsy.
Inherited disorders of metabolism may have
no morphologic impact on the liver, or the manifestation may be minimal, as, for example, in most
urea cycle disorders where trivial steatosis, congestion, or very occasional hepatocellular necrosis
*Corresponding author
occurs. Under these circumstances, the pathologist
has no opportunity to offer a diagnosis, except an
exclusionary one. Many metabolic diseases affecting the liver do create typical histopathologic patterns that assist the pathologist in the diagnostic
workup (Table 1). Others show a limited number of
patterns, while some evolve from one pattern to
another. The interpretation, therefore, requires
knowledge of the histologic mosaic and is improved by understanding the pathogenesis, pace,
and course of metabolic diseases.
The presentation here highlights light- and
electron-microscope manifestations in the liver at
the time of biopsy, with emphasis on the value of
morphology in guiding the investigation of metabolic diseases.
THE LIVER BIOPSY
The liver biopsy allows for the diagnosis of metabolic diseases as well as other clinical entities that
may mimic it, such as neonatal hepatitis; but most
importantly, it provides tissue for a focused investigation using multiple ancillary techniques.
In experienced hands, percutaneous biopsy
(PCB) of the liver in selected children safely produces adequate tissue for diagnostic purposes. A
review of the recent literature indicates that most
Table 1.
Major pathologic manifestations
1. Hepatitic
2. Cholestatic
3. Ductopenic
4. Storage
5. Steatotic
6. Fibrotic/cirrhotic
7. Neoplastic
biopsists use a Menghini, Jamshidi, or Trucut
needle in an intercostal approach to the liver in
sedated children who do not have ascites or a
coagulopathy. Pre-biopsy ultrasound examination
is considered mandatory to exclude focal lesions
that may cause hemorrhage. Biopsy under ultrasound guidance is used for focal lesions. In the
pediatric population, major complications include
hemorrhage into the abdomen or chest requiring
transfusions (incidence 1.1% to 2.2%), which is not
predicted by coagulation studies; pneumothoraces
or bile leaks are less frequent. Less severe complications, such as skin hematomas, oversedation requiring respiratory monitoring, or pain treated by
medication, were recorded in 11.7% of 483 biopsies
in one review. Three deaths reported in three
studies with a total of 811 biopsies occurred in
children with malignancy or post–bone marrow
transplant recipients who developed septicemia or
hemorrhage [1–3]. Transjugular biopsies can be
performed safely by a skilled interventional radiologist in children with ascites or coagulopathy who
are under general anaesthesia [4].
At our institution, percutaneous needle biopsies are generally performed with a 16-gauge (1.5
mm diameter) Jamshidi needle. A single pass usually offers a 20-mm-long core to the pathologist in
attendance. In theory, this should yield approximately 45 mg of tissue. Approximately 20 mg of
liver is adequate for the biochemical diagnosis of
most diseases, especially those lysosomal storage
diseases diagnosed by fluorometric enzyme assay.
Notable exceptions include investigations of glycogen storage disease of unknown type or nonketotic
hyperglycinemia, which may require 100 mg of
tissue, usually obtained by open biopsy. It is of
critical importance that the history and differential
diagnosis be discussed with the pathologist prior to
the procedure so that the tissue can be processed
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optimally. In cases where a particular metabolic
disorder is suspected clinically, the precise liver
weight necessary for enzyme assay is usually available from the laboratory performing the diagnostic
assay. The pathologist should communicate the
expected number of passes to the biopsist.
Generally, we require at least two cores of
liver, one of which is dissected for formalin fixation, submitted in glutaraldehyde for electron microscopy, frozen in OCT for ancillary histochemistry—especially for the demonstration of lipids or
compounds that may be dissolved during fixation
or processing—or polarizing microscopy, and stored
in OCT at 2207C. At the discretion of the pathologist, for consideration of the disease history, at least
half of the other core is usually frozen in liquid
nitrogen and stored at 2707C while portions may
be submitted to virology or used for fibroblast
culture or additional morphology, especially when
examination of portal tracts is more important.
Tissue is often fixed in glutaraldehyde at the time of
biopsy, especially if optimal organelle ultrastructure is desirable. This approach allows for a versatile diagnostic workup and is especially useful
when the histopathologic picture suggests a disorder not clinically suspected. Open or wedge biopsies are rarely undertaken at our hospital (unless
the child undergoes a laparotomy for some other
reason).
HEPATITIC PATTERN
A pattern of hepatitis, resembling either neonatal
hepatitis or chronic (active) hepatitis in infants and
older pediatric patients, will in most instances be
either idiopathic or due to autoimmune, infectious,
or toxic agents, rather than a primary metabolic
disorder (Table 2a,b).
Neonatal hepatitis as a pathologic entity displays ‘‘unrest’’ of hepatocytes through the lobule,
necrosis (usually focal), foci of extramedullary
erythropoiesis, cytoplasmic and canalicular cholestasis, giant cell transformation of hepatocytes, and
portal and lobular inflammation with lymphocytes,
but not in excessive numbers. Lobular or portal
fibrosis is absent or initially minimal. Ductal and
ductular cholestasis is usually absent and there is
no or minimal proliferation of these structures.
Giant cell transformation of hepatocytes alone is
not diagnostic of hepatitis and has many associa-
Table 2a.
Neonatal hepatitides (histologic)
Idiopathic
Idiopathic neonatal hepatitis
Infectious
Viral hepatitides: cytomegalovirus; rubella virus; reovirus 3
Table 3. Giant cell transformation
of hepatocytes
Neonatal hepatitis
a-1-Antitrypsin deficiency
Biliary atresia
Cystic fibrosis
Choledochal cyst
Galactosemia
Bile duct paucity
Niemann-Pick disease
Hepatitis viruses (A, B, C): ECHO virus; Coxsackie
virus; herpes virus; varicella virus; adenovirus;
parvovirus B19; HIV virus
Cytomegalovirus
Gaucher disease
Herpes virus
Neonatal hemochromatosis
Coxsackievirus
Mucolipidosis
Bacterial hepatitides: Listeria monocytogenes; Mycobacterium tuberculosis; Treponema pallidum; bacterial sepsis-associated hepatitis
Toxoplasmosis
Cerebrohepatorenal syndrome
Syphilis
Familial intrahepatic cholestatic
syndromes
Protozoan hepatitis: Toxoplasma gondii
Bacterial sepsis
Bile acid synthetic disorders
Trisomy 21
Immunohemolytic disorders
Trisomy 18
Tyrosinemia, acute
Metabolic
a-1-Antitrypsin deficiency; Niemann-Pick disease type
C; cystic fibrosis; bile acid synthesis disorders
(some); mitochondriopathies (some); peroxisomopathies (some); tyrosinemia
Monosomy X
Table 4.
Hepatitic pattern
Table 2b. Childhood chronic hepatitides
(histologic)
a-1-Antitrypsin deficiencya
Infectious
Cerebrohepatorenal syndrome of Zellwegera
Hepatitis viruses
Autoimmune
Autoimmune hepatitis
Niemann-Pick disease type Ca
Familial intrahepatic cholestatic syndromesa,b
Bile acid synthetic disordersa,b
Neonatal hemochromatosisa
Drug induced
Cystic fibrosisa
Metabolic: Wilson disease; a-1-antitrypsin deficiency;
Indian childhood cirrhosis; ornithine transcarbamylase deficiency
Wilson disease
Idiopathic
Acute tyrosinemia
Indian childhood cirrhosis
Ornithine transcarbamyl transferase deficiency
aMay
bSee
tions (Table 3). Hepatitis histopathology in older
infants and children is well known to pathologists.
