This document discusses the epidemiology, pathogenesis, and histopathology of fatty liver disease. It covers both non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD). For NAFLD, key points include its increasing prevalence due to the rise in obesity and diabetes. The pathogenesis involves insulin resistance leading to lipid accumulation in the liver. Histologically, NAFLD is graded based on steatosis, lobular inflammation, and hepatocyte ballooning. For ALD, heavy drinking can lead to steatosis in most people, while a subset develop alcoholic steatohepatitis. The pathogenesis of ALD also involves oxidative stress and cytokine pathways. Liver biopsy remains the gold standard

1. THE EPIDEMIOLOGY, PATHOGENESIS
AND HISTOPATHOLOGY OF FATTY
LIVER DISEASE
Adam P Levene and Robert D Goldin
Department of Histopathology,
Imperial College Faculty of Medicine at St Mary’s Hospital,London.UK
(Histopathology 2012, 61, 141-152)

2. INTRODUCTION
• Fatty liver disease consists of alcoholic liver
disease (ALD) and non-alcoholic fatty liver
disease (NAFLD), with a subset of these groups
developing alcoholic steatohepatitis (ASH) and
non-alcoholic steatohepatits (NASH),
respectively.
• The increasing prevalence of obesity and
diabetes, and the associated metabolic syndrome
is leading to an increased incidence of NAFLD.

3. • The increasing prevalence of NAFLD is
particular worrying because patients
appear to have a higher mortality from
non-liver related and recent data have
also highlighted an increased risk for
cardiovascular disease.

4. • Underlying pathogenesis of NAFLD is still incompletely
understood.
• Majority of patients with NAFLD have a stable disease, with
isolated steatosis and an indolent course with no progression
to advanced liver disease or clinical sequelae.
• However, a subset develop NASH and are at risk progressive
fibrosis, cirrhosis and liver failure, or hepatocellular
carcinoma (HCC).
• The distinction between isolated steatosis and NASH is
important, as their prognoses and management are different

5. • The gold standard for identification of
patients with NASH is liver biopsy.

6. EPIDEMIOLOGY AND DISEASE
NATURAL HISTORY
NAFLD:
• Estimates of NAFLD prevalence based on
‘cryptogenic’ abnormal liver function test results,
autopsy samples, ultrasound and magnetic
resonance spectroscopy range between 3% and
37%, with the usual figure quoted being around
30%.

7. • Studies suggest that ethnic background plays a
role in the incidence of NAFLD were provided by
a study that revealed a significantly higher
prevalence of NAFLD in Hispanics than in non-
Hispanic whites, even after controlling for
obesity and body fat distribution.

8. • Studies show that NAFLD is not always associated
with raised alanine transaminase (ALT) or gamma-
glutamyl transpeptide positivity.
• NAFLD was more likely in the presence of obesity,
hyperglycemia,hyperinsulinaemia,
hypertriglyceridaemia, and systolic hypertension, all
features of the metabolic syndrome.
• Although total fat consumption is not significantly
associated with the risk of cirrhosis or liver cancer,
cholesterol consumption is.

9. • Recent clinical and experimental studies
have shown that coffee protects the liver
against the development of fat-induced
liver damage.

10. • The prevalence of NASH is difficult to
determine, as large population-based
studies are not possible, because a liver
biopsy is still, currently, required for
diagnosis.
• An autopsy study found NASH in 18.5% of
markedly obese patients and 2.7% of lean
patients.
• This highlights the possible effect that
weight has on the incidence of NASH.

11. Normal liver
30%
Fatty liver Stable
disease
30%
NASH
20%
Progressive fibrosis
cirrhosis
30-40% 0-10%
Liver failure Liver cell cancer

12. • A well recognized complication of cirrhosis
resulting from NAFLD is HCC.
• HCC has also been noted as a rare complication of
NAFLD prior to cirrhosis.
• This may be explained by the association of
diabetes and obesity with the development of
HCC, as well as the carcinogenic factors
associated with cirrhosis in general.

13. ALD:
• Studies in unselected heavy drinkers of
alcohol suggest that 80% develop
steatosis, which is the earliest and most
common histopathological manifestation of
ALD.
• Steatosis occurs in most people consuming
alcohol in excess of 80 g/day, and can
resolve within 2-4 weeks of abstinence.

14. Normal liver
Alcohol intake
80% Alcohol abstinence
Fatty liver ?Abstinence effect
20-40%
ASH
fibrosis
40%
cirrhosis

15. • Severe ASH has a very poor prognosis,
whereas patients with mild and moderate
ASH can improve with abstinence.

