Adv Lab Med 2021; 2(2): 199–208 Guidelines and Recommendations Armando R. Guerra-Ruiz*, Gregori Casals, Paula Iruzubieta, Marta Lalana, Alba Leis, Rosa María López, Javier Crespo and Manuel Morales-Ruiz Biochemical assessment of metabolic associated fatty liver disease https://doi.org/10.1515/almed-2021-0009 This document provides an overview of the most relevant Received June 29, 2020; accepted October 18, 2020; serological markers in the characterization and diagnosis of published online March 15, 2021 MAFLD. An example is provided of a routine diagnostic algorithm that incorporates serological testing. A range of Abstract: Metabolic-associated fatty liver disease (MAFLD) useful serological scores are currently available for the is defined as fat accumulation in the liver in the presence of management of MAFLD patients, especially for the stratifi- metabolic alterations. This disorder is generally asymp- cation of patients at risk of fibrosis. A large proportion of tomatic and may progress to severe liver disease, which are the population is at risk of developing severe liver disease. linked to inflammation and/or fibrosis. MAFLD has a high The integration of non-invasive serological markers in prevalence (26%) and therefore a considerable number of the stratification of patients at risk for liver fibrosis may patients are at high risk of having advanced liver disease. contribute to improve the control and management of MAFLD patients. *Corresponding author: Armando Raúl Guerra Ruiz, Commission on Keywords: hepatic steatosis; liver fibrosis; metabolic-asso- Biochemistry of Liver Disease, Spanish Society of Laboratory Medicine ciated fatty liver disease; serum markers; steatohepatitis. (SEQC-ML), Barcelona, Spain; and Service of Clinical Analysis, Hospital Universitario Marqués de Valdecilla, Avenida Valdecilla, s/n. CP 39008, Santander, Spain, E-mail: a.raulguerra@gmail.com. https://orcid.org/0000-0001-8896-8611 Definition and introduction Gregori Casals, Commission on Biochemistry of Liver Disease, Spanish Society of Laboratory Medicine (SEQC-ML), Barcelona, Spain; Metabolic-associated fatty liver disease (MAFLD), previ- and Service of Biochemistry and Molecular Genetics, CDB, Hospital ously known as non-alcoholic fatty liver disease (NAFLD) Clinic de Barcelona, IDIBAPS, CIBEREHD, Barcelona, Spain [1] encompasses a wide spectrum of liver lesions associ- Paula Iruzubieta and Javier Crespo, Spanish Society of Digestive Pathology (SEPD), Madrid, Spain; and Service of Digestive System, ated with the accumulation of fat in the liver, which is Hospital Universitario Marqués de Valdecilla, Clinical and Traslational known as steatosis. In the past, diagnosis of NAFLD was Research Group on Digestive Disorders, IDIVAL, Santander, Spain only established after considerable alcohol consumption Marta Lalana, Commission on Biochemistry of Liver Disease, Spanish or drug-induced liver disease had been excluded. How- Society of Laboratory Medicine (SEQC-ML), Barcelona, Spain; and ever, the new criteria for the diagnosis of MAFLD elimi- Service of Clinical Analysis, Hospital de Barbastro, Huesca, Spain nate these exclusion criteria and incorporate “positive” Alba Leis, Commission on Biochemistry of Liver Disease, Spanish Society of Laboratory Medicine (SEQC-ML), Barcelona, Spain; and criteria. Thus, diagnosis of MAFLD [2] is currently based Service of Clinical Analysis and Biochemistry, Laboratori Clínic on histological (biopsy), imaging, or serological evidence Metropolitana Nord, Hospital Universitari Germans Trias i Pujol, of liver fat accumulation (presence of steatosis), accom- Badalona, Spain panied by one of the following criteria: overweight or Rosa María López, Commission on Biochemistry of Liver Disease, obesity, type 2 diabetes mellitus, and/or evidence of Spanish Society of Laboratory Medicine (SEQC-ML), Barcelona, Spain; and Departments of Biochemistry and Microbiology, Unit of Liver metabolic disorders. The latter, which are also criteria for Disease, Hospital Universitari Vall d’Hebron, Universitat Autònoma de the diagnosis of metabolic syndrome, are described in Barcelona, Barcelona, Spain Table 1. Manuel Morales-Ruiz, Commission on Biochemistry of Liver Disease, MAFLD has become the leading cause of chronic liver Spanish Society of Laboratory Medicine (SEQC-ML), Barcelona, Spain; disease in Western countries, with a prevalence of 24% in and Department of Biomedicine of the Faculty of Medicine and Health the general population [3]. MAFLD is strongly associated Sciences, Service of Biochemistry and Molecular Genetics, Hospital Clínic de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, with the signs and symptoms of metabolic syndrome, with Barcelona, Spain a higher prevalence among patients with this condition. Open Access. © 2021 Armando R. Guerra-Ruiz et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 200 Guerra-Ruiz et al.: Biochemical assessment of MAFLD Table : Evidence of metabolic alterations, defined as the presence of at least two of the following findings. The terms related to non-alcoholic fatty liver disease (NAFLD) must be replaced with those of – Waist circumference ≥102 cm in men and ≥88 cm in women. metabolic-associated fatty liver disease (MAFLD), – Blood pressure ≥130/85 mmHg, or specific pharmacological treatment. as this term better reflects current knowledge of the – Plasma triglycerides ≥150 mg/dL (≥1.70 mmol/L), or specific pathologic process and allows for a better pharmacological treatment. categorization and management of patients. – Plasma HDL cholesterol <40 mg/dL (<1.0 mmol/L) for men and <50 mg/dL (<1.3 mmol/L) for women, or specific pharmacological treatment. – Prediabetes (fasting glycemia: 100–125 mg/dL [5.6–6.9 mmol/L], or 2 h after glucose tolerance test: 140–199 mg/dL [7.8– Histopathology and natural history 11.0 mmol/L] or HbA1c: 5.7–6.4% [39 a 47 mmol/mol]). – HOMA-IR (Homeostasis model assessment for assessing insulin MAFLD is categorized into two histological types: a) simple resistance) ≥2.5. steatosis, which includes patients with hepatic steatosis – High-sensitivity C-reactive protein>2 mg/L. with or without mild inflammation; and b) steatohepatitis, characterized by the presence of inflammation and hepa- tocyte injury (ballooning) with or without concomitant fibrosis [5, 6]. By histological analysis, fatty liver is char- The prevalence of steatohepatitis is 3–5% in the general acterized by: 1) simple steatosis, 2) steatosis with concom- population, which frequently has metabolic comorbidities itant lobular or portal inflammation without ballooning, such as diabetes and obesity [4]. About, 25% of these or 3) steatosis with concomitant ballooning but without patients will develop cirrhosis and/or hepatocellular car- inflammation. Diagnosis of steatohepatitis requires the cinoma (CHC). presence of steatosis, ballooning, and lubular inflamma- In general terms, long-term mortality is higher in tion. Chronic liver inflammation may progress to fibrosis MAFLD patients as compared to the general population, and eventually to cirrhosis. with cardiovascular disease, cancer, and liver disease Figure 1 includes a diagram of the natural history of being leading causes of mortality, in that order. Consid- MAFLD. Although the mechanisms that induce the devel- ering the morbidity and mortality from cardiovascular and opment and progression of MAFLD are still unclear, it is liver disease secondary to MAFLD, simple early diagnosis widely accepted that initial events are contingent on is essential to ensure the adequate management and obesity and insulin resistance [7]. However, since not all treatment of these patients and reduce all-cause mortality. MAFLD patients are insulin-resistant or obese, it is evident Figure 1: Natural history of metabolic associated fatty liver disease (MAFLD). Guerra-Ruiz et al.: Biochemical assessment of MAFLD 201 that both environmental and genetic factors play a role in finding that shows alterations in liver function markers. the etiopathogenia of MAFLD [8]. Additionally, cases of Another cause of suspicion is an alteration in liver fibrosis regression have been documented in patients with morphology or echogenicity detected by an imaging study MAFLD, with reported percentages ranging from 15 to 33% performed for another reason [15, 16]. according to the particularities of the group studied [9]. MAFLD patients may exhibit elevated concentrations of More specifically, 7% of cases steatohepatitis pro- aminotransferases, being MAFLD the main cause of gresses to hepatocellular carcinoma (HCC) in a timeframe persistent elevation of liver function markers. However, of 6.5 