**4. Diagnosis of hepatic fibrosis**

The stage of fibrosis ranges from absent (F0) to cirrhosis (F4), with stages F2–F4 considered to be clinically significant and stages F3–F4 considered to be advanced fibrosis. Apart from liver biopsy, there are two broad categories of non-invasive markers used to determine the stage of liver fibrosis: serum and radiological markers. This stratification based on markers of fibrosis is more tractable than those used for NASH and so it is currently used to identify patients who are at risk of disease progression.

#### **4.1. Serum biomarkers**

There are two large groups of predictive models of advanced fibrosis: 'simple bedside models', which use a combination of routine blood tests and clinical variables, and 'complex models', which use serum markers of fibrosis (measures of extracellular matrix deposition and turnover).

Although several of these predictive models of advanced fibrosis have been evaluated (**Table 3**) [61, 66, 115], two of the tests have been more widely studied and have easily available parameters, *Fibrosis-4 index* (FIB-4) and *NAFLD Fibrosis Score* (NFS). FIB-4 is based on age, levels of AST, ALT and platelet count. Values of this index below −1.30 enable the exclusion of the presence of advanced fibrosis with a sensitivity of 74% and a specificity of 71%, while values above 2.67 indicate advanced fibrosis with a sensitivity and specificity of 33 and 98%, respectively [115]. NFS is another formula developed and validated for the detection of advanced fibrosis that includes age, BMI, presence of diabetes or hyperglycemia, platelet count, albumin and AST/ALT ratio (http://nafldscore.com/). In a meta-analysis of 13 studies with more than 3000 patients, a value of NFS < −1.455 had a sensitivity of 90% and a specificity of 60% to exclude advanced fibrosis, while a value of >0.676 identified the presence of it with a sensitivity of 67% and a specificity of 97% [116].

The principal drawback of all these biomarkers is that none of them is specific of the liver and their results can be influenced by co-morbidities of patients, so a critical interpretation of the

AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; TIMP-1, tissue inhibitor of

haptoglobin, GGT, 0.7 (high cut-off) 15 90

**Model Variables Cut-offs Sensitivity (%) Specificity (%)**

levels, platelets 2.67 (high cut-off) 33 98

1 (high cut-off) 67 97

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13

FIB-4 Age, AST and ALT 1.30 (low cut-off) 74 71

NAFLD Age, hyperglycemia, −1.455 (low cut-off) 90 60 fibrosis score BMV, AST/ALT ratio, 0.675 (high cut-off) 67 97

AST to AST, platelet 1 27 89

AST/ALT ratio AST, ALT 0.8 (low cut-off) 90 60

BAAT score BMI, age, ALT, serum 2 71 80

BARD score BMI, AST/ALT ratio, 2 89 44

Enhanced Age, hyaluronic acid, 8.5 (low cut-off) 80 90

FibroTest α2-macroglobulin, 0.3 (low cut-off) 77 77

Hepascore Age, gender, bilirubin, 0.44 75 84

metalloproteinase *1*; PIIINP, procollagen III amino-terminal peptide; GGT, gamma glutamyl transferase.

**Table 3.** Predictive models for significant and advanced fibrosis in NAFLD patients.

albumin, platelet

triglycerides

liver fibrosis TIMP-1, PIIINP 11.3 (high cut-off)

diabetes

bilirubin, apolipoprotein

hyaluronic acid, α2 macroglobulin

platelet ratio index (APRI)

(ELF) test

With respect to image techniques, transient elastography (FibroScan®) is the most widely used technique in the diagnosis of liver fibrosis, not only in NAFLD but also in different chronic

result is necessary.

**4.2. Imaging methods to measure fibrosis**


AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; TIMP-1, tissue inhibitor of metalloproteinase *1*; PIIINP, procollagen III amino-terminal peptide; GGT, gamma glutamyl transferase.

**Table 3.** Predictive models for significant and advanced fibrosis in NAFLD patients.

The principal drawback of all these biomarkers is that none of them is specific of the liver and their results can be influenced by co-morbidities of patients, so a critical interpretation of the result is necessary.