Fatty change in idiopathic neonatal hepatitis is rare
in our experience, and its presence implies that
other conditions that may show overlapping hepatitic and steatotic patterns, such as fructose intolerance or galactosemia, be considered.
Metabolic disorders that may manifest a hepatitic pattern are listed in Table 4. Not all are proved
metabolic diseases, for example, neonatal hemochromatosis (neonatal iron storage syndrome), but
these are included for purposes of developing a
differential diagnosis.
Alpha-1-antitrypsin deficiency
Alpha-1-antitrypsin (a1-AT) deficiency in children is
mostly manifested by liver disease in the form of
have pathologic changes of neonatal hepatitis.
Table 5.
neonatal hepatitis, chronic hepatitis, and cirrhosis,
and in adults by hepatocellular carcinoma [5]. It may
rarely have a ductopenic pattern associated with bile
duct paucity. This disease then may span several
patterns encountered in metabolic liver disease (Figs.
1–3).
Most a1-AT is synthesized in the liver where it
is translocated into the endoplasmic reticulum
(ER) [6]. Here it associates with a polypeptide
chain–binding protein and folds into its native
conformation which allows it to traverse the secretory pathway. In Pi ZZ deficiency, the amino acid
substitution results in misfolded molecules. Only
15% dissociate and enter the secretory pathway.
METABOLIC LIVER DISEASE
181
Figure 1. a-1-antitrypsin deficiency (piZZ) in a neonate
with neonatal hepatitis syndrome. Electron microscope
examination of the liver biopsy. Note the accumulations
of granular material distending the endoplasmic reticulum.
Figure 2. a-1-antitrypsin deficiency, PiZZ, in a child with
clinical chronic hepatitis. The liver biopsy shows portal
septal fibrosis with a predominantly lymphocytic infiltrate of the portal tract with involvement of the adjacent lobule. There is moderate steatosis. Two hematoxylin and eosin stains, 3100.
Most of the protein remains bound in the ER and is
later degraded. Hepatocyte injury is thought to be
secondary to these accumulations, possibly because of the effects of released lysosomal enzymes,
disordered cell metabolism, or a cellular response [7].
Liver disease usually presents in the first few
months of life with clinical features of neonatal
hepatitis syndrome; it is estimated that up to 29%
of cases are due to a1-AT deficiency [5,8–10]. The
liver biopsy shows pathologic changes of neonatal
hepatitis and in some cases, sufficient bile duct
proliferation to consider the diagnosis of extrahepatic bile duct obstruction [11,12]. A few cases
have bile duct paucity. A significant proportion of
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Figure 3. Liver biopsy from an infant with chronic
cholestasis and a-1-antitrypsin deficiency (PiZZ). The
biopsy shows a portal tract lacking a bile duct, and in the
adjacent lobule there is a cytoplasmic and cannalicular
cholestasis. A biopsy in Alagille syndrome would also
demonstrate a paucity of bile ducts. Hematoxylin and
eoxin, 3100.
children with PiZZ a1-AT deficiency and neonatal
cholestasis develop childhood cirrhosis (25/45 in
one report), but some cases of cirrhosis have also
been reported with other (SZ and MZ) phenotypes
[13–15]. These phenotypes may be associated with
transient liver enzyme increases in childhood liver
disease, although a1-AT inclusions in association
with chronic active hepatitis and cirrhosis is reported in a minority of affected adults [16–18]. In
the newborn, the histologic diagnosis may be difficult. We use electron microscopy to show the
distended endoplasmic reticulum to suggest a diagnosis of a1-AT deficiency (Fig. 1). a1-AT immunohistochemistry may show striking positivity in nonaffected infants in this age-group, and we do not use it on
its own for diagnostic purposes. The PAS-D stain may
be misinterpreted in cholestatic biopsies, as it also
highlights bile.
Morphologically, the a1-AT collections form characteristic eosinophilic, hyalin, intracytoplasmic globules seen mostly in periportal hepatocytes but also in
bile duct epithelium [5,19]. They measure up to 15 mm
in diameter and are periodic acid–Schiff (PAS) positive
and diastase resistant. Electron micrographs show
distension of sacs of rough endoplasmic reticulum by
electron-dense, finely granular or homogenous material frequently separated from the membrane by a
lucent halo. The inclusions tend to increase in number
with age and may not be seen in biopsies from
children less than 3 months of age.
a1-AT accumulations are not specific for a
diagnosis of deficiency [20]. They have also been
described in conditions such as focal nodular hyperplasia, liver cell adenoma, and hepatocellular carcinoma in which globular collections within focal
cells and similar-appearing inclusions that do not
stain as a1-AT have been reported in centrilobular
locations in adult autopsy material. In spite of
these observations, identification of typical periportal inclusions by histochemistry, immunomarkers,
or electron microscopy are diagnostically useful in
the context of pediatric liver disease. The diagnosis
is made by measurement of a1-AT serum activity,
phenotyping, and liver biopsy.
Niemann-Pick disease type C
In the neonatal period, Niemann-Pick disease type
C (type II) may present with neonatal jaundice,
hepatosplenomegaly, failure to thrive, and death
between 3 and 9 months. The liver pathology
resembles neonatal hepatitis [21–24]. Foamy macrophages or Kupffer cells (Niemann-Pick cells)
may be infrequent or absent in this form of the
disease [25]. As the disease progresses, the liver
shows more storage material and fibrosis. Ultrastructural appearances of whorled and irregular
lamellar inclusions, clefts and lipid cytosomes in
macrophages, and Kupffer cells especially, and to a
lesser extent, hepatocytes, are more specific and
diagnostically useful. These appearances in liver
biopsies determine selection of cases for diagnosis
by demonstration of impaired cholesterol esterification and accumulation within lysosomes in fibroblast culture. The pathogenesis of liver injury is
unknown.
Cerebrohepatorenal syndrome of Zellweger
Primary peroxisomal disorders can be classified
into two groups: those with defective assembly of
the organelle and those with enzyme deficiencies
but formed peroxisomes. Of the group of primary
peroxisomopathies, the cerebrohepatorenal syndrome of Zellweger most commonly has an hepatitic pattern that often evolves rapidly to fibrosis
and cirrhosis. One patient with infantile Refsum
disease and one with di- and trihydroxy cholestanoic acidemia had giant cell hepatitis [26,27].
In cerebrohepatorenal syndrome initially there
is hepatocellular unrest, variability in nuclear size,
nucleolar enlargement, focal necrosis, steatosis,
and canalicular and cytoplasmic cholestasis, with
pseudoacinar and giant cell transformation of hepatocytes [28–33]. In time, lymphocytes and PASpositive, diastase-resistant macrophages may accumulate in sinusoids and portal tracts. Excess but
inconstantly present hemosiderin in hepatocytes
and Kupffer cells may relate to age, with greatest
prominence occurring between 5 and 18 weeks.
Intrahepatic bile ducts may be normal, deficient, or
hyperplastic. By 20 weeks of age the liver is usually
enlarged, firm, and diffusely nodular with portal
and intralobular fibrosis (see Fig. 7 in a second
paper on histopathologic approach to metabolic
liver disease, to be published in the next issue of
Pediatric and Developmental Pathology).
The pathogenesis of liver injury is unknown.
