**2. Screening and diagnostic criteria**

The mechanisms leading to the development and progression of NAFLD are not completely known, but it is widely accepted that the initial events are dependent on the development of obesity and insulin resistance (IR) [11]. For this reason, NAFLD has a strong association with the factors constituting the metabolic syndrome, the prevalence in this group of patients being considerably heightened. This relation is especially close in morbid obesity, where NAFLD is present in more than 90% of the cases, this condition taking the form of steatohepatitis in a third of the cases, while in up to 5–10% of the subjects, the liver disease has progressed to cirrhosis [12, 13]. The association between NAFLD and IR or diabetes mellitus type 2 (DM2) has also been clearly established [14]. It has been demonstrated that DM2 is associated with a greater hepatic content of triglycerides independently of the body mass index (BMI) [15, 16]. Thus, the prevalence of NAFLD in DM2 patients can reach up to 70% [11, 17, 18]. Moreover, both prediabetes (glucose intolerance and altered glucose when fasting) and DM2 are related to the severity of liver damage, the presence of steatohepatitis, fibrosis and even HCC [1, 19, 20]. Overall, 80% of the NAFLD cases present with some of the cardiovascular risk factors that constitute the metabolic syndrome (IR, obesity, dyslipidemia and arterial hypertension), and its prevalence directly increases the number of these factors that are present [21].

As a consequence of its high prevalence, especially in subjects with the abovementioned risk factors, its prognostic implications, and given that NAFLD is generally an asymptomatic disease, some authors recommend the implantation of an NAFLD-screening programme within the risk population [22, 23]. However, this topic is at present controversial given the great load on the national health systems that could be caused by these screening programmes and the lack of efficacious treatments currently available. In fact, the principal associations for the study of liver diseases (American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of the Liver (EASL)) in their guidelines for clinical practice do not recommend this screening in any population [24] or they recommend it, with an A2 level of evidence, only for patients with DM2 independently of the levels of hepatic enzymes [25]. To attempt to answer this question, valid cost-utility studies are necessary in screening programmes. There is no discussion, however, about the need to act when faced with a patient suspected of having NAFLD and not to underestimate its discovery due to the limited clinical and analytical repercussion manifested at first. In our opinion, patients with NAFLD and suspected to have advanced disease must be evaluated in specialist units for their correct characterization in case of a prompt availability of specific treatment.

#### **2.1. Clinical and analytical manifestations**

(HCC) [4]. However, several studies with paired liver biopsies have demonstrated that both patients with NASH and those with NAFL have the potential to develop a progressive hepatic disease, and in this risk of progression there are some key factors such as diabetes mellitus [5, 6]. In general, patients with NAFLD have a higher long-term mortality than the general population, cardiovascular disease (CVD) being the principal cause of death, followed by different types of cancer [7–9] and liver-related complications, as well as the cardiovascular risk caused by the different factors of the metabolic syndrome, very frequent in this type of subjects; NAFLD is itself an independent risk factor for CVD [10]. Liver-related mortality is increased up to 10-fold in patients with NAFLD. In this sense, it should be emphasized that cirrhosis and HCC are the fifth most prevalent cause of mortality in the world. Therefore, given the hepatic and cardiovascular morbi-mortality generated by NAFLD, the early identification of these patients is important to provide suitable management that can lower the

The mechanisms leading to the development and progression of NAFLD are not completely known, but it is widely accepted that the initial events are dependent on the development of obesity and insulin resistance (IR) [11]. For this reason, NAFLD has a strong association with the factors constituting the metabolic syndrome, the prevalence in this group of patients being considerably heightened. This relation is especially close in morbid obesity, where NAFLD is present in more than 90% of the cases, this condition taking the form of steatohepatitis in a third of the cases, while in up to 5–10% of the subjects, the liver disease has progressed to cirrhosis [12, 13]. The association between NAFLD and IR or diabetes mellitus type 2 (DM2) has also been clearly established [14]. It has been demonstrated that DM2 is associated with a greater hepatic content of triglycerides independently of the body mass index (BMI) [15, 16]. Thus, the prevalence of NAFLD in DM2 patients can reach up to 70% [11, 17, 18]. Moreover, both prediabetes (glucose intolerance and altered glucose when fasting) and DM2 are related to the severity of liver damage, the presence of steatohepatitis, fibrosis and even HCC [1, 19, 20]. Overall, 80% of the NAFLD cases present with some of the cardiovascular risk factors that constitute the metabolic syndrome (IR, obesity, dyslipidemia and arterial hypertension), and

its prevalence directly increases the number of these factors that are present [21].

