Joanna K Dowman
email: j.k.dowman@bham.ac.uk

Jeremy W Tomlinson

Phil Newsome

MRC Senior Clinical Fellow Honorary Consultant

Endocrinologist Pn Newsome

Mbchb ( Hons

Diagnosis and Staging of Non-Alcoholic Fatty