There is no evidence of hepatocellular degeneration, necrosis, or bile duct injury in fetuses at
mid-gestation, but because hepatomegaly may be
present at birth, the liver is probably abnormal late
in gestation. Hepatic pathology develops actively in
the first few weeks of life and usually progresses
rapidly in cerebrohepatorenal syndrome. Abnormal amounts of intermediary bile acids, produced
in a number of the peroxisomal disorders, may
possibly injure hepatocytes and bile duct epithelium [27,34]. Hemosiderin accumulation is too
inconsistent in cerebrohepatorenal syndrome to
support a hypothesis that iron-induced injury is
responsible for the hepatic pathology.
Diagnosis of peroxisome assembly disorders
can be made by documenting absence or paucity of
the organelle in hepatocytes. Standard electron
microscopy may be adequate but morphometric
quantitation and histochemical or immunohistochemical identification of catalase, other peroxisomal enzymes, or organelle membrane proteins is
preferable.
The rare peroxisome assembly disorders, neonatal adrenoleukodystrophy and infantile Refsum
disease are discussed in a second paper on histopathologic approach to metabolic liver disease, to
be published in the next issue of Pediatric and
Developmental Pathology.
Familial intrahepatic cholestatic syndromes
Familial cholestatic disorders (Table 5) may have
an hepatitic pathology in biopsies taken early in the
METABOLIC LIVER DISEASE
183
Table 5.
Cholestatic pattern
a-1-Antitrypsin deficiency
Niemann-Pick disease type C
Cystic fibrosis
Bile acid synthetic disorders
3b-Hydroxy-C27-steroid dehydrogenase/isomerase
deficiency
D4-3-Oxosteroid 5b-reductase deficiency
Familial intrahepatitic cholestasis syndromes
Byler disease
Alagille syndrome
Aagenaes syndrome (Norwegian)
North American Indian cholestasis
Greenland Eskimo cholestasis
course of the disease. Neonatal hepatitis and giant
cell transformation have been noted in Byler syndrome, Alagille syndrome, Aagenaes syndrome
(Norwegian cholestasis), and North American Indian and Greenland Eskimo cholestasis. Metabolic
defects have yet to be identified for these entities,
and pathogenesis is unknown.
Byler disease is characterized by progressive
familial cholestasis that may have an hepatitic
pattern including giant cell transformation, duct
paucity, and fibrosis arising in the perivenular zone
and evolving to cirrhosis. A locus is mapped to
chromosome 18q21-22 in original Byler pedigree,
but not in those with Byler syndrome [35]. Ultrastructural changes include canalicular distention
by particulate bile, diminished microvilli, and interruption of the canalicular basement membrane [36].
Aagenaes syndrome (Norwegian cholestasis),
which is probably autosomal recessive, initially has
a hepatitic pattern that may evolve to fibrosis.
North American Indian cholestasis, which is also
autosomal recessive, has abnormally prominent
peri-canalicular actin filaments. An initial hepatitic
pattern progresses to cirrhosis [37,38].
Cholestasis and giant cell transformation particularly in the perivenular region progress to fibrosis and then periportal fibrosis in Greenland Eskimo cholestasis. Like the other disorders, this one
is probably autosomal recessive [39]. ‘‘Byler’’-like
bile and thickening of pericanicular filaments are
described.
Alagille syndrome is autosomal dominant with
variable penetrance and expression. The gene has
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been mapped to coding mutations of chromosome
20p12 [40–42]. Pathology is characterized by paucity of interlobular bile ducts and there may be
diminished portal tracts [43] (Fig 3). Biopsies taken
early in infancy may show an hepatitic appearance
with giant cell transformation, and normal numbers of interlobular bile ducts. Duct inflammation
and degeneration have also been found. Later
diminished interlobular ducts and absence of inflammation have been noted. Portal fibrosis and
even cirrhosis do occur. Originally considered a
failure of development of the interlobular bile
ducts, Alagille syndrome may be due to an insult,
infectious or otherwise, to the duct, beginning in
utero [44].
Inborn errors of bile acid synthesis
Disorders of bile acid synthesis manifest as hepatitis and progressive cholestasis. It is estimated that
they account for 1–2.5% idiopathic cholestatic liver
diseases. To date, they are not known to cause bile
duct paucity. 3b-hydroxy-C27-steroid dehydrogenase/isomerase deficiency involves a deficiency of
this microsomal enzyme which leads to the accumulation of substrate bile acids. The age of onset is
between 3 months and 14 years and the occurrence
is often familial. The liver shows hepatitis with
giant cell transformation of hepatocytes, canalicular, bile stasis, and inflammatory changes with
progression to cirrhosis [45,46].
The D4-3-oxosteroid 5b-reductase enzyme deficiency presents in the neonatal period with progressive cholestasis and liver disease. Liver biopsies have shown lobular disarray with giant cell and
pseudoacinar transformation, hepatocellular and
canalicular bile stasis, and extramedullary hematopoiesis. Electron microscopy has shown small,
slit-like bile canaliculi without usual microvilli and
containing electron-dense material [47]. Histologic
improvement is reported with ursodeoxycholic acid
therapy.
Wilson disease
Wilson disease is an inborn error of copper metabolism characterized by impaired copper incorporation into ceruloplasmin, decreased excretion into
bile, and copper retention in the liver and other
organs including brain, kidneys, and corneas. The
disease is transmitted through a recessive gene on
Figure 4. A liver biopsy from a child with chronic
hepatitis and Wilson disease shows prominent portal
fibrosis and lymphocytic inflammation with both fibrosis
and inflammation extending into the adjacent lobule.
Hematoxylin and eosin, 3100.
chromosome 13q14-21. About 1 in 90 persons are
heterozygous carriers. In homozygotes the hepatic
copper threshold is usually exceeded by the early
teens, and it is this unbound copper ion that is thought
to damage cell and organelle membranes, acting as a
free radical [48]. Most children will present in the first
two decades with hepatic liver disease, and more than
half of these have no neurologic abnormality.
The liver shows progressive changes in asymptomatic children, beginning with nuclear hyperglycogenation in periportal hepatocytes and steatosis,
followed by periportal mononuclear inflammatory,
fibrosis and perivenular fibrosis, hepatocyte swelling, necrosis, and cholestasis (Fig. 4). There may be
loss of the limiting plate and periportal piecemeal
necrosis. Some progress to micronodular cirrhosis
with bands of fibroconnective tissue separating
regenerative nodules with pseudoacini, periportal
fat, nuclear hyperglycogenation, and perivenular fibrosis. Hepatocytes may be pigmented. Hepatocellular
carcinoma is a very rare conclusion in adults [49–53].
Some children presenting with fulminant
hepatitis have areas of coagulative necrosis. The
remaining hepatocytes contain microvesicular fat
with areas of cell dropout, Mallory hyaline, scant
multinucleated giant cells, and bile duct proliferation. Kupffer cells may be laden with pigment. In
these children the liver is often, but not always,
cirrhotic [54,55]. The characteristic mitochondrial
changes are enlargement and pleomorphism, with
dilated intracristal spaces, large granules, and crystalline, vacuolated, or dense matrix inclusions [56].