As a consequence of its high prevalence, especially in subjects with the abovementioned risk factors, its prognostic implications, and given that NAFLD is generally an asymptomatic disease, some authors recommend the implantation of an NAFLD-screening programme within the risk population [22, 23]. However, this topic is at present controversial given the great load on the national health systems that could be caused by these screening programmes and the lack of efficacious treatments currently available. In fact, the principal associations for the study of liver diseases (American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of the Liver (EASL)) in their guidelines for clinical practice do not recommend this screening in any population [24] or they recommend it, with an A2 level of evidence, only for patients with DM2 independently of the levels of hepatic

mortality for all causes.

4 Liver Research and Clinical Management

**2. Screening and diagnostic criteria**

A diagnosis of NAFLD is very often reached through a casual analytical discovery during a health examination after an alteration in tests of liver function, or an alteration in hepatic morphology detected through an image study done with another objective, given that it is generally an asymptomatic disease. In the cases in which the patient reports symptoms, they are usually mild and unspecific, asthenia and abdominal problems being frequent, especially in the right hypochondria. The physical exploration may be normal or detect a soft, painless hepatomegaly, although occasionally it is difficult to evaluate as these patients very often present with central-type obesity, and in the patients with advanced fibrosis and cirrhosis, we may find signs of portal hypertension such as ascites, splenomegaly or jaundice [26].

Analytically, most of the patients present tests with normal or discretely altered liver function, with a predominance of ALT (alanine aminotransferase) compared to AST (aspartate aminotransferase). On specific occasions, a discrete elevation can be appreciated in the markers of cholestasis, especially GGT (gamma-glutamyl transpeptidase), which has been related to obesity and IR [27]. Another frequent analytical discovery is the elevation of the levels of ferritin in blood and of the transferrin saturation index without having demonstrated a corresponding increase in the deposits of hepatic iron [28]. Something similar occurs with the presence of elevated autoantibodies, which appear quite frequently in NAFLD and are considered an epiphenomenon [29].

#### **2.2. Diagnosis of steatosis**

Hepatic steatosis is defined histologically as the deposit of fat ≥5% of the hepatocytes and is classified in four grades depending on the percentage of hepatocytes with steatotic vacuoles. The normal liver (S0) contains fat in less than 5% of the hepatocytes while grade 1 steatosis (S1) corresponds to less than 33% of the steatotic hepatocytes. In grade 2 and 3 steatosis, fat is present in at least 33 or 66% of the hepatocytes, respectively.

The presence of risk factors such as DM2, metabolic syndrome and obesity with the elevation of the hepatic enzymes, especially ALT, increases the possibility of fatty liver presenting. Nevertheless, although the ALT is a useful test, it is not valid for predicting the presence of this disease, or even the risk of progression, given that it can occur with normal hepatic enzymes [30]. In fact, in patients with DM2 and normal levels of ALT, a high prevalence of NAFL and NASH has been reported [31].

In clinical practice, ultrasound scan is a first-rate image technique if NAFLD is suspected due to its wide availability, low cost and safety [32]. The sensitivity of this technique is 93% when the steatosis is greater than 33%; however, this sensitivity decreases considerably when the steatosis affects less than 30% of the hepatocytes [33, 34]. Steatosis can also be diagnosed through computerized tomography (CT), but its cost and the patient's exposure to radiation make its systematic use in long-term follow-up unadvisable in this pathology; moreover, its sensitivity does not improve substantially if the steatosis is mild [32]. Magnetic resonance imaging (MRI), including spectroscopy, can diagnose content levels of hepatic fat >5% and it is reliable to determine changes (≥0.5%) in the grade of steatosis after weight loss. Although its use has widened in many studies, its use in clinical practice is limited by its cost and duration [35, 36].