years. In 11% of cases, steatohepatitis progresses to normal levels of aminotransferases do not exclude the cirrhosis in a period of 15 years [10]. Indeed, the presence of presence of MAFLD. Indeed, most patients have normal cirrhosis secondary to steatohepatitis in patients waiting aminotransferase concentrations [17, 18]. When amino- for a liver transplant has tripled in the last decade, being transferases are elevated, levels of alanine aminotrans- the second leading cause of inclusion in the waiting list in ferase (ALT) and aspartate aminotransferase (AST) are the USA [11]. slightly above the upper limit of normality. In the initial The main prognostic factor of MAFLD is the presence stages of steatosis, the AST/ALT ratio generally is<1. of liver fibrosis, since fibrosis determines the risk of Reversal of this ratio may be suggestive of progression cirrhosis, hepatic decompensation, and/or development of to fibrosis. Nevertheless, the degree of elevation of amino- HCC. In other words, the presence of fibrosis determines transferases is not related to the degree of fibrosis or liver the risk of mortality from liver disease. The degree of inflammation. As it occurs with aminotransferases, gamma- fibrosis is independently associated with all-cause mor- glutamyltransferase (GGT) is frequently elevated in MAFLD tality, including cardiovascular mortality, especially in the patients, and its elevation has been associated with a higher case of steatohepatitis [12, 13]. risk of fibrosis [19]. Alkaline phosphatase can also be In MAFLD, the most widespread histological staging slightly elevated, in parallel to other liver function markers. system for fibrosis is the one described by Brunt et al. [14], Another frequent analytical finding is increased serum where S1 is defined as perisinusoidal or periportal fibrosis; ferritin concentrations and transferrin saturation index. S2 as perisinusoidal fibrosis with portal or periportal No evidence has yet been provided of a concurrent in- fibrosis; S3 as bridging fibrosis; and S4 as cirrhosis. The crease in hepatic iron deposition [20]. Likewise, patients term “significant fibrosis” refers to S2 or a higher stage, and may also frequently exhibit elevated serum autoantibody advanced fibrosis to S3 or a higher stage. Hepatic biopsy is level, which is considered an epiphenomenon [21]. Some the gold standard for the accurate diagnosis of MAFLD authors credit a prognostic value to this marker in liver as well as for differential diagnosis of simple steatosis disease [22, 23]. Bilirubin and albumin are rarely altered, and steatohepatitis. This method categorizes the disease except for patients with cirrhosis, who also exhibit pro- according to the level of activity (inflammation and cellular longed prothrombin time, thrombocytopenia, and damage) and the stage of fibrosis. Biopsy, however, is an neutropenia. invasive method with an associated risk of complications On suspicion of MAFLD, ultrasonograpy is the first- besides some limitations, such as potential sampling errors line imaging study employed in routine practice because of and within- and inter-subject variability. For this reason, its wide availability, low cost, and safety. The main limi- efforts are increasingly focused on the development of non- tation of this imaging study is that it has a limited sensi- invasive diagnostic methods to detect steatohepatitis and tivity for the detection of mild steatosis. Thus, steatosis is fibrosis as first-line screening for identifying patients with not detected if it is<20% or in subjects with a BMI>40 [24]. severe liver disease and higher risk of mortality. Non- In addition, ultrasonography is not useful for differenti- invasive methods include serological markers based on ating simple steatosis from steatohepatitis. analytical parameters, as detailed below. Differential diagnosis of simple steatosis vs. steato- hepatitis along with staging of liver fibrosis are key factors Determining the presence and degree of fibrosis in in the diagnosis of MAFLD, as patients with steatohepatitis MAFLD is crucial, as this parameter is associated and fibrosis are at the highest risk of developing liver with all-cause mortality. complications and cardiovascular disease. As it was mentioned above, biopsy is the gold standard for the his- tological staging