Interpretation of the Shikata orcein stain for
copper-associated protein and the rhodamine or rubeanic stains for copper may be difficult, especially in
a needle biopsy of a cirrhotic liver [48,57]. The distribution of stainable copper in the nodules may be variable
with some nodules laden, others not. The orcein and
copper stains show a panlobular distribution when
positive. The absence of stainable copper in some
nodules is difficult to explain, but it may be related to
copper release from injured cells. Cytosolic copper in
Wilson disease is more difficult to identify than granular lysosomal copper seen in other conditions, such as
cholestatic disorders, or in the newborn where it is
seen normally in periportal hepatocytes [58]. Copper
staining is often less intense than that for copperassociated protein. A negative copper stain does not
exclude Wilson disease, especially on a needle biopsy,
and in these circumstances, biochemical determination of tissue copper using the biopsy specimen is
essential.
The entire biopsy specimen may be processed in
copper-free wax and examined prior to biochemical
determination of copper levels [59]. A false-negative
result will occur if the analysis is performed on fibroconnective tissue scars rather than on hepatic parenchyma. Normal hepatic copper content is less than 50
mg/g dry weight of liver. In Wilson disease it is almost
always in excess of 250 mg/g dry weight [60]. Liver
copper content may be borderline in young, asymptomatic affected children, but a normal concentration
excludes this diagnosis in older children or adults. In
Indian childhood cirrhosis, an uncommon disease
outside India, the copper content is usually higher,
whereas in heterozygotes or cholestatic disorders the
concentration is lower.
No single test can be used to diagnose Wilson
disease in all circumstances. Diagnosis is best done
in consideration of the history, physical examination, and laboratory tests, including measurements
of ceruloplasmin, urinary copper excretion, and
liver biopsy for light microscopy, electron microscopy, and quantitative copper determination. Histopathology findings with the electron-microscopic,
mitochondrial-characteristic changes are specific
for Wilson disease.
Indian childhood cirrhosis
Indian childhood cirrhosis (ICC) is a rapidly progressive disease with a non specific hepatitic patMETABOLIC LIVER DISEASE
185
Figure 5. Indian childhood cirrhosis. Note the portal and
lobular fibrosis, modest chronic inflammation, and mild bile
duct proliferation. Hematoxylin and eosin, 3100.
tern in the early stage. It is common in India where
it accounts for more than half of the chronic liver
diseases, but it is seldom reported in emigrant
children. Occasional cases of ICC-like disease have
been described in non-Indians. Most cases present
between one and three years of age [61,62].
The liver biopsies characteristically have enormously elevated copper concentrations, suggesting
that its accumulation is involved in the pathogenesis of the disease [63,64]. Excessive copper may
alter microtubule assembly, leading to accumulations of intermediate filaments as Mallory’s hyalin,
that interfere with intracellular transport, retention of secretory proteins, and ballooning of hepatocytes [65]. Brass and copper household utensils
have been suggested as a possible source of copper
exposure. Boiling and storage of milk in these
unplated utensils is common practice in rural
India. A recent observation that boiled animal milk
stored in brass utensils with a high casein-bound
copper content may be relevant, but in one report,
almost half of the patients with ICC did not use
brass utensils [66,67]. Other studies point to a
possible genetic component, implying there may be
an associated disorder of copper metabolism.
There are three stages of progressive change that
correlate with the clinical early, intermediate, and late
phases of disease: early injury characterized by ballooning degeneration, Mallory’s hyalin, and portal fibrosis;
progressive degeneration and necrosis with cholestasis, inflammation and creeping fibrosis; cirrhosis with
extensive fibrosis and Mallory’s hyalin in over threequarters of hepatocytes [62] (Fig. 5).
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Histology reviewed by Joshi includes active
hepatocellular injury, inflammation, fibrosis, nodularity, and a paucity or absence of regenerative
activity [62]. There is ballooning or necrosis of
hepatocytes. These may have a ‘‘birds eye’’ appearance with a centrally located nucleolus in a vesicular nucleus. Mallory’s hyalin is seen in all biopsies
and affects a progressively larger proportion of
cells. Chronic active inflammation in the portal
tracts, lobules, or septa is variable. Polymorphonuclear leukocytes may surround necrotic hepatocytes. Hepatocellular and canalicular cholestasis
with bile duct proliferation is present. Creeping
fibrosis surrounds individual hepatocytes or small
groups of cells. There is perivascular and subendothelial fibrosis of central veins. Glycogen depletion
and multinucleated giant cells are characteristic of
the advanced cirrhotic lesion. Kupffer cells may
contain iron and lipofuscin, while hepatocytes show
coarse brown aggregates of copper-associated protein and panlobular cytoplasmic copper with Shikata’s orcein and rhodamine stains, respectively.
Electron microscopy reveals Mallory’s hyalin,
indistinct mitochondria, dilated rough endoplasmic reticulum, and irregular dense cuprosomes.
The liver in ICC has very high copper concentrations with a reported average of 1389 6 525 mg/g dry
weight (expected range, 5–15 mg/g) [68]. Although
elevated hepatic copper content is found in neonatal
livers and accompanies hepatitic disorders such as
Wilson disease, primary biliary cirrhosis, cholestatic
hepatitis, and extrahepatic biliary obstruction, the ICC
copper has a more diffuse intralobular distribution
and the concentrations are higher [69].
Ornithine transcarbamylase deficiency
In heterozygous female patients an hepatitic pattern may develop. Inflammation and hepatocellular necrosis, with a piecemeal pattern, are associated with steatosis and fibrosis [70]. This
uncommon manifestation enters the differential
diagnosis of hepatitis in the older child or adult.
The diagnosis is made by demonstration of markedly reduced enzyme activity in frozen liver samples.
CHOLESTATIC PATTERN
Cholestasis dominates in this histologic category
(Table 5). There is overlap particularly with the
neonatal hepatitic group. This is not surprising,
Table 6.
Ductopenic pattern
a-1-Antitrypsin deficiency
Cerebrohepatorenal syndrome of Zellweger
Coprostanic acidemia
Alagille syndrome
Byler disease
Cystic fibrosis
given the frequency with which cholestasis and
giant cell transformation occur in pediatric liver
disease. The entities in this group are discussed in
the sections on hepatitic and storage patterns.
There are two other histologic variations within
this group. One with biliary tract obstructive features (ductular and ductal proliferation and lumenal bile plugging) is exemplified by alpha-1antitrypsin deficiency and occasionally cystic
fibrosis, in which anatomic obstruction exists. This
cholestatic pattern requires distinction from other
forms of biliary tract obstruction, such as atresia,
and from the pathologic changes induced by parenteral alimentation. The other variation is ductopenic cholestasis (see Table 6) [71,72]. Note that of
metabolic disorders, alpha-1-antitrypsin deficiency
in particular and cystic fibrosis produce neonatal
hepatitic cholestatic and ductopenic patterns (Fig.
4), although for cystic fibrosis the hepatitic and
duct paucity expressions are very rare.
DUCTOPENIC PATTERN
A ductopenic group (Table 6) warrants separate
identification in view of the observation that although usually associated with prominent cholestasis, the latter may resolve, as exemplified by syndromic Alagille and nonsyndromic bile duct paucity
(Fig. 3). The entities in this group are discussed
above in the sections on cholestatic and hepatitic
patterns.
Paucity of intrahepatic bile ducts arises as a
primary developmental defect, presumably in some
instances from atrophy, ischemia, toxic or infectious and inflammatory mechanisms. Identification of duct paucity requires proper quantitative
documentation of deficient numbers of ducts.