The recently developed CAP (controlled attenuation parameter), an application of transient elastography (TE), which will be discussed later, available in the latest generation devices, enables the immediate and easy quantification of steatosis. CAP measures the degree of attenuation of the ultrasound wave transmitted through the liver, which is proportional to the amount of hepatic fat, and is less influenced by the sampling error than the liver biopsy, since it explores a liver volume approximately 100 times greater. Its values oscillate between 100 and 400 dB/m and it is possible to measure the liver stiffness used for the evaluation of fibrosis simultaneously. The studies published to date indicate that CAP is capable of diagnosing steatosis in chronic liver diseases of diverse causes even in mild stages (>10%) and has a good correlation with the degree of steatosis [37–44]. These studies show different cut-offs of CAP for the different grades of steatosis, but all of them demonstrate that the cut-offs do not differ among the different causes of liver diseases, in contrast with what happens with transient elastography [40]. In this sense, a recent meta-analysis including 2735 patients has established a series of cut-offs for the different grades of steatosis: 248 dB/m for S1, 268 dB/m for S2 and 280 dB/m for S3, with a sensitivity of 69, 77 and 88%, respectively, and a specificity of 82, 81 and 78%, respectively. In this meta-analysis, etiology, BMI and diabetes showed a significant influence on the value of the CAP, so the authors suggested the cut-offs established but subtracting 10 dB/m from the value of the CAP for the patients with NAFLD, 10 dB/m for diabetics, and subtracting/adding 4.4 dB/m for each unit of BMI above/below 25 kg/m<sup>2</sup> in the interval of 20–30 kg/m<sup>2</sup> [45].

Another image technique is magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), which is based on chemical shift-based water fat separation methods. MRI-PDFF has shown good correlation with histology-determined steatosis grade in NAFLD patients, so it could be used for follow-up and treatment-response evaluation [46].

Lastly, within the non-invasive diagnosis of steatosis, various serological tests of biomarkers have been developed to predict the existence of hepatic fat (**Table 1**) [47–51]. However, all these biomarkers may be influenced by inflammation and fibrosis, and given that they do not provide great advantages compared to image techniques and routine analysis, their use in clinical practice is not widespread; even so, the Fatty Liver Index (FLI) that uses easily available parameters could be considered, although CAP has demonstrated better performance than this test for the diagnosis of grade 2–3 steatosis [44]. Nevertheless, FLI has been associated independently to liver-related mortality, as well as to the mortality rates due to

cardiovascular disease and cancer, but it seems that these associations are interfered by the

**Indices Formula Cut-offs Sensitivity (%) Specificity (%)**

60 (high cut-off) 44 91

Diagnosis and Characterization of Non-Alcoholic Fatty Liver Disease

http://dx.doi.org/10.5772/intechopen.72668

7

Hepatic 8 × ALT/AST ratio + BMI 30 (low cut-off) 93 40 Steatosis (+2, if DM; +2, if female) 36 (high cut-off) 45 93

Fatty liver exp(*n*)/1 + exp (*n*) × 100 10 (low cut-off) 95 29

SteatoTest Proprietary formula 0.3 (low cut-off) 90 54

NAFLD liver −2.89 + 1.18 × metabolic −0.640 86 71

Lipid LAP (men) = Waist 20 (low cut-off) 99 16 accumulation circumference – 65 80 (high cut-off) 43 94

(a2-macroglobulin, 0.7 (high cut-off) 46 88

Index (HSI)

Index (FLI) (*n*) = 0.953 × ln(TG) +

0.139 × BMI + 0.718 ×

haptoglobin,

bilirubin, ALT,

gender)

Fat score Syndrome (yes: 1, no: 0)

product (LAP) LAP (women) = Waist

**Table 1.** Indices for diagnosis of steatosis.

ln(GGT) + 0.053 × waist circumference – 15.745

apolipoprotein A1, GGT,

cholesterol, triglycerides, glucose, BMI, age,

+ 0.45 × Type 2 diabetes (yes: 2, no: 0) + 0.15 × insulin + 0.04 × AST − 0.94 × AST/ALT

