**5. Histology of NAFLD**

NAFLD represents a histopathologic spectrum ranging from steatosis alone, to necroinflam‐ mation, summarized as NASH; and progression to advanced fibrosis and cirrhosis.

The histologic characterization of NAFLD and NASH may include description of steatosis and cell injury in addition to inflammation and fibrosis. Kleiner and Brunt [7] propose cate‐ gorizing the histologic changes when studying NAFLD as follows in table 2.

The main histological characteristic of NAFLD is the accumulation of fat in the form of tri‐ glyicerides within hepatocytes, lesion termed steatosis (Figure 1 and 2); this term is defined by the guideline [1] as NAFL – non-alcoholic fatty liver, where the risk of progression to cir‐ rhosis and liver failure is minimal. The presence of >5% steatoic hepatocytes in a liver biopsy is accepted as the minimum criterion for thehistological diagnosis of NAFLD [8].


**Figure 2.** Steatosis. Hematoxylin-eosin stain.

Steatosis in NAFLD is usually macrovesicular, which refers to hepatocytes with single large intracytoplasmatic fat droplet or smaller well defined droplets displacing the nucleus to the cell periphery. This macrovesicularsteatosis is usually present in a zone 3 or panacinar dis‐ tribution; it differs from zone 1 steatosis that is a common distribution in chronic hepatitis C.

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The extent of steatosis can be evaluated and classified semi-quantitative. The most reprodu‐ cible method follows the acinararchiqueture dividing the liver parenchyma in thirds and as‐

NASH, under this concept is the histology pattern of NAFLD, which is at risk of developing advance fibrosis. The minimal criteria for the histopathological diagnosis of adult NASH in‐ clude steatosis, hepatocyte injury, usually in form of ballooning, and lobular inflammation,

The key feature for the diagnosis of NASH is the ballooning injury (Figures 3 and 4), and it is considered a marker of apoptosis [12]. This type of cell injury is characterized by a cell that becomes enlarged and the cytoplasm becomes irregularly clumped with optically clear, nonvesiculated areas. Ballooned cells are seen most frequently in zone 3 near the hepatic veins, and lose this localization, becoming portal inflammation more prominent when the disease progresses and in severe cases. Immunostaining of hepatocyte keratins 8 and 18

Azonal steatosis is most often seen in biopsies with advanced fibrosis [9].

sessing percentage involvement bay steatoic hepatocytes [8] – table 3.

**Table 3.** Steatosis semi-quantification according to acinar architecture [8].

typically localized in acinar zone 3 [10, 11].

**STEATOSIS SEMI-QUANTIFICATION** Mild 0 – 33% Moderate 33 – 66% Severe > 66%.

**Table 2.** Histologic Categorization of NAFLD [7].

**Figure 1.** Steatosis. Hematoxylin-eosin stain.

**Figure 2.** Steatosis. Hematoxylin-eosin stain.

**CATEGORY DEFINITION**

Steatosis:

Steatohepatitis:

Steatosis with inflammation. Steatosis with nonspecific fibrosis

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**Table 2.** Histologic Categorization of NAFLD [7].

**Figure 1.** Steatosis. Hematoxylin-eosin stain.

No significant evidence of fatty liver disease. Insufficientsteatosis for diagnosis of steatosis, without

Zone 1, borderline pattern Form of steatohepatitis that occurs mainly in young

Cryptogenetic fibrosis/ cirrhosis Presence of fibrosis (usually advanced) or cirrhosis, with

steatohepatitis.

significance.

other changes (ballooning, fibrosis) that would suggest

Steatosis without specific changes to suggest a form of steatohepatitis. This category may include spotty lobular inflammation and/or mild degrees of fibrosis of uncertain

Form of steatoshepatitis most common in adults; defined as a zone 3 centered injury pattern that includes steatosis, inflammation, ballooning injury, (often with Mallory-Denk

steatohepatitsis has some, but not all ofthe features that

little ton o steatosis and no changes (ballooning, Mallory-Denk bodies) that would suggest borderline or definite steatohepatitis. Other explanations for fibrosis (besides

bodies) with or without fibrosis. Borderline

would allow a diagnosis of steatohepatitis.

ballooning injury in zone 1 if present).

steatohepatitis) should be considered.

children, characterized by zone 1-centered (portal inflammation, portal-based fibrosis, zone 1 steatosis, Steatosis in NAFLD is usually macrovesicular, which refers to hepatocytes with single large intracytoplasmatic fat droplet or smaller well defined droplets displacing the nucleus to the cell periphery. This macrovesicularsteatosis is usually present in a zone 3 or panacinar dis‐ tribution; it differs from zone 1 steatosis that is a common distribution in chronic hepatitis C. Azonal steatosis is most often seen in biopsies with advanced fibrosis [9].