#### **4.2. Imaging methods to measure fibrosis**

of more than 500 serum metabolites through liquid chromatography coupled with mass spectrometry (LC–MS) in NAFLD patients obtaining a metabolomic profile that enables the differentiation between NAFL and NASH with good specificity and sensitivity [112]. Moreover, the same group thanks to the study of metabolomic profiles at the serum level observed two different subtypes of NAFLD according to the involvement of the methionine metabolism, subtype M and subtype no M, distinguishing those patients that could benefit from therapy

Recently, studies in rodents suggest that epigenetic events, inheritable events not caused by changes in DNA sequence, may influence susceptibility to NASH. The three most commonly described epigenetic mechanisms are DNA (CpG) methylation, post-translational histone modifications and microRNAs (miRNAs). Several miRNAs have been identified in serum/ plasma of NAFLD patients that show diagnostic potential for distinguishing NAFL from

The stage of fibrosis ranges from absent (F0) to cirrhosis (F4), with stages F2–F4 considered to be clinically significant and stages F3–F4 considered to be advanced fibrosis. Apart from liver biopsy, there are two broad categories of non-invasive markers used to determine the stage of liver fibrosis: serum and radiological markers. This stratification based on markers of fibrosis is more tractable than those used for NASH and so it is currently used to identify patients

There are two large groups of predictive models of advanced fibrosis: 'simple bedside models', which use a combination of routine blood tests and clinical variables, and 'complex models', which use serum markers of fibrosis (measures of extracellular matrix deposition

Although several of these predictive models of advanced fibrosis have been evaluated (**Table 3**) [61, 66, 115], two of the tests have been more widely studied and have easily available parameters, *Fibrosis-4 index* (FIB-4) and *NAFLD Fibrosis Score* (NFS). FIB-4 is based on age, levels of AST, ALT and platelet count. Values of this index below −1.30 enable the exclusion of the presence of advanced fibrosis with a sensitivity of 74% and a specificity of 71%, while values above 2.67 indicate advanced fibrosis with a sensitivity and specificity of 33 and 98%, respectively [115]. NFS is another formula developed and validated for the detection of advanced fibrosis that includes age, BMI, presence of diabetes or hyperglycemia, platelet count, albumin and AST/ALT ratio (http://nafldscore.com/). In a meta-analysis of 13 studies with more than 3000 patients, a value of NFS < −1.455 had a sensitivity of 90% and a specificity of 60% to exclude advanced fibrosis, while a value of >0.676 identified the presence of it with

with SAMe (S-adenosyl methionine) [113].

12 Liver Research and Clinical Management

NASH and advanced fibrosis [114].

**4. Diagnosis of hepatic fibrosis**

who are at risk of disease progression.

a sensitivity of 67% and a specificity of 97% [116].

**4.1. Serum biomarkers**

and turnover).

With respect to image techniques, transient elastography (FibroScan®) is the most widely used technique in the diagnosis of liver fibrosis, not only in NAFLD but also in different chronic liver diseases [117]. TE measures the propagation velocity of low-frequency waves (50 Hz) through the hepatic parenchyma using ultrasounds and is expressed in kilo Pascal (kPa); the higher the propagation velocity, the greater the stiffness of the tissue. The advantages provided by this technique are its speed, the immediacy of the results and the ease of handling. However, proper results require careful interpretation of data, based on at least 10 successful measurements, a success rate above 60% and an interquartile range (IQR) of <30% of the median value. A limitation of TE in NAFLD is the high rate of technical failure due to the attenuation of the elastic wave by interposition of adipose tissue secondary to the central obesity, very frequent in these patients. Although an XL probe has been developed, which enables greater penetration of the wave, this difficulty is often insurmountable [118, 119]. Moreover, this technique has been initially validated in patients with chronic infection by VHC [120], while the studies focusing on evaluating its use in NAFLD are smaller and have often used different cut-offs [42, 118, 121–129] (**Table 4**). According to the results of several studies, the cut-offs with M probe accepted for NAFLD patients are 7.0 kPa for significant fibrosis (≥F2), 8.7 kPa for advanced fibrosis (≥F3) and 10.3 kPa for cirrhosis (F4) [124, 126, 128]. When using the XL probe, these cut-offs differ as the measure of liver stiffness with this probe is less than that with the M probe in the same patient; in this case, 6.2, 7.2 and 7.9 kPa are the cut-offs for significant fibrosis, advanced fibrosis and cirrhosis, respectively [119, 130, 131].