of liver lesions. Nevertheless, we should not forget that liver biopsy is an invasive procedure with Analytical profile and diagnosis potential complications and its characteristics coupled with the high prevalence of fatty liver disease hinder the Most patients with MAFLD remain asymptomatic, and routine use of liver biopsy in a high proportion of patients. suspicion of steatosis is raised by an incidental analytical As a result, this technique is restricted to some clinical 202 Guerra-Ruiz et al.: Biochemical assessment of MAFLD settings. Liver biopsy is only recommended to assess the with insulin-resistance, and predict future metabolic, presence of fibrosis, disease severity, co-occurrence of liver, and cardiovascular events with variable accuracy. other liver diseases, and/or in patients at a higher risk of These scores indicate the presence of steatosis with ac- steatohepatitis and/or advanced fibrosis [5, 25, 26], who curacy, although they do not allow for the quantification can be previously identified by using other less invasive of liver fat [31]. techniques. This highlights the relevance of non-invasive The most widespread and extensively validated tools methods in the diagnosis and staging of MAFLD, as they are FLI and HSI. FLI values<30 exclude the presence of are useful in the characterization of patients and identifi- steatosis, with a likelihood ratio of 0.2; values≥60 are cation of high-risk patients [16], and spare low-risk patients suggestive of steatosis, with a likelihood ratio of 4.3. from undergoing a liver biopsy. An HSI<30 excludes MAFLD (with a negative likelihood Non-invasive methods are categorized into two ratio of up to 0.186), whereas an HSI≥36 confirms a diag- groups: those based on “biological” or serum markers, and nosis of MAFLD (with a minimum positive likelihood ratio those based on “physical” or radiological markers. The of 6.069). latter include transient elastography (FibroScan®) and These scores may be influenced by the presence of liver magnetic resonance elastography. Serum biochemical inflammation and fibrosis and, as they do not offer ad- markers are gaining ground because of their validity, vantages over routine imaging and analytical studies, their reproducibility, and easy use, emerging as a first-line use is scarcely spread [16]. However, their wide availabil- technique in primary and secondary care for determining ity, low cost and accessibility in primary care make them whether further studies are needed. Next, we will discuss candidate to becoming a first-line screening tool for the main serum markers identified to date for the detection MAFLD. Some authors advocate FLI as first-line screening of the three histological components of MAFLD (steatosis, for advanced liver disease in the general population, as it inflammation, and fibrosis). they identify patients with steatosis [32]. • The inclusion of GGT in basic liver panel may help • Because of their wide availability, serum scores in establishing the diagnosis and prognosis of should be the first-line screening test for steatosis MAFLD. in high-risk population. • In the management of MAFLD, non-invasive • FLI and HSI emerge as the best option, as they methods are useful to assess the probability that have been widely validated in our population. the patient has a severe histological lesion and requires a liver biopsy. Serum markers of steatohepatitis Steatosis scores The serum markers investigated so far as potential markers of steatohepatitis are involved in the pathophysiological Several serum scores have been developed to predict the pathways of the disease (apoptosis/cell death, inflamma- presence or not of hepatic steatosis: Fatty liver index tion, and oxidative stress). The most extensively studied (FLI) [27], SteatoTest [28], NAFLD-Liver Fat Score [29] marker is cytokeratin-18 fragments (CK18-F), a product of and Hepatic Steatosis Index (HSI) [30]. Table 2 contains a hepatocyte apoptosis [33]. A meta-analysis reported an summary of the variables that compose each index. AUROC of 0.82 for CK18-F and a sensitivity and specificity These scores have been validated both, for the general of 75 and 77%, respectively, for the prediction of steato- population and population with obesity, are associated hepatitis, which indicates a very limited diagnostic Table : Predictive biochemical scores for hepatic steatosis. Biochemical markers Other variables Ref. Fatty Liver Index (FLI) Triglycerides, GGT BMI, waist (cm) [] Steatotesta α-macroglobulin, haptoglobin, apo A, bilirubin, Age, sex, DM [] GGT, ALT, glucose, triglycerides, cholesterol NAFLD – Liver Fat Score Insulin, AST, ALT DM, metabolic syndrome [] Hepatic Steatosis Index (HIS) ALT, AST BMI, DM [] a Registered score. Apo A, apolipoprotein A; BMI, body mass index; DM, diabetes mellitus. Guerra-Ruiz et al.: Biochemical assessment of MAFLD 203 accuracy. In addition, a wide variety of cut-off values were addition, especially in the case of MAFLD, the degree of reported [34]. However, when this marker was incorpo- fibrosis has been found to be independently associated rated to several panels, their diagnostic efficacy increased with cardiovascular risk and all-cause mortality [12, 13]. by up to 0.92 [35]. Further studies are needed to determine Diagnosis of cirrhosis is easy when the patient has the utility of this marker. already exhibited signs of decompensated liver disease Other markers studied include hormones such as the (ascites, gastrointestinal bleeding due to esophageal vari- fibroblast growth factor 21 (FGF21) and adiponectin, with a ces, hepatic encephalopathy). However, it is in the early very low diagnostic accuracy. Oxidative stress and stages (S2 fibrosis or significant fibrosis) when prognosis can inflammation markers such as interleukin 6 and tumor be changed the most. As patients with cirrhosis are generally necrosis factor-α (TNFα) have also been studied. These asymptomatic, serum markers play a crucial role. markers have been assessed in small case series or pilot Serum markers of liver fibrosis are categorized into: 1) studies in heterogeneous groups of patients, with con- indirect markers of liver function, such as albumin, bili- flicting results. None of the markers studied so far could rubin, AST and ALT; and 2) direct markers, which are differentiate NASH from simple steatosis with a high components of the extracellular matrix, including hyal- sensitivity and specificity [36]. To improve the diagnostic uronic acid, matrix metalloproteinases, and collagen accuracy of markers, several predictive models have been subtypes [44, 45]. A large proportion of these markers have developed that combine some of these serum biomarkers been evaluated in patients with MAFLD, but none has with analytical parameters and clinical variables, including shown a good diagnostic efficacy. The most effective genetic polymorphisms. However, these models have not approach is based on the use of predictive models that been adequately validated and are not recommended in combine serum markers with the clinical characteristics of clinical practice [37]. the patient [46–68], which are described in Table 3. The evidence provided so far by metabolomic studies There are two large groups of predictive models of helped a Spanish research group to develop a test (OWL advanced fibrosis: a) simple models, which use a combi- Liver Test) that differentiates steatohepatitis from simple nation of routine analytical parameters and clinical vari- steatosis, with good sensitivity and specificity (ROC>0.8). ables; and b) complex models, which use serum markers This test was developed using samples from a cohort of 465 involved in the processing and degradation of the extra- patients [38] and has been validated in blind studies in two cellular matrix (direct). It is important to note that the independent cohorts [39]. Limitations to the use of this test markers included in these predictive models of fibrosis can in other ethnic groups and patients with poor diabetes be influenced by other causes, such as thrombocytopenia control were recently solved by the inclusion of trans- unrelated to liver disease, elevated levels of GGT induced aminases and glycosylated hemoglobin (HbA1C) in the by antiretroviral therapies, alcohol consumption, or the diagnostic algorithm [40]. The test is validated and mar- use of medications with liver toxicity, Gilbert syndrome, keted with the CE mark. The Spanish Society of Digestive hemolysis and cholestasis, to name a few [25, 56, 69, 70]. Disorders (SEPD) recently launched a multicentric study, Direct markers better reflect extracellular matrix turnover the “NASH