*2.1. Steatosis*

Hepatic steatosis is the key feature of NAFLD. Steatosis is diagnosed when more than 5% of hepatocytes contain fat or when the total amount of intrahepatic triglycerides is bigger than 5.5% without having any other liver disease in the patient's history [23,24]. Today there is no specific serum marker to assess hepatic steatosis available. However, several reproducible blood biomarker panels and scores were developed to help diagnose NAFLD (Table 1).


**Table 1.** Blood biomarker panels to identify inflammatory liver disease.

Abbreviations used are: α2M, α2-macroglobulin; γ-GT, γ-glutamyltransferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HA, hyaluronic acid; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; M30, antigen of the serum cytokeratin 18; PIIINP, amino-terminal propeptide of type III procollagen; TIMP-1, tissue inhibitor of metalloproteinases 1.

Most of these multiparametric panels include biochemical markers indicating liver damage or dysfunction (AST, ALT, bilirubin, γ-GT, platelet count, haptoglobin), lipid metabolism disorders (cholesterol, triglycerides), diabetes (HbA1c, fasting insulin level), inflammation (α2M, ferritin), or provide information about matrix expression and turnover (TIMP-1, PIIINP, HA) (Figure 3).

**Figure 3.** Significance of laboratory parameters in the diagnosis NAFLD and NASH. In the scheme, the biological source and alteration of individual biochemical markers during progression of NAFLD/NASH are indicated. High caloric intake and elevated quantities of fat result in hepatic steatosis, triggering steatohepatitis. The inflammatory response is triggered by infiltrating immune cells and liver-resident Kupffer cells releasing a plenitude of inflammatory triggers. As a consequence, the expression of acute phase response proteins (α<sup>2</sup> macroglobulin (α2M) and ferritin) is increased in hepatocytes. In addition, the increase of cholesterol and triglycerides provokes cellular fat accumulation, damage, and cellular leakage of hepatocytes. This is indicated by elevated quantities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, and γ-glutamyltransferase (γ-GT). Subsequently, the overall capacity of these cells to synthesize typical liver proteins (haptoglobuin, thrombopoietin) decreases. Lower quantities of thrombopoietin results in reduced formation of platelets within the bones. Dysfunction of synovial lining cells (reduced capacity to degrade hyaluronic acid (HA)) and ongoing fibrogenesis lead to elevated levels of HA. In addition, the transdifferentiation of hepatic stellate cells (HSC) to myofibroblast (MFB) is associated with the occurrence of typical biomarkers (tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP)), which correlate to extracellular matrix formation and/or turnover. The metabolic syndrome associated with NAFLD/NASH results in higher quantities of fasting insulin and basal glucose triggering the non-enzymatic formation of glycated hemoglobin (HbA1c). All these parameters are diagnostically relevant in the diagnosis or scoring of NAFLD/NASH and are the basis of various blood biomarker panels to identify inflammatory liver disease.

With an AUROC (area under the receiver-operating characteristic curve) accuracy value of 0.87, the NAFLD ridge score is currently one of the most efficient panel based on laboratory parameters. The NAFLD ridge score was developed as a machine learning algorithm facilitating registry research. It includes serum levels of alanine aminotransferase (ALT), high-density lipoprotein (HDL) cholesterol, triglycerides, hemoglobin A1c (HbA1c), leukocyte count, and the presence of hypertension [43,44]. With proton magnetic resonance spectroscopy (H-MRS) as reference, the NAFLD ridge score has a negative predictive value of 96%. However, this score is good to detect NAFLD but yet limited to the research setting and does not give the opportunity to distinguish between different steatosis grades or to assess changes during the development of steatosis over time.

A quantitative and by this more sensitive score to be calculated is the NAFLD Liver Fat Score (NLFS). This score includes the measurement of the liver fat content as determined by H-MRS, the presence or absence of the metabolic syndrome together with type 2 diabetes mellitus, aspartate aminotransferase (AST) levels, the AST:ALT ratio, and the fasting insulin serum level. With a sensitivity of 86% and a specificity of 71% the NLFS defines a liver fat content of more than 5.56% [45]. A recent study from Ruiz-Tovar and colleagues tested the accuracy of the NLFS in patients one year after bariatric surgery and considered it to be the most accurate biochemical score to assess liver steatosis at the moment [61]. The Hepatic Steatosis Index (HIS) also considers the AST/ALT ratio, BMI, diabetes and sex and has a sensitivity of 66% and a specificity of 69% [46].

The fatty liver index (FLI) includes BMI, waist circumference and serum levels of triglycerides and the γ-glutamyltransferase (γ-GT). It could be shown that the FLI significantly correlates with insulin resistance [47,48]. The major drawback when rating HIS and FLI is that ultrasonography is used as the reference standard to diagnose fatty liver. This technique is in general dependent on the operator and thereby to some extent biased and insensitive if only mild steatosis is present. The lipid accumulation product index (LAP) first established by Bedogni et al. takes into account sex, serum triglyceride levels and weight circumference to evaluate lipid overaccumulation [49].

A comparison of the accuracy in predicting NAFLD in a cross-sectional NAFLD cohort showed that NLFS is the best score to reliably predict NAFLD with an AUC of 0.771 [62]. Although the presented scores are capable to indicate the presence of hepatic steatosis, there are several limitations given. To be critically considered are the facts that using these indices it is not possible to distinguish between different steatosis grades and detect and trace changes over time is not possible.