**Study Patients,** *n* **Probe Fibrosis** 

**Stage**

Yoneda et al. [122] 67 M *F* ≥ 2 6.65 82 91

Yoneda et al. [125] 97 M *F* ≥ 2 6.65 74 97

Nobili et al. [129] 52 M *F* ≥ 2 7.4 100 92

Wong et al. [128] 246 M *F* ≥ 2 7.0 79 76

Lupsor et al. [124] 72 M *F* ≥ 2 6.8 67 84

Petta et al. [118] 169 M *F* ≥ 2 7.25 69 70

Kumar et al. [126] 205 M *F* ≥ 2 7.0 78 79

Pathik et al. [121] 110 M *F* ≥ 2 9.1 – –

Cassinotto et al. [123] 291 M *F* ≥ 2 6.2 90 –

Imajo et al. [42] 142 M *F* ≥ 2 11.0 62 100

**Table 4.** Comparative studies of FibroScan with liver biopsy in the detection of fibrosis in NAFLD.

*F* = 4 –

*F* = 4 –

*F* = 4 –

**Cut-off (kPa) Sensitivity** 

Diagnosis and Characterization of Non-Alcoholic Fatty Liver Disease

*F* ≥ 3 8.0 87 84 *F* = 4 17.0 100 98

*F* ≥ 3 9.8 85 81 *F* = 4 17.5 100 97

*F* ≥ 3 10.2 100 100

*F* ≥ 3 8.7 84 83 *F* = 4 10.3 92 88

*F* ≥ 3 10.4 100 97

*F* ≥ 3 8.75 76 78

*F* ≥ 3 9.0 85 88 *F* = 4 11.8 90 88

*F* ≥ 3 12.0 90 80 *F* = 4 20.0 90 80

*F* ≥ 3 8.2 90 – *F* = 4 9.5 90 –

*F* ≥ 3 11.4 86 84 *F* = 4 14.0 100 76

**(%)**

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**Specificity (%)**

15

Another liver elasticity-based imaging technique is ARFI (*acoustic radiation force impulse imaging*). Although for the time being there are few studies that have evaluated its utility in NAFLD patients, its great advantage is that it can be easily connected to traditional ultrasound scan enabling the positioning of the zone of interest under visual control [132, 133]. Another method suitable for studying the elastic properties of the hepatic parenchyma is magnetic resonance elastography (MRE). MRE can be more reliable than TE to diagnose advanced fibrosis; moreover, it has the advantage of being able to evaluate the whole hepatic parenchyma even in obese patients, but the technique is expensive and not widely available [134, 135]. Magnetic resonance imaging is more widely available and is the basis of new software called DEMILI (*Detection of Metabolic-Induced Liver Injury*), which through computerized optical analysis of its images determines a series of optical biomarkers enabling the detection of the presence of NASH (NASHMRI) and predicting significant fibrosis (FibroMRI) in NAFLD patients. For the detection of NASH, a cut-off has been established with NASHMRI of >0.5, presenting a sensitivity and specificity of 87 and 60%, respectively. In the case of FibroMRI, the cut-off is also >0.5 for the prediction of significant fibrosis with a sensitivity of 77% and a specificity of 80% [136]. Given that this technique enables the analysis of the total volume of the liver, as well as its use in the diagnosis of NASH and significant fibrosis, it enables the potential effects of a therapy to be monitored.

A recently developed technique is multiparametric magnetic resonance (MR) that includes *T*<sup>1</sup> mapping for fibrosis/inflammation imaging, *T*<sup>2</sup> ∗ mapping for liver iron quantification and proton magnetic resonance spectroscopy (<sup>1</sup> H-MRS) for liver fat quantification. In a recent study, it has demonstrated good correlation with disease severity in NAFLD patients, showing excellent accuracy in quantifying both the inflammatory and fibrotic components of NAFLD [137].

A summary of the approach to the management and characterization of NAFLD patients is shown in **Figure 1**.



liver diseases [117]. TE measures the propagation velocity of low-frequency waves (50 Hz) through the hepatic parenchyma using ultrasounds and is expressed in kilo Pascal (kPa); the higher the propagation velocity, the greater the stiffness of the tissue. The advantages provided by this technique are its speed, the immediacy of the results and the ease of handling. However, proper results require careful interpretation of data, based on at least 10 successful measurements, a success rate above 60% and an interquartile range (IQR) of <30% of the median value. A limitation of TE in NAFLD is the high rate of technical failure due to the attenuation of the elastic wave by interposition of adipose tissue secondary to the central obesity, very frequent in these patients. Although an XL probe has been developed, which enables greater penetration of the wave, this difficulty is often insurmountable [118, 119]. Moreover, this technique has been initially validated in patients with chronic infection by VHC [120], while the studies focusing on evaluating its use in NAFLD are smaller and have often used different cut-offs [42, 118, 121–129] (**Table 4**). According to the results of several studies, the cut-offs with M probe accepted for NAFLD patients are 7.0 kPa for significant fibrosis (≥F2), 8.7 kPa for advanced fibrosis (≥F3) and 10.3 kPa for cirrhosis (F4) [124, 126, 128]. When using the XL probe, these cut-offs differ as the measure of liver stiffness with this probe is less than that with the M probe in the same patient; in this case, 6.2, 7.2 and 7.9 kPa are the cut-offs for significant fibrosis, advanced fibrosis and cirrhosis, respectively [119, 130, 131].