The ratio of interlobular bile ducts to portal
tracts is normally 0.9:1.8 and there are 8.2 1 4.3
portal tracts in 10 mm2. In Alagille syndrome, the
interlobular duct-to-portal tract ratio is 0:0.4; por-
tal tracts may be reduced to 4.5 6 2.9 per 10 mm2
[43,73]. Accurate quantitation requires that a sufficient number of portal tracts ranging from 5 to 20
be examined [74]. It is important to remember that
the development of the intrahepatic biliary system
continues through and beyond full gestation. Thus
reference to established normal values or use of
age-matched normal controls is necessary when
evaluating the liver of premature and neonatal
infants [75].
STORAGE PATTERN
In this group, the cells of the liver exhibit a bloated
appearance because of expansion of the cytoplasm
by accumulated material (Table 7). Hepatocytes,
Kupffer cells, portal macrophages, and duct epithelium may be involved, and depending on the disease, singly or in combinations. The intracellular
storage sites vary, as they are cytoplasmic or within
lysosomes, endoplasmic reticulum, and Golgi
vesicles. Storage in its broadest sense may include
extracellular locales of canaliculus or bile duct
lumen. The accumulated material may be sufficiently distinct by appearance and locale to be
diagnostic. In some instances, however, a definitive
diagnosis is not possible, although the pattern may
suggest a category of metabolic disease.
The storage pattern, as one might expect, is
mimicked frequently by physiologic or acquired
pathologic changes. This potential confusion is
especially relevant with lipid or glycogen accumulation (see also Steatotic Pattern, in Histological
Approach to Metabolic Disease, Part 2, to be published in the next issue of Pediatric and Developmental Pathology). Drug-induced mimickers include
phospholipidosis, lipid deposition, and lipofuscin
accumulation; the latter two are exemplified by the
consequences of intravenous alimentation. In ischemia and hypoxemia, hepatocellular swelling occurs. There may be steatosis and eosinophilic protein aggregates in the endoplasmic reticulum
simulating a storage disease. Chronic cholestasis
may produce foamy cells (pseudoxanthoma). Cells
of Ito (perisinusoidal cells) with excessive vitamin
A storage become foamy. The liver of normal
fetuses in late gestation may have abundant glycogen and nuclear glycogenation of periportal hepatocytes is seen in normal children.
METABOLIC LIVER DISEASE
187
Table 7.
Storage pattern
Disorder
Hepatocyte
storagea
Macrophage
storagea
Site
Mucopolysaccharidoses
11
12
Lysosome
Mannosidosis/fucosidosis
11
12
Lysosome
Sialidosis
11
12
Lysosome
Mucolipidosis
11
12
Lysosome
GMI general gangliosidosis
11
12
Lysosome
12
Lysosome
Metachromatic leukodystrophy
12 biliary epithelia
12
Lysosome
Farber disease
11
12
Lysosome
Gaucher disease
11
12
Lysosome
Niemann-Pick disease
11
12
Lysosome
Wolman disease
12
12
Lysosome
11
12
Lysosome/cytoplasm
Cystinosis
Cystic fibrosis
Duct lumen
Peroxisomopathy (some)
a-1-antitrypsin deficiency
12
Endoplasmic reticulum
Glycogen storage disease I
12
Cytoplasm
Glycogen storage disease II
12
Glycogen storage disease III
12
Cytoplasm
Glycogen storage disease IV
12
Cytoplasm
Glycogen storage disease VI
12
Cytoplasm
Wilson disease
12
11
Cytoplasm/lysosome
Indian childhood cirrhosis
12
11
Cytoplasm/lysosome
Neonatal hemochromatosis
12
11
Cytoplasm/lysosome
Genetic hemochromatosis
12 1 and biliary epithelium
11
Cytoplasm/lysosome
Protoporphyria
12
11
Cytoplasm/lysosome/duct lumen
aThe
12
Lysosome
score approximately expresses the degree of storage for cell type.
Lysosomal storage sites
Lysosomal storage diseases (LSD) are characterized by accumulations of exogenous endocytic
substrate or endogenous metabolite in membranebound vesicles. In the liver biopsy, LSDs may
manifest as an expansion of cytoplasm in some
cells, but other histologically ‘‘normal’’ cells can
also be seen to be involved using special stains or
electron microscopy, which are very important
diagnostic tools in this group of disorders. Hepatocytes in many of the LSDs resemble one another
histologically, especially when the stored product is
extremely water soluble as in the mucopolysaccharidoses and in glycogen storage disease, type II.
Since many of the accumulated metabolites may be
dissolved during routine fixation or processing,
examination of frozen or specially fixed tissues is
often enormously helpful. Lysosomes may be iden188
G.P. JEVON
AND
J.E. DIMMICK
tified in frozen sections with an acid phosphatase
reaction, which may highlight increased or abnormal activity.
Kupffer cells and macrophage cells in the
reticuloendothelial system are rich in lysosomes.
These Golgi apparatus derivatives contain acid
hydrolases (including phosphatases, glycosidases,
phospholipases, sulphatases, etc.) synthesized in
the endoplasmic reticulum. Mannose-6-phosphate
(M-6-P) binds the enzyme to the Golgi membrane,
preventing leakage into the cytoplasm while polypeptide folding and oligosaccharide chains protect
it from autodigestion. Lysosomes merge with phagosomes and autophagosomes. Digestive products
usually pass the lysosomal lipid membrane freely
or require carrier-mediated transport.
Most lysosomal enzyme deficiencies are secondary to genetic mutations. The failure of M-6-P
enzyme incorporation (I-cell disease, pseudoHurler polydystrophy), absence of protective proteins (galactosialidosis), or defective transport of
digested products from the lysosome (cystinosis,
sialidosis) may cause functional enzyme deficiency.
Substrates may cause cell injury by mechanical
disruption of the cytoplasmic circulation, metabolite imbalance, or metabolite toxicity. The pace of
evolution of pathologic change varies widely in
LSD, from rapid, as in Wolman disease, to slow, as
in some forms of Gaucher disease.
Most LSDs involve glycolipid, phospholipid,
or mucopolysaccharide metabolism. Cell membrane glycolipids and phospholipids are arranged
in bilayers and may form lamellar configurations
such as myelin or zebra bodies. Mucopolysaccharides (glycosaminoglycans) are mostly secreted by
fibroblasts in the extracellular spaces in epithelial
tissues. They are dissolved by conventional fixation, leaving empty spaces or sparse reticular granular structures in lysosomes. Glycoprotein products
are incompletely degraded oligosaccharides, glycopeptides, and glycolipids. Many of these are hydrophilic and cause changes similar to those in mucopolysaccharidoses.
The mucopolysaccharidoses (MPS) are caused
by degradative MPS (glycosaminoglycan) enzyme
deficiency. Catabolism of dermatan-, heparan-, keratan- or chondroitan-sulfate may be blocked. They
share many features, including chronic, progressive multiorgan involvement, particularly hepatosplenomegaly, with stored, undegraded MPS in
lysosomes. Liver biopsy in types I, II, III, and VII
(Hurlers, Hunter, Sanfilippo, and Sly syndromes,
respectively) shows marked vacuolization and swelling of some Kupffer cells and hepatocytes. This
may have a lobular distribution of progressive
decrease in size from periportal to perivenular
regions. These and other cells, which do not appear
enlarged, may be filled with small vesicles. These
are demonstrated especially well in 0.5–1.0 mm
osmium tetroxide–uranyl acetate fixed sections
stained with toluidine blue. The storage product
may be demonstrated with a variety of stains, but
the colloidal iron stain is favored by some pathologists [76]. Liver fibrosis and cirrhosis have been
described [77,78]. Electron microscopy shows a
scant intralysosomal fibrillogranular material [79].