When steatosis is suspected in the aforementioned non-invasive methods, the liver biopsy is still the gold-standard method to conclusively diagnose NAFLD and the only one capable of distinguishing between NAFL and NASH, thus enabling the classification of the disease

risk conferred by the state of insulin resistance [52].

circumference − 58


**Table 1.** Indices for diagnosis of steatosis.

in the

when the steatosis is greater than 33%; however, this sensitivity decreases considerably when the steatosis affects less than 30% of the hepatocytes [33, 34]. Steatosis can also be diagnosed through computerized tomography (CT), but its cost and the patient's exposure to radiation make its systematic use in long-term follow-up unadvisable in this pathology; moreover, its sensitivity does not improve substantially if the steatosis is mild [32]. Magnetic resonance imaging (MRI), including spectroscopy, can diagnose content levels of hepatic fat >5% and it is reliable to determine changes (≥0.5%) in the grade of steatosis after weight loss. Although its use has widened in many studies, its use in clinical practice is limited by its cost and dura-

The recently developed CAP (controlled attenuation parameter), an application of transient elastography (TE), which will be discussed later, available in the latest generation devices, enables the immediate and easy quantification of steatosis. CAP measures the degree of attenuation of the ultrasound wave transmitted through the liver, which is proportional to the amount of hepatic fat, and is less influenced by the sampling error than the liver biopsy, since it explores a liver volume approximately 100 times greater. Its values oscillate between 100 and 400 dB/m and it is possible to measure the liver stiffness used for the evaluation of fibrosis simultaneously. The studies published to date indicate that CAP is capable of diagnosing steatosis in chronic liver diseases of diverse causes even in mild stages (>10%) and has a good correlation with the degree of steatosis [37–44]. These studies show different cut-offs of CAP for the different grades of steatosis, but all of them demonstrate that the cut-offs do not differ among the different causes of liver diseases, in contrast with what happens with transient elastography [40]. In this sense, a recent meta-analysis including 2735 patients has established a series of cut-offs for the different grades of steatosis: 248 dB/m for S1, 268 dB/m for S2 and 280 dB/m for S3, with a sensitivity of 69, 77 and 88%, respectively, and a specificity of 82, 81 and 78%, respectively. In this meta-analysis, etiology, BMI and diabetes showed a significant influence on the value of the CAP, so the authors suggested the cut-offs established but subtracting 10 dB/m from the value of the CAP for the patients with NAFLD, 10 dB/m for diabetics, and subtracting/adding 4.4 dB/m for each unit of BMI above/below 25 kg/m<sup>2</sup>

Another image technique is magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), which is based on chemical shift-based water fat separation methods. MRI-PDFF has shown good correlation with histology-determined steatosis grade in NAFLD

Lastly, within the non-invasive diagnosis of steatosis, various serological tests of biomarkers have been developed to predict the existence of hepatic fat (**Table 1**) [47–51]. However, all these biomarkers may be influenced by inflammation and fibrosis, and given that they do not provide great advantages compared to image techniques and routine analysis, their use in clinical practice is not widespread; even so, the Fatty Liver Index (FLI) that uses easily available parameters could be considered, although CAP has demonstrated better performance than this test for the diagnosis of grade 2–3 steatosis [44]. Nevertheless, FLI has been associated independently to liver-related mortality, as well as to the mortality rates due to

patients, so it could be used for follow-up and treatment-response evaluation [46].

tion [35, 36].

6 Liver Research and Clinical Management

interval of 20–30 kg/m<sup>2</sup>

[45].

cardiovascular disease and cancer, but it seems that these associations are interfered by the risk conferred by the state of insulin resistance [52].