The extent of steatosis can be evaluated and classified semi-quantitative. The most reprodu‐ cible method follows the acinararchiqueture dividing the liver parenchyma in thirds and as‐ sessing percentage involvement bay steatoic hepatocytes [8] – table 3.


**Table 3.** Steatosis semi-quantification according to acinar architecture [8].

NASH, under this concept is the histology pattern of NAFLD, which is at risk of developing advance fibrosis. The minimal criteria for the histopathological diagnosis of adult NASH in‐ clude steatosis, hepatocyte injury, usually in form of ballooning, and lobular inflammation, typically localized in acinar zone 3 [10, 11].

The key feature for the diagnosis of NASH is the ballooning injury (Figures 3 and 4), and it is considered a marker of apoptosis [12]. This type of cell injury is characterized by a cell that becomes enlarged and the cytoplasm becomes irregularly clumped with optically clear, nonvesiculated areas. Ballooned cells are seen most frequently in zone 3 near the hepatic veins, and lose this localization, becoming portal inflammation more prominent when the disease progresses and in severe cases. Immunostaining of hepatocyte keratins 8 and 18 might help to identify ballooned hepatocytes [13]. Ballooned degeneration is difficult to di‐ agnose even by trained pathologist, for that reason it can show significant inter-observer variation [14].

Kupffer or other inflammatory cells. Apoptosis has been validated as an accurate marker for

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Inflammatory infiltrates (Figure 5) can be seen in the hepatic acini/lobules or the portal tract. Lobular inflammation is usually mild, consists of a mixed inflammatory cell infiltrate, com‐ posed of lymphocytes, some eosinophils, and a few neutrophils. Polymorphs can be ob‐ served around ballooned hepatocytes that are called "satellitosis" (Figure 6). Kuppfer cells

diagnosis of NASH based on immunochemistry in liver tissue [17].

**Figure 5.** Mononuclear inflammatory infiltration. Hematoxylin-eosin stain.

**Figure 6.** Polymorph around ballooned hepatocytes, "satelitosis".

Hematoxylin-eosin stain.Portal chronic mononuclear cell inflammation in adult NASH is common and mild. When portal inflammation is greater than lobular other aetiologies should be ruled out, such as chronic hepatitis C [18]. On the other side, a greater portal in‐ flammation than lobular inflammation can be seen in successfully treated patients [19]. In a large database of liver biopsies from the NASH Clinical Research Network, including adults

aggregates as lobular microgranulomas and lipogranulomas may appear [10]

**Figure 3.** Hepatocyte ballooned. Hematoxylin-eosin stain.

Ballooning degeneration is associated with an increased liver-related mortality [15].

**Figure 4.** Ballooning hepatocyte. Hematoxylin-eosin stain.

Mallory-Denk Bodies (MDB), also known as Mallory bodies, are eosinophilic, ropey cyto‐ plasmatic inclusion bodies in the hepatocyte of patients with chronic liver disease. This type of lesion contains abnormal cytokeratin 8 and 18 filaments that have been ubiquinated.

Mallory bodies have an importance in disease progression and it is suggested a possible prognostic role in steatohepatitis [16]. In a recent study, the presence of MBD was signifi‐ cantly associated with liver-related mortality [15].

Both ballooning degeneration and MDB can trigger the development of apoptosis. Apoptot‐ ic (acidophil) bodies are common in NASH. They can be identified as rounded, eosinophilic cytoplasmic fragments, which appear to be free within the sinusoids or surrounded by Kupffer or other inflammatory cells. Apoptosis has been validated as an accurate marker for diagnosis of NASH based on immunochemistry in liver tissue [17].