Storage products are typically difficult to demon-
strate unless great care is taken to minimize aqueous extraction during fixation or processing. Use of
special fixatives such as tetrahydrofuran or frozen
sections may prevent this problem. A negative
result does not rule out MPS, especially in younger
age-groups or in those types with some enzyme
activity. The definitive type of MPS is decided by
fluorometric enzyme assay.
The sphingolipidoses are associated with incomplete lysosomal degradation of gangliosides,
sphingomyelin, and glycosphingolipids. Gaucher
disease is caused by glycocerebrosidase deficiency
resulting in accumulation of glucosylceramide, a
glycoprotein found with fatty acids in cell membranes. The liver is affected by all types of Gaucher
disease. The diagnostic features are produced by
sphingolipid engorged Kupffer cells and macrophages in sinusoids and portal areas (Fig. 6). These
cells are large with an eccentric nucleus and eosinophilic, corrugated cytoplasm. Tightly packed lysosomes are filled with tubular glucocerebroside structures (Fig. 6). Hepatocytes are not involved in
storage. Fibrosis or cirrhosis may evolve in the
lobules and portal tracts as a consequence of
progressively advancing storage, probably causing
pressure atrophy (Fig. 6).
Liver disease is a common feature of earlyand later-onset forms of Niemann-Pick disease.
Types A and B are due to sphingomyelinase deficiency whereas Type C is caused by a defect of
intracellular trafficking of cholesterol. Type C may
present in early life with a neonatal hepatitis-like
picture including giant cell transformation (see
Hepatitis pattern). In Niemann-Pick disease there
is a progressive increase of storage cells within
hepatic sinusoids. These are reticuloendothelial
cells with uniform birefringent cytoplasmic droplets, which, especially in the acute forms, may be
infrequent in very young infants. There is also
hepatocellular vacuolation staining as sphingolipids and cholesterol [80] (Fig. 7). Hepatic lobules
become progressively distorted with portal septal
fibrosis and cholestasis. Ultrastructurally, cytosomes with typical whorled, concentric membranes are diagnostically useful. Sea-blue histiocytes may be present in the more chronic forms
[81]. Lipopigment or ceroid gives the reticuloendothelial cells a pale yellow discoloration and granules stain blue-green with the Wright-Giemsa stain.
METABOLIC LIVER DISEASE
189
Figure 7. Niemann-Pick disease in an older child with
hepatomegaly. Note the swollen appearance with some
vacuolation of hepatocytes which creates an irregular
pattern to the lobule. Also present are foamy histiocytes
within the sinusoids.
Figure 8. Infant with hepatomegaly and mucolipidosis
type II. In this autopsy liver specimen, sinusoidal foamy
histiocytes are particularly abundant. There is some
vacuolation in hepatocytes. Hematoxylin and eosin,
3400.
Figure 6. Gaucher disease. a: Electron microscope appearance of the liver in Gaucher disease. The distended
lysosomes contain many tubular profiles. b: Liver biopsy
from a child with Gaucher disease and hepatosplenomegaly. Note the abundant storage histiocytes that have
displaced presumably through compression atrophy.
Fibrosis is also present. Hematoxylin and eosin, 3100.
c: Liver at autopsy from a young adult with Gaucher
disease. Note the irregular and nodular outline. The pale
areas represent large zones of storage histiocytes with
fibrosis following compression atrophy of hepatocytes.
They represent intralysosomal accumulation of
sudanophilic, PAS-positive, autofluorescent material with ultrastructural fingerprint lamellae. Seablue histiocytes have also been reported in neuro190
G.P. JEVON
AND
J.E. DIMMICK
nal ceroid lipofuscinosis and in other nonstorage
diseases in which lysosomes are filled with undigestable materials.
The mucolipidoses have hydrolase deficiencies due to abnormal lysosomal enzyme transport,
especially in the mesenchymal cells [82]. The group
includes I-cell disease (mucolipidosis II) and
pseudo-Hurler polydystrophy (mucolipodosis IV).
The histologic appearance is similar in both [83].
There is vacuolation of fibrocytes in the portal
tracts, foamy histiocytes, and involvement of
Kupffer cells (Fig. 8). There may be vacuolation of
hepatocytes, which stain positively for neutral lipid.
Ultrastructurally, the vacuoles are single-membrane bound and are often electron lucent or
Figure 9. Neonate with mild Hurler-type dysmorphic
features, hepatomegaly, and sialidosis. a: The liver shows
prominent vacuolation of hepatocytes. Vacuolated histiocytes are seen as well in small numbers in the sinusoids.
Hematoxylin and eosin, 3400. b: Ultrastructural appearance of the lysosomes in the hepatocytes. The lysosomes
are sparsely populated by nonspecific fibrillogranular
inclusions.
contain reticulogranular or membranous material
[84]. Involvement of mesenchymal structures, particularly fibrocytes and endothelial cells, predominates.
Disorders of glycoprotein degradation are
caused by deficiencies of enzymes that sequentially
remove glycoprotein oligosaccharide side chains.
There is hepatosplenomegaly in fucosidosis, mannosidosis, and the congenital and infantile forms of
sialidosis. The sialidoses, formerly mucolipidosis 1,
involve storage of sialic acid, free and bound. The
infantile dysmorphic types may present in the
neonatal period with hydrops fetalis or congenital
ascites. These infants have biochemical evidence of
liver dysfunction, hepatomegaly, and prominent
vacuoles in Kupffer cells and hepatocytes with
PAS-positive, diastase-resistant staining [85] (Fig.
9). Ultrastructurally, lysosomes contain sparse, non-
specific granular and fibrillar material (Fig. 9).
Lamellar membranous arrays may be present. Portal fibrosis may be marked, causing nodularity
approaching cirrhosis.
Lysosomal membrane transfer defects include
a failure to transport sialic acid or cystine from the
lysosome. Sialic acid storage results in a spectrum
of disorders. In the severe infantile free-acid storage disease, there is hepatosplenomegaly and the
liver biopsy appears similar to that seen in sialidosis, but the vacuoles stain with colloidal iron.
Electron microscopy shows lysosomes containing
small amounts of fibrillar granular material [86].
Most are empty. In cystinosis liver histology has
shown cystine crystals in Kupffer cells, sinusoidal
fibrosis and hepatocyte atrophy, and hepatic venoocclusive disease [87,88].
Infantile GM1 gangliosidosis, an acute neurovisceral disorder, presents in the neonatal period, or
earlier with hydrops fetalis or ascites [89,90].
b-galactosidase deficiency leads to GM1 ganglioside
accumulation within lysosomes. The liver is enlarged
and vacuolation of Kupffer cells and hepatocytes is
present. Ultrastructurally, nonspecific fibrillogranular
material is present in the vacuoles. The liver in TaySachs or Sandhoff disease in the GM2 group of gangliosidoses is histologically normal, but zebra bodies may
be seen on ultrastructural examination.
Wolman disease and cholesterol ester storage
disease are caused by deficiency of acid lipase. The
liver becomes enlarged and yellow. Hepatocytes and
Kupffer cells are swollen and vacuolated (Fig. 10). The
portal and periportal areas are filled with foamy histiocytes and portal and periportal fibrosis progresses to
cirrhosis, which occurs frequently and rapidly in Wolman disease. There may be cholestasis with bile duct
proliferation in Wolman disease [91]. The availability
of frozen tissue is of critical importance to the demonstration of birefringent cholesterol ester crystals in
Kupffer cells and portal macrophages. Ultrastructurally, most of the lipid is found in lysosomes and with
cholesterol crystal profiles.