Another liver elasticity-based imaging technique is ARFI (*acoustic radiation force impulse imaging*). Although for the time being there are few studies that have evaluated its utility in NAFLD patients, its great advantage is that it can be easily connected to traditional ultrasound scan enabling the positioning of the zone of interest under visual control [132, 133]. Another method suitable for studying the elastic properties of the hepatic parenchyma is magnetic resonance elastography (MRE). MRE can be more reliable than TE to diagnose advanced fibrosis; moreover, it has the advantage of being able to evaluate the whole hepatic parenchyma even in obese patients, but the technique is expensive and not widely available [134, 135]. Magnetic resonance imaging is more widely available and is the basis of new software called DEMILI (*Detection of Metabolic-Induced Liver Injury*), which through computerized optical analysis of its images determines a series of optical biomarkers enabling the detection of the presence of NASH (NASHMRI) and predicting significant fibrosis (FibroMRI) in NAFLD patients. For the detection of NASH, a cut-off has been established with NASHMRI of >0.5, presenting a sensitivity and specificity of 87 and 60%, respectively. In the case of FibroMRI, the cut-off is also >0.5 for the prediction of significant fibrosis with a sensitivity of 77% and a specificity of 80% [136]. Given that this technique enables the analysis of the total volume of the liver, as well as its use in the diagnosis of NASH

and significant fibrosis, it enables the potential effects of a therapy to be monitored.

mapping for fibrosis/inflammation imaging, *T*<sup>2</sup>

ton magnetic resonance spectroscopy (<sup>1</sup>

14 Liver Research and Clinical Management

shown in **Figure 1**.

A recently developed technique is multiparametric magnetic resonance (MR) that includes *T*<sup>1</sup>

has demonstrated good correlation with disease severity in NAFLD patients, showing excellent accuracy in quantifying both the inflammatory and fibrotic components of NAFLD [137]. A summary of the approach to the management and characterization of NAFLD patients is

∗ mapping for liver iron quantification and pro-

H-MRS) for liver fat quantification. In a recent study, it

**Table 4.** Comparative studies of FibroScan with liver biopsy in the detection of fibrosis in NAFLD.

phosphatase, bilirubin, globulin, albumin, serum insulin, hematocrit, INR and platelets, to predict advanced fibrosis. However, further studies are still necessary to externally validate this model. Other metabolic factors described with more evidence for the disease progression are central obesity, arterial hypertension and high levels of LDL cholesterol [7, 148–150]. No study shows cost-efficacy in the monitoring of the progression in these at-risk patients, but we recommend carrying out NFS and/or FIB-4 every 2 or 3 years in these patients with nonsignificant fibrosis, and if NASH and/or significant fibrosis is presented in the initial diagno-

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17

The other factor having the greatest effect on the disease progression is liver fibrosis [151]. In general, in a period of 15 years, 13% of the patients with stage F2 and 25% of those presenting with F3 will develop cirrhosis [6, 7, 62]. These patients with significant fibrosis should be considered for pharmacological treatment, besides lifestyle modifications (diet and exercise). Moreover, NAFLD patients may develop HCC even in the absence of cirrhosis [152], given that it is the continuous hepatocyte injury that leads to a compensatory proliferation, key driver of the development of HCC [153]. Therefore, patients with NASH and significant fibrosis, which is indicative of important cellular damage, are also at risk of developing this liver tumor.

With all this information, we recommend recalculating NFS and/or FIB-4 every 4–5 years for patients with NAFL without risk factors or if the patient develops DM2; in patients with NASH without significant fibrosis, we recommend an annual follow-up with a calculation of NFS and/or FIB-4 and carrying out TE and ultrasound, and in patients with significant fibrosis, a 6-monthly follow-up is recommended with special interest in screening for HCC. The management and follow-up of the patients with advanced fibrosis/cirrhosis due to NASH does not

Another important question is the evaluation of the response to the therapy provided. The non-invasive methods available currently have not been reliable or have not been validated to document efficacy of the treatments, so liver biopsy is still necessary to determine this effi-

In recent years, several studies have confirmed that the morbimortality associated with NAFLD is not limited only to hepatic injury, yet it is a disease with multisystemic behavior

As was previously mentioned, the concurrent characteristics of metabolic syndrome increase the risk of developing NAFLD, and a recent study of the HepaMet group relates the severity of NAFLD with the number of factors of the metabolic syndrome present (publication pending). However, the presence of NAFLD in itself also increases the risk of developing complications such as dyslipidemia and insulin resistance [154–156]. In this sense, the diagnosis and

sis, the follow-up will not differ from the rest of the patients.

differ from the rest of etiologies [25].

cacy, especially in a clinical trial setting.

**6. Screening of associated diseases**

**6.1. Insulin resistance and metabolic syndrome**

with affectation of different organs.

**Figure 1.** Practical approach to the management of patient with NAFLD.