Registry” aimed at assessing the usefulness of as compared to matrix deposition. Concentrations of direct this tool. markers increase as the level of inflammation increases, whereas elevated matrix deposition can be underestimated Further studies are needed to validate the impact of when inflammation is minimal. However, none of these serum markers of steatohepatitis in clinical markers is specific to liver function and all can be influ- practice. enced by other comorbidities and states (postoperative) associated with fibrosis. Therefore, concurrent extrahe- patic inflammation can increase serum concentrations of a marker. Additionally, the release of some markers can be Serum markers of liver fibrosis influenced by sinusoidal endothelial cell dysfunction or reduced bile excretion secondary to liver disease. Never- Chronic liver disease is characterized by inflammation, theless, the accessibility, low cost and high negative pre- ballooning, hepatocyte necrosis or apoptosis and fibrosis. dictive value of simple models make them a useful tool for However, fibrosis has been widely demonstrated to be the initial diagnosis based on a critical interpretation of re- most determinant factor of liver disease progression sults. Table 4 shows the diagnostic value of high cut-off [12, 41–43]. For this reason, staging the degree of fibrosis is values for the main serological indices of advanced fibrosis mandatory in a patient with chronic liver disease. In in patients with MAFLD. 204 Guerra-Ruiz et al.: Biochemical assessment of MAFLD Table : Scores for non-invasive screening for liver fibrosis. Variables are categorized into direct and indirect biochemical markers and other variables. Indirect biochemical markers Direct biochemical markers Other Ref. variables NAFLD Fibrosis Score (NFS) Glucose, platelets, AST, ALT, albumin – Age, BMI [, ] Fibrosis- (FIB-) Platelets, AST, ALT – Age [, ] Enhanced Liver Fibrosis (ELF)* Hyaluronic acid, PIIINP, [, ] TIMP- BARD score AST, ALT BMI, DM [] AST to platelet ratio index AST, platelets [, ] (APRI) FibroTesta α-macroglobulin, haptoglobin, apo A, GGT, Age, sex [] bilirubin ActiTest α-macroglobulin, haptoglobin, apo A, GGT, [] bilirubin Hepascorea Bilirubin, GGT, α-macroglobulin Hyaluronic acid Age and sex [, ] PGA Prothrombin index, GGT, apo A [] FibroIndex Platelets, AST, Ύ-globulin [] Forns GGT, cholesterol, platelets Age [] Fibrometer NAFLD Glucose, AST, ALT, ferritin, platelets Age, weight [, ] FibroSpect IIa α-macroglobulin Hyaluronic acid, TIMP- [] SHASTA AST, albumin Hyaluronic acid [] Hepamet Fibrosis Score (HFS) AST, albumin, platelets, HOMA (glucose and insulin) Age, sex, DM [] ADAPT Platelets PRO-C Age, DM [] a Registered scores. Apo A, apolipoprotein A; BMI, body mass index; DM, diabetes mellitus; PIIINP, amino-terminal propeptide of procollagen type III; TIMP-, tissue inhibitor of metalloproteinases type . The most extensively studied predictive models of diagnostic Odds Ratio for the low (<0.12) and high (>0.47) fibrosis that have been validated for different populations of cut-off values than FIB-4 and NFS to exclude/diagnose patients with MAFLD are Fibrosis-4 (FIB-4) and NAFLD advanced fibrosis irrespective of the age, BMI, and the Fibrosis Score (NFS). FIB-4 values<1.30 help exclude the presence of diabetes [60]. Thus, HFS emerges as the most presence of advanced fibrosis with a NPV of 95%; whereas reliable indirect marker of liver fibrosis at this moment. values>3.25 indicate advanced fibrosis with a PPV of 75% ADAPT is another promising score that was recently [63]. With regard to NFS, a multicentric study in more than published, which formula includes age, the presence of 700 patients reported a NPV of 88–93% to exclude diabetes, platelet count, and PRO-C3. PRO-C3 is a marker of advanced fibrosis for values <−1,455; and a PPV of 82–90% collagen III formation that has not only been documented to for values>0.676 [71]. The influence of comorbidities such as be involved in the etiology of MAFLD, but also to increase advanced age, diabetes and obesity on the results of these with fibrosis [61]. The reported AUROC for this score is 0.89 scores should always be considered. Another index, the both, for the derivation and the validation cohort, which Hepamet fibrosis score (HFS) was recently developed and exceeds the diagnostic accuracy of FIB-4 and NFS. validated in Spain. HFS has shown a significantly higher • Serum marker scores of fibrosis are a first screening approach for advanced fibrosis in Table : Diagnostic value of high cut-off values for the main patients with MAFLD. serological scores of advanced fibrosis in patients with MAFLD. • Both, FIB-4 and NFS have been validated for MAFLD and show a high diagnostic performance. Cut-off AUROC Sens, % Spec, % PPV, % PNV, % Ref The use of HFS is also recommended. NFS ≥. .     [] • When serum marker scores are suggestive of ELF >. .     [] advanced fibrosis, further biochemical, FIB- ≥. .     [] radiological, and/or biopsy studies should be APRI ≥ .     [] performed. HFS ≥. .     [] • During the critical evaluation of the scores AUROC, area under the ROC curve; Sens, sensitivity; Spec, specificity; obtained, the potential influence of other factors PPV, positive predictive value; NPV, negative predictive value; NFS, unrelated to metabolic liver disease must be NAFLD Fibrosis Score; ELF, Enhanced Liver Fibrosis; HFS, Hepamet taken into account. Fibrosis Score. Guerra-Ruiz et al.: Biochemical assessment of MAFLD 205 Use in clinical practice reduces the need to perform a liver biopsy significantly. However, the studies included should not be performed On suspicion of MAFLD, non-invasive risk assessment simultaneously, as, although this combination provides a should be performed in routine practice. The choice will high sensitivity and specificity, the area of uncertainty depend on the local availability of the test. In Primary Care, increases [75]. In contrast, when these studies are per- the simplicity, low cost and wide availability of simple formed sequentially, the area of uncertainty decreases serum scores of fibrosis with a high negative predictive while keeping a fine sensitivity and specificity [76]. value make them the best first-line screening test. The development of sequential diagnostic algo- As mentioned above, serum markers have some limi- rithms of MAFLD should start with widely-validated, low- tations or may be affected by a number of confounding cost serum scores with a higher negative predictive value factors that should be taken into account in the interpre- such as FIB-4 or NFS. Patients with FIB-4<1.3 or tation of results. NFS<−1.455 are considered to be at a low risk of having With regard to radiological markers, acoustic radiation advanced fibrosis and further diagnostic testing is not force impulse (ARFI), Shear Wave elastography (SWE), and required. However, patients at an intermediate (FIB-4 transient elastography with measurement of controlled between 1.45 and 3.25 or NFS from −1.455 to 0.675) or high attenuation parameter (CAP) can assess steatosis and risk for advanced fibrosis (FIB-4>3.25 or NFS>0.675) fibrosis simultaneously [72]. The limitations of these tech- should be referred to a center of reference for further niques are similar to those of serum markers. The efficacy of testing of liver function with radiological or direct serum FibroScan® is limited by the presence of morbid obesity or a markers. For diagnosis of advanced fibrosis in patients narrow intercostal space. Results can be influenced by any with steatohepatitis, the combination of FIB-4 or NFS with process that alters liver tenderness such as steatosis, ELF is recommended by the NICE (The National Institute venous congestion induced by heart failure, or acute of Health, UK) [77]. inflammation secondary to acute hepatitis. Limitations of Figure 2 proposes an approach to MAFLD based on the MRI-based methods include their limited availability and clinical evidence currently available. This model initiates high cost. For this reason, some authors propose the com- with a suspicion of MAFLD roused by the presence of bination of serum markers and radiological markers, being steatosis detected by ultrasound, or the presence of meta- the most widely used FibroScan [73–75]. This combination bolic risk factors (obesity, type 2 diabetes mellitus or increases the diagnostic accuracy of advanced fibrosis and metabolic syndrome) [37, 78]. Figure 2: Proposal for an algorithm for the detection of liver fibrosis based on risk stratification using non-invasive markers in patients with suspected metabolic fatty liver. 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