Farber disease, a ceramidase deficiency, is
characterized by collections of histiocytes, some
foamy, and systemic lipogranulomata with multinucleated cells progressing to fibrosis. Ultrastructural studies show comma-shaped curvilinear, tubular structures or Farber bodies, probably representing
intralysosomal ceramide [92,93].
METABOLIC LIVER DISEASE
191
Figure 10. Neonate with hepatomegaly, liver dysfunction, and Wolman disease. The liver biopsy shows marked
varying-sized vacuoles in hepatocytes and sinusoidal
histiocytes. Developing portal and sinusoidal fibrosis
also occurred. Hematoxylin and eosin, 3100.
Figure 11. Pompe disease (glycogen storage type II) in a
hypotonic infant. The electron microscopic appearance
of the liver illustrates multiple lysosomes distended with
granular material (glycogen).
In the late infantile and juvenile forms of
metachromatic leukodystrophy, a deficiency of arylsulfatase A leads to collections of cerebroside sulfate seen in periportal hepatocytes, biliary epithelium, and in Kupffer cells and macrophages [94].
The accumulations stain as brown metachromatic
granules in 1% cresyl violet frozen sections at low
pH. They also stain with alcian blue, colloidal iron,
and PAS. Prismatic and tuffstone inclusions are
diagnostic ultrastructural findings.
Glycogen storage disease type II, Pompe disease, is due to acid maltase deficiency [27,95].
Intralysosomal accumulation of glycogen occurs
generally. The liver enlarges as all cells participate
in the storage that is especially well observed
ultrastructurally. Glycogen-engorged lysosomes are
readily apparent and diagnostic (Fig. 11). By light
microscopy hepatocytes will appear mildly enlarged and may have a delicately vacuolated cytoplasm (Fig. 11); this is best appreciated in PASstained sections after diastase digestion of glycogen.
To exclude microvesicular lipid as a cause, a frozen
section stained for neutral lipid is helpful.
tion. Glycogen accumulation in many organs including liver, heart, kidneys, and skeletal muscle
may result in marked organomegaly and dysfunction presumably by interfering with intracellular
transportation and metabolism. The clinical and
biochemical findings are diverse, but types I, III, IV,
VI, and IX have clinical and pathological expressions that involve the liver. Type II, Pompe disease,
is a lysosomal storage disease that can readily be
distinguished from other glycogenoses by light
microscopy (see above).
Type I GSD is clinically the most severe. Type
Ia is due to a deficiency of glucose-6 phosphatase,
whereas type 1b has normal glucose-6 phosphatase
but a deficiency in a transmembrane protein (translocase A1), required for glucose-6-phosphate transport into microsomes. Large quantities of glycogen
enlarge especially the liver and kidneys. Deficiency
of amylo-1, 6-glucosidase, a debranching enzyme,
causes type III GSD, whose manifestations are
similar to those of type I but are often less severe.
Liver disease in some of these children may progress
to cirrhosis. Children with type VI GSD (liver
phosphatase deficiency) or type IX (phosphorylase
b-kinase deficiency) usually have hepatomegaly
without symptomatology.
In GSD, the liver biopsy shows a panlobular
regular or irregular mosaic pattern formed by
glycogen-swollen hepatocytes with accentuated cell
membranes and pale cytoplasm, causing sinusoidal compression [95] (Fig. 12). Nuclear hyperglycogenation is characteristic of types I and III and its
Endoplasmic reticulum storage site
a-1-antitrypsin deficiency is the foremost example
in this category (see Hepatitic Patterns, above, for
discussion; Fig. 1).
Cytoplasmic storage site
The glycogen storage diseases (GSDs) are characterized by a deranged glycogen degradation or forma192
G.P. JEVON
AND
J.E. DIMMICK
presence separates these from types VI and IX (Fig.
12). Type 1 GSD hepatocytes usually show lipid
vacuoles of small or medium size. Types III, VI, and
IX may have portal septal fibrosis. The glycogen
can best be demonstrated by a PAS stain performed
on frozen tissue fixed in ethanol and is completely
digestible by diastase. None of these changes is
definitely diagnostic of a particular type of GSD.
For instance, fibrosis may also occur in type 1 GSD,
and the other GSDs may contain small amounts of
intracytoplasmic fat. Nevertheless, the morphologic pattern is usually sufficiently distinctive to
offer direction for biochemical investigations
[96,97]. Ultrastructurally, the hepatocytes in these
GSDs contain dense pools of glycogen, mostly in a
monoparticulate form, displacing organelles (Fig.
12). Hepatocellular adenoma, which are usually
multiple, occur in type I GSD patients who survive
beyond the first decade. Hepatocellular carcinoma
has also been reported [98–100].
Type IV GSD is very uncommon. Deficient
branching enzyme, 1,4-glucan: 1-4-glucan,6-glycosyl
transferase, causes formation and an accumulation of
abnormal glycogen with reduced branch points and
increased chain length. This amylopectin-like polysaccharide is relatively insoluble. Congenital and infantile
presentations occur. There is hepatosplenomegaly and
portal fibrosis progresses quickly to cirrhosis, probably because of the intracellular toxic nature of the
abnormal glycogen. The biopsy shows large, pale,
eosinophilic cytoplasmic inclusions, often surrounded
by a halo in the majority of hepatocytes, that are
especially prominent in the periportal zone [95,101]
(Fig. 13). Other organs, particularly heart, may also be
involved. The inclusions resist diastase digestion, but
they are removed by pectinase or a-amylase and stain
either brown, pale blue, or not at all with Lugol’s
iodine, depending on the chain length. Ultrastructurally, the inclusions consist of cental fibrillar glycogen
surrounded by polyparticulate glycogen rosettes.
Extracellular storage site
Figure 12. a: Glycogen storage disease type III. Irregular
swollen hepatocytes give a mosaic appearance to the
lobule and there is a mild degree of portal septal fibrosis.
b: In this biopsy from a child with glycogen storage
disease type I, nuclear hyperglycogenation of hepatocytes (arrow) is evident. Hematoxylin and eosin. a, 3100;
b, 3400. c: Electron microscope appearance of a liver in
glycogen storage disease type I illustrates abundant
cytoplasmic glycogen and intranuclear glycogen.
In cystic fibrosis (CF), the major liver disease
occurring in some portal areas was described by
Bodian as ‘‘focal biliary fibrosis’’ that may progress
to cirrhosis. Other hepatobiliary complications include steatosis, neonatal cholestasis, and hepatitis,
intrahepatic bile duct paucity, extrahepatic bile
duct obstruction, and gallstones [71,102,103]. The
METABOLIC LIVER DISEASE
193
Figure 13. Glycogen storage disease type IV in an infant
with hepatomegaly and liver dysfunction. Note the
aggregates (arrows) of so-called amylopectin accumulating in the cytoplasm of hepatocytes.
Figure 14. The biopsy in this child with cystic fibrosis
illustrates portal fibrous expansion, ductular proliferation, and inspisation of (eosinophic) material within the
ducts (arrows). There is also steatosis. Hematoxylin and
eosin, 3100.
much improved prognosis of patients with cystic
fibrosis has accentuated the complications of liver
disease. In a recent study, liver disease, including
portal hypertension and failure, was the second
most common cause of death [104]. We now consider CF as an inherited metabolic disease of biliary
epithelium complicated by liver disease due to duct
obstruction.