Inflammatory infiltrates (Figure 5) can be seen in the hepatic acini/lobules or the portal tract. Lobular inflammation is usually mild, consists of a mixed inflammatory cell infiltrate, com‐ posed of lymphocytes, some eosinophils, and a few neutrophils. Polymorphs can be ob‐ served around ballooned hepatocytes that are called "satellitosis" (Figure 6). Kuppfer cells aggregates as lobular microgranulomas and lipogranulomas may appear [10]

**Figure 5.** Mononuclear inflammatory infiltration. Hematoxylin-eosin stain.

might help to identify ballooned hepatocytes [13]. Ballooned degeneration is difficult to di‐ agnose even by trained pathologist, for that reason it can show significant inter-observer

Ballooning degeneration is associated with an increased liver-related mortality [15].

Mallory-Denk Bodies (MDB), also known as Mallory bodies, are eosinophilic, ropey cyto‐ plasmatic inclusion bodies in the hepatocyte of patients with chronic liver disease. This type of lesion contains abnormal cytokeratin 8 and 18 filaments that have been ubiquinated.

Mallory bodies have an importance in disease progression and it is suggested a possible prognostic role in steatohepatitis [16]. In a recent study, the presence of MBD was signifi‐

Both ballooning degeneration and MDB can trigger the development of apoptosis. Apoptot‐ ic (acidophil) bodies are common in NASH. They can be identified as rounded, eosinophilic cytoplasmic fragments, which appear to be free within the sinusoids or surrounded by

variation [14].

166 Liver Biopsy – Indications, Procedures, Results

**Figure 3.** Hepatocyte ballooned. Hematoxylin-eosin stain.

**Figure 4.** Ballooning hepatocyte. Hematoxylin-eosin stain.

cantly associated with liver-related mortality [15].

**Figure 6.** Polymorph around ballooned hepatocytes, "satelitosis".

Hematoxylin-eosin stain.Portal chronic mononuclear cell inflammation in adult NASH is common and mild. When portal inflammation is greater than lobular other aetiologies should be ruled out, such as chronic hepatitis C [18]. On the other side, a greater portal in‐ flammation than lobular inflammation can be seen in successfully treated patients [19]. In a large database of liver biopsies from the NASH Clinical Research Network, including adults and children, portal chronic inflammation was associated with clinical and histologic fea‐ tures of severity and advance disease [20].

Vascular alterations in NAFLD. Recent paper has focused the study of NASH in microves‐ sels of the liver [21]. This work has found an intraacinar branch of the hepatic artery in the perivenular region in active steatohepatitis. This finding is important because it can lead to confusion for a portal tract resulting in an equivocal diagnosis. Likewise, the presence of this vessel correlates with higher stage of fibrosis.

Fibrosis in adult NASH usually starts in acinar zone 3 and has characteristic "chicken wire" pattern due to deposition of collagen an other extracellular matrix fibres along the sinusoids of zone 3 and around the hepatocytes (Figure 7 and 8). Portal fibrosis has been reported in cases of morbid obesity-related NASH and in pediatric NASH. Fibrosis predicts clinical out‐ comes in NASH [22]. there was noter from this study that the progression of the fibrosis is accompanied of steatosis reduction. Approximately 37% to 41% of patients with NAFLD have fibrosis progression over 3 to 10 years [22, 23]. The higher rates of fibrosis progression were related to: body mass index, diabetes and low initial fibrosis [22]. When periportal fib‐ rosis was not present, there was a 100% of negative predictive value in predicting liver-relat‐ ed outcomes [23]. Steatosis, inflammation, ballooning and Mallory hyaline were not associated with liver-related mortality after adjusting for the presence of fibrosis [15]. The inclusion of fibrosis explains why the recent classifications for NASH used by Younossi [15] and Matteoni [16], independently correlated with liver-related mortality. This observation shows the importance of fibrosis in NAFLD, patients with NASH and fibrosis portends a higher risk of death [24].

**Figure 8.** Zone 3 fibrosis perivenular/pericellular. Masson trichrome stain.

ated nuclei and iron deposition.

tion, ballooning and definite NASH [27].

**6. Histologic scoring systems**

alcoholic injury [26].