16. • It is extremely difficult to predict histological
stage clinically in ALD patients before the
development of decompensated cirrhosis.
• A small subset of ALD patients presenting with
decompensated chronic liver disease has severe
ASH without cirrhosis on biopsy.
• Conversely, some ALD patients who are clinically
well but have abnormal liver function test results
and undergo liver biopsy are found to have
advanced ALD with severe ASH.

17. • Staging Of ALD is most accurately
performed histologically by performing
liver biopsy, which may need to be via the
transjugular route in patients with
impaired clotting.

18. PATHOGENESIS
NAFLD:
• NAFLD is closely linked to obesity, insulin
resistance, and the metabolic syndrome.
• The initial theory for the pathogenesis of
NAFLD was the ‘two-hit’ hypothesis.
• The first hit, steatosis, sensitives the liver to
the induction of inflammation by a second
insult that promotes oxidative stress and
steatohepatitis.

19. • This model has subsequently been revised,
in recognition that a combination of
‘second hits’ (both environmental and
genetic) may lead to the development of
steatohepatitis.

20. • Currently, it is not fully understood why some
patients develop isolated steatosis and others
develop steatohepatitis.
• However, it appears that insulin resistance and
increased levels of free fatty acids in the liver
are strongly associated with NASH.
• When insulin resistance develops, free fatty
acids are inappropriately moved to non-adipose
tissues such as the liver by decreased inhibition
of lipolysis and increased de novo lipogenesis.

21. • There is currently increasing work on the
interaction between cytokines and
adipokines (cytokines secreted by adipose
tissue) in an attempt to understand the
mechanisms involved in NAFLD
development.

22. • Insulin resistance is thought to be
regulated by proinflammatory cytokines,
such as tumour necrosis factor-a (TNF-a),
and adipokines such as adiponectin and
leptin.
• Oxidative stress and apoptosis also appear
to contribute to the development and
progression of NASH.

23. ALD:
• Pathogenesis is still incompletely understood.
• Recent discoveries - Many pathways leading to
oxidative stress, and the new mechanism of
endoplasmic reticulum stress.
• There are many similarities with NAFLD, such as
the proinflammatory cytokine TNF-a and the
adipokines adiponectin and leptin.
• Obesity being implicated as a risk factor for the
development of ASH and cirrhosis in people with
heavy alcohol consumption.

24. HISTOPATHOLOGY
NAFLD
• Histological analysis of a liver biopsy remains the
gold standard for and only accurate way of
assessing the degree of steatosis,
necroinflammatory changes and fibrosis of
NASH, and therefore distinguishing NASH from
isolated steatosis.

25. • The pathology committee of the NASH CRN
group developed and histological scoring system
for use in NAFLD.
• For each case, an NAFLD activity score (NAS)
and a separate fibrosis stage was given.
• The NAS comprised 14 histological features, nine
of which were recorded as present or absent
(such as Mallory-Denk bodies), and three of
which were semiquantitatively [steatosis (0-3),
lobular inflammation (0-3), and hepatocellular
ballooning (0-2)], to give a score of between 0
and 8.

26. Steatosis grade (0-3) Lobular inflammation (0-3) Hepatocyte ballooning (0-2)
0: <5% 0: None 0: None
1: 5-33% 1: <2 foci/*20 field 1: Mild, few
2: 34-66% 2: 2-4 foci/*20 field 2: Moderate-many
3: >66% 3: >4 foci/*20 field
NAFLD activity score (NAS): 0-8 0-2 not NASH
3-4 uncertain for NASH
5-8 NASH
Non-alcoholic steatohepatitis (NASH) Clinical Research Network Fibrosis Staging System

27. Fibrosis: based on the use of Masson’s trichrome stain
0: None
1a: Mild zone 3 perisinusoidal fibrosis
Requires trichrome stain to identify
1b: Moderate zone 3 perisinusoidal fibrosis
May be appreciated on haemotoxylin and eosin
1c: Portal fibrosis only
2: Zone 3 perisinusoidal fibrosis andd periportal fibrosis
3: Bridging fibrosis
4: Cirrhosis
Non-alcoholic steatohepatitis (NASH) Clinical Research Network Fibrosis Staging System

28. • The lobular inflammatory infiltrate is usually
composed predominantly of neutrophils, but
lymphocytes and macrophages are commonly seen.
• In practice, the presence of ballooned
hepatocytes is a sine qua non for the diagnosis of
NASH rather than steatosis.
• In a meticuluos histological study, serial staining
consistently demonstrated that hepatocellular
ballooning was associated with fat droplets, as
shown by oil red O positivity and CK-18-positive
Mallory-Denk bodies.