When steatosis is suspected in the aforementioned non-invasive methods, the liver biopsy is still the gold-standard method to conclusively diagnose NAFLD and the only one capable of distinguishing between NAFL and NASH, thus enabling the classification of the disease according to the grade of activity (inflammation and hepatocyte injury) and the stage of fibrosis, the best predictors of the disease progression [53, 54]. The advantages and, especially, the drawbacks of the liver biopsy are dealt with later.

changes produced on initiating specific treatment. Nowadays, we do not have a test available that has these characteristics, so these non-invasive methods are based on diverse complementary approaches: clinical factors, genetics, serological markers, image tests and transient

Diagnosis and Characterization of Non-Alcoholic Fatty Liver Disease

http://dx.doi.org/10.5772/intechopen.72668

9

The best predictor of the evolution of NAFLD is the presence of necroinflammation and fibrosis in liver biopsy; however, there are more and more studies reporting no insignificant rates of progression of simple steatosis [5, 6, 62]. A first study that analyzed patients with NAFLD and paired biopsies demonstrated that even patients with simple steatosis can progress to NASH and advanced fibrosis, especially in the presence of metabolic risk factors [6]. Therefore, there is a series of non-modifiable and modifiable factors in patients associated

Various transversal studies have demonstrated that the disease is more severe in older patients, although this phenomenon could be due to the sum of pathogenic factors and a greater duration of the liver disease itself and the associated diseases [8, 63, 64]. In fact, the longitudinal studies have not managed to demonstrate that age is a factor that aggravates the disease per se [65]. The association between sex and fibrosis progression is controversial; two transversal studies show that men and post-menopause women have a greater risk of fibrosis in comparison with pre-menopause women; moreover, precocious menopause is associated with a greater risk of fibrosis [66–69]. Other non-modifiable factors are genetic; dozens of genes with multiple polymorphisms associated with NAFLD have been discovered thanks to genome-wide association studies (*GWAS*), but the number of strongly validated genes in large independent cohorts is limited to two, *patatin-like phospholipase domain containing 3* (PNPLA3) and *transmembrane 6 superfamily member 2* (TM6SF2) [70]. The presence of the single nucleotide polymorphisms (SNPs) rs738409 and rs58542926 of the genes PNPLA3 and TM6SF2, respectively, has been associated with a greater risk of NAFLD, as well as a more severe disease [71–76]. Recently, an SNP of IL28b (also implicated in the response to interferon in chronic hepatitis C patients (VHC)) has been associated with an increment in fibrosis in NAFLD patients [77]. Moreover, in a controlcase study carried out by our working group, we have observed that the presence of the variants rs1421085 and rs1558902 of the fat mass and obesity-associated (FTO) gene confer a high risk of liver inflammation particularly in patients of normal weight with NAFLD (unpublished).

On the other hand, NAFLD tends to be more severe in patients with various factors of the metabolic syndrome, particularly DM2 and obesity. In fact, the reduction in weight and good glycemic control are associated with an improvement in inflammation and liver fibrosis [11, 78, 79]. However, it is known that NASH can also be present in slim subjects although it is unknown whether the natural history of the disease in these slim subjects is similar to that present in obese subjects. As for arterial hypertension, it is arguable whether its treatment improves the histology of NASH [5, 80]. Another factor of the metabolic syndrome, frequent in NAFLD patients, is dyslipidemia, fundamentally in the form of hypertriglyceridemia and atherogenic dyslipidemia [64, 81]; but moreover, a recent study has related the very low-density lipoprotein (VLDL) profile with the NAFLD severity,

**3.2. Risk factors associated with non-alcoholic steatohepatitis and progressive disease**

with a greater risk of development of NASH and more progressive disease.

elastography [60, 61].

#### **2.3. Initial diagnosis of NAFLD**

From what has been mentioned so far, we can specify a series of characteristics that indicate a patient with NAFLD: (1) radiological evidence of steatosis or CAP >248 dB/m ± Abnormal liver blood test, (2) the presence of insulin resistance or another component of the metabolic syndrome, (3) consumption of alcohol of <30 g/d in men and <20 g/d in women and (4) exclusion from other causes of chronic liver disease (viral hepatitis, cholestatic diseases, autoimmune hepatitis, hemochromatosis, α1 antitrypsin deficiency, Wilson's disease, drug-induced liver injury and celiac disease) [24, 25]. Once the initial diagnosis of NAFLD has been made, our next step is to evaluate the stage of disease and the necessity of carrying out a liver biopsy.