The hepatobiliary system in CF may be compared to an exocrine gland [105]. In the liver, the
cystic fibrosis transmembrane conductance regulator (CFTR) lies in the apical portion of the bile duct
epithelial membrane [106]. This protein functions
as a cyclic AMP–dependent chloride channel. The
active chloride secretion allows paracellular passage of sodium and water into the duct lumen
which modifies bile concentration. In CF, multiple
gene mutations, including LF508, may result in
CFTR defects. Consequently, bile volume is reduced, bile acid concentration is increased, and
malabsorption of taurine-conjugated bile acids increases the proportion of hydrophobic, potentially
hepatotoxic, glycoconjugated bile acids.
Focal biliary fibrosis is the diagnostic hepatic
lesion in cystic fibrosis (Fig. 14). Its precise incidence is unknown. Definitive longitudinal biopsybased studies have not been reported. In the autopsy study of CF infants done by Oppenheimer
and Esterly, those younger than 3 months had an
incidence of 10.6% which increased to 26.8% in
children over 1 year of age [103]. The lesion describes portal tracts with ducts filled by inspissated,
pale eosinophilic material, perhaps due to increased mucin mixed with bile, ductular proliferation, inflammation, and portal fibrosis. It is frequently accompanied by cholangitis. Other portal
tracts may be normal or show nonspecific changes.
Multilobular biliary cirrhosis may evolve unpredictably from expansion of focal biliary fibrosis in
some patients. Others, including ourselves, have
found that significant liver disease may be present
at biopsy in the absence of other predictive biochemical or radiological changes [107,108]. The
portal scarring is probably secondary to inflammation of the obstructed ducts, which can often
extend into the lobule. The underlying epithelial
chloride transport abnormality is a major component in the formation of obstructive plugs. However, other contributors that remain unconfirmed,
such as extrahepatic bile duct narrowing, may
explain the variable occurrence of liver disease and
the focality of these lesions. In our biopsy study, the
occurrence of liver disease was not restricted to
certain genotypes.
Steatosis may occur in isolation in an otherwise normal liver or in association with focal
biliary fibrosis or cirrhosis. In some patients it may
be relatively mild, whereas in others it may be quite
diffuse. The cause of the steatosis is uncertain; it
may be related to malnutrition, but some patients
are well nourished and comply with enzyme replacement therapy. Steatosis may also be related to
low circulating lipoprotein levels, or essential fatty
acid, vitamin E, or choline deficiency [109]. Mas-
194
G.P. JEVON
AND
J.E. DIMMICK
sive steatosis may be associated with secondary
carnitine deficiency. Hepatotoxic therapeutic agents
may also be implicated. Steatosis alone is a relatively benign condition without any proven relationship to the subsequent development of cirrhosis.
Pigment storage
A subgroup of the storage pattern comprises those
few conditions with accumulated pigment. In pediatric patients one may encounter neonatal hemochromatosis, Indian childhood cirrhosis, porphyria, genetic hemochromatosis, Dubin-Johnson
syndrome (intralysosomal melanin-like pigment
accumulation beginning in early life), and Gilbert
disease (lipofuscin accumulation). In Wilson disease the copper storage does not appear obvious,
but a pale lipofuscin tint may be present. The
differential considerations for increased iron (hemosiderin) in pediatric-age biopsies must include
iron overload from transfusions, and hemoglobin
and erythrocyte disorders. Note that the neonatal
liver normally has easily identifiable hemosiderin
and copper in periportal hepatocytes. Copper accumulation accompanies long-standing cholestasis,
as, for instance, in biliary atresia, and lipofuscin
may become increased with intravenous alimentation.
Neonatal hemochromatosis, Indian childhood cirrhosis, and Wilson disease are discussed
elsewhere (see sections on hepatitic and cirrhotic
patterns above).
Genetic hemochromatosis
Genetic (idiopathic familial) hemochromatosis
(GH) is a common disorder of unknown biochemical etiology that leads to systemic iron overload,
especially in the liver, heart, joints, and endocrine
organs. Although GH is usually thought of as an
adult disease, it has been recognized increasingly
in adolescents, and it has been reported in children
as young as 2 years. A candidate gene has been
described on chromosome 6 and is strongly associated with the HLA-A3 allele. Those with GH absorb
iron at a rate approximately 10 times greater than
normal, despite elevated iron stores [110,111]. In
some Caucasian populations as many as 1 person
in 200 is a homozygote. Iron-induced peroxidative
injury to organelle membrane phospholipids underlies several cellular injury theories. Fibrosis and
cirrhosis are present when the hepatic iron concen-
Figure 15. Liver biopsy from a 7-year-old girl with
genetic hemochromatosis. The biopsy shows portal septal fibrosis and marked hemosiderin deposition, which is
particularly prominent in the periportal region and
extends through the lobule. Prussian blue, 340.
tration exceeds 22 mg/g dry liver weight [112]. It is
important to collect the liver with iron-free utensils
and containers in the fresh state for quantitative
studies.
The precirrhotic liver shows progressive iron
accumulation in hepatocytes, beginning in the
periportal areas and becoming panlobular [113–
116] (Fig. 15). Steatosis may occur in parallel with
the progression of iron deposition in the lobule.
Periportal fibrosis develops into a septal pattern
and evolves to cirrhosis. There are occasional acidophilic bodies or small foci of necrosis and variable
degrees of ductular proliferation. Iron in bile duct
epithelium may be seen in the precirrhotic stage
but becomes more prominent later, when it is also
found in reticuloendothelial cells, vessel walls, and
in the portal fibroconnective tissue. Ultrastructurally, siderosomes and ferritin granules are present
in hepatocytes, Kupffer, endothelial, and biliary
epithelial cells. Hepatocellular carcinoma may occur in cirrhotic livers of adult patients.
Porphyria
Cytoplasmic or extracellular porphyrin deposition
in the liver may precipitate significant liver disease.
The porphyrias are a group of diseases caused by
inherited deficiencies of enzymes in the heme
synthesis pathway. Each is characterized biochemically by accumulation of substrate proximal to the
enzyme block and has a specific pattern in blood,
urine, and feces. Most porphyrias do not present
before puberty, although a few have been reported
METABOLIC LIVER DISEASE
195
in infancy or early childhood. Traditionally, they
have been divided into two groups, hepatic and
erythroid, depending on the organ that is the major
site of expression of the enzyme defect. Five are
expressed exclusively in the liver, two both in liver
and bone marrow, and one uncommon type in the
bone marrow alone.
Liver disease has most frequently been noted
in protoporphyria, one of the most common forms.
Severe disease occurs in 5–10% of patients, usually
in adulthood, but has been reported in children.
Progression to liver failure may be rapid. The liver
disease is attributed to insoluble precitates of protoporphyrin pigment that obstructs bile flow in canaliculi and ducts and collects in hepatocytes and
Kupffer cells where it probably has a hepatotoxic
effect [117]. Cholelithiasis is also common. The
livers are enlarged, nodular, and black. There is
fibrosis or a macronodular cirrhosis, with inflammation and cholestasis. Hepatocytes, Kupffer cells,
and small bile ducts are filled with dark brown
birefringent protoporphyrin pigment, which gives
an intense red fluorescence under ultraviolet light.
Ultrastructurally, protoporphyrin occurs as aggregates of slender, straight, or curved, needle-shaped
electron-dense material, in a radiating ‘‘starburst’’
pattern [118–121]. The incidence of hepatocellular
carcinoma has increased, but it is most significant
in those patients over 60 years of age.
10.
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