29. • Histological features recorded but not scored in
NAS include Mallory-Denk bodies,
megamitochondria, and nuclear vacuolation.
• In NAFLD, Mallory-Denk bodies are often small
and poorly formed, and may be difficult to detect
in routinely stained sections.
• Immunohistochemistry for ubiquitin, p62, CK-8
and CK-18 can be used to demonstrate antigens
associated with Mallory-Denk bodies and
ballooned hepatocytes.

30. • Summary, Histological spectrum of NAFLD is
characterized by steatosis, lobular inflammation,
ballooning of hepatocytes, fibrosis, and other
features that may or may not be present, such as
Mallory-Denk bodies and portal inflammation.
• The NASH CRN grading and staging system of
NASH is based on the use of haematoxylin and
eosin and Masson’s trichrome stain, so it can be
used routinely by histopathologies.

31. Nonspecific steatosis, predominantly macrovesicular, with
occasional foci of inflammatory cells in the hepatic lobules and
many hepatocytes with glycogenated nuclei (H&E, ×200).

32. Steatohepatitis with several hepatocytes showing ballooning degeneration
intermixed with steatosis and foci of inflammatory cells in the hepatic
lobules (H&E, ×200).

33. Mallory hyaline showing eosinophilic and ropy inclusions in the
cytoplasm (H&E, ×400).

34. Perivenular/pericellular fibrosis in zone 3 (Masson trichrome,
×200).

35. ALD:
• The diagnosis of ALD is usually easy to make with
a clear history of excessive alcohol consumption
and negative markers for other chronic liver
diseases.
• However, one needs to remember that the liver
disease seen in patients who drink excessively is
not always caused by alcohol

36. • The role of Liver biopsy in ALD is controversial
but it is still the gold standard investigation and
used to
• clarify cases with an unusual clinical course,
• to better define the contribution of alcohol in
patients with possible non-alcohol-related
comorbidity (e.g. in hepatitis C or use of lipid-
lowering medications),
• and in some patients, to determine the
severity of liver disease.

37. • It can also be used to specifically assess
the amount neutrophils within the liver
parenchyma to act as a guide to whether
the patient would benefit from steroid
treatment.
• Liver biopsy is not used in advanced ALD
where there is evidence of
decompensated cirrhosis, as the risk
outweigh any potential benefits.

38. • The histopathology of ALD is similar to that of
NAFLD, and occurs mainly in the liver
parenchyma in the perivenular areas.
• Characteristic early lesions in ALD include
perivenular and pericellular fibrosis.
• The development of ASH is dominated by a
neutrophilic infiltrate, ballooning degeneration,
Mallory-Denk bodies, and hepatocyte necrosis.

39. • In most cases, a distinction between ALD and
NAFLD cannot be made on morphological criteria
and the diagnosis has to rely on
clinicopathological correlation.
• Overall features in favour of ALD include
canalicular cholestasis, cholangiolitis, florid zone
3 changes such as dense infiltration by
neutrophils, prominent Mallory-Denk bodies, and
extensive zone 3 fibrosis associated with
sinusoidal obliteration and hepatic veno-occlusive
lesions.

40. • NAFLD tends to have more marked steatosis and
less severe steatohepatitis changes
• One of the most helpful changes, and one that
strongly favours NAFLD, is nuclear vacuolation,
which is seen in 70-80% of NAFLD cases and in
only 5-10% of ALD cases.
• The glycogenated nuclei can be seen in NAFLD-
related cirrhosis even when other changes of
NASH have disaappeared.

41. • Immunohistochemistry for protein tyrosine
phosphate 1B (PTP1B) and insulin receptor on
hepatocytes has been suggested to be useful in
differentiating NAFLD from ALD.
• PTP1B was shown to be upregulated in the
cytoplasm of hepatocytes in NASH biopsies as
compared with ASH biopsies.
• Insulin receptor showed loss of membranous
staining in hepatocytes from NASH biopsies, but
was still present on ASH biopsies.

42. CONCLUSION
• NAFLD and ALD are increasingly common and recognized
diseases.
• The risk factors and possible courses of the two diseases
are well known, with many areas of ovelap, although it is
still unclear why some people progress to NASH/ ASH and
cirrhosis and others do not.
• There is an increasing knowledge of the pathogenesis of
both diseases.
• In NAFLD especially, and in ALD to a lesser degree, liver
biopsy and histopathological assessment are key for
determining whether a patient has steatosis or
steatohepatitis, and also for assessing the degree of liver
damage and fibrosis.

43. THANK YOU