Other histological lesions that may be seen in NASH include megamitochondria, glycogen‐

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Megamitochondria (giant mitochondria) are round or needle-shaped, eosinophilic, intracy‐ toplasmatic inclusions more commonly observed in hepatocytes with microvesicularsteato‐ sis. This abnormal mitochondria is a result of injury from lipid peroxidation or represent an adaptive change [25]. Glycogenated nuclei are vacuolated nuclei usually observed in peri‐ portal hepatocytes. Their presence is more frequent in non-alcoholic etiology and it is rare in

Finally, hepatic siderosis might be seen in NAFLD. One study of 293 liver biopsies (34,5% of patients with NAFLD) investigates the relationship between iron deposition and NAFLD [27]. Stainable hepatic iron described three histological patterns: hepatocellular pattern, re‐ ticuloendothelial system cell – RES - (mainly Kupffer cell) pattern and mixed. RES pattern was associated with advanced fibrosis and higher histological features of portal inflamma‐

NAFLD histologic criteria requires an accumulation of more than 5% of fat deposition, mainly in form of triglycerides. NAFLD was first described by Ludwig and colleagues [28],

In 1999, Matteoni and colleagues characterized histologic subtypes that correlate with clini‐ cal outcomes [16] – table 4. In 2005, NASH Clinical Research Network developed NAFLD activity score (NAS) [8]. This score comprises four features evaluated semi-quantitatively: steatosis, lobular inflammation, hepatocellular ballooning and fibrosis. Fibrosis was classi‐ fied separately – table 5. When NAS is >5 sensitivity and specificity for definite NASH were 0,75 and 0,83, respectively. Finally, a recent classification for NAFLD has been proposed by

and since then several systems for grading and staging NAFLD have been proposed.

**Figure 7.** Fibrosis pattern: around hepatocytes. Masson trichrome stain.

**Figure 8.** Zone 3 fibrosis perivenular/pericellular. Masson trichrome stain.

and children, portal chronic inflammation was associated with clinical and histologic fea‐

Vascular alterations in NAFLD. Recent paper has focused the study of NASH in microves‐ sels of the liver [21]. This work has found an intraacinar branch of the hepatic artery in the perivenular region in active steatohepatitis. This finding is important because it can lead to confusion for a portal tract resulting in an equivocal diagnosis. Likewise, the presence of this

Fibrosis in adult NASH usually starts in acinar zone 3 and has characteristic "chicken wire" pattern due to deposition of collagen an other extracellular matrix fibres along the sinusoids of zone 3 and around the hepatocytes (Figure 7 and 8). Portal fibrosis has been reported in cases of morbid obesity-related NASH and in pediatric NASH. Fibrosis predicts clinical out‐ comes in NASH [22]. there was noter from this study that the progression of the fibrosis is accompanied of steatosis reduction. Approximately 37% to 41% of patients with NAFLD have fibrosis progression over 3 to 10 years [22, 23]. The higher rates of fibrosis progression were related to: body mass index, diabetes and low initial fibrosis [22]. When periportal fib‐ rosis was not present, there was a 100% of negative predictive value in predicting liver-relat‐ ed outcomes [23]. Steatosis, inflammation, ballooning and Mallory hyaline were not associated with liver-related mortality after adjusting for the presence of fibrosis [15]. The inclusion of fibrosis explains why the recent classifications for NASH used by Younossi [15] and Matteoni [16], independently correlated with liver-related mortality. This observation shows the importance of fibrosis in NAFLD, patients with NASH and fibrosis portends a

tures of severity and advance disease [20].

168 Liver Biopsy – Indications, Procedures, Results

vessel correlates with higher stage of fibrosis.

**Figure 7.** Fibrosis pattern: around hepatocytes. Masson trichrome stain.

higher risk of death [24].

Other histological lesions that may be seen in NASH include megamitochondria, glycogen‐ ated nuclei and iron deposition.

Megamitochondria (giant mitochondria) are round or needle-shaped, eosinophilic, intracy‐ toplasmatic inclusions more commonly observed in hepatocytes with microvesicularsteato‐ sis. This abnormal mitochondria is a result of injury from lipid peroxidation or represent an adaptive change [25]. Glycogenated nuclei are vacuolated nuclei usually observed in peri‐ portal hepatocytes. Their presence is more frequent in non-alcoholic etiology and it is rare in alcoholic injury [26].

Finally, hepatic siderosis might be seen in NAFLD. One study of 293 liver biopsies (34,5% of patients with NAFLD) investigates the relationship between iron deposition and NAFLD [27]. Stainable hepatic iron described three histological patterns: hepatocellular pattern, re‐ ticuloendothelial system cell – RES - (mainly Kupffer cell) pattern and mixed. RES pattern was associated with advanced fibrosis and higher histological features of portal inflamma‐ tion, ballooning and definite NASH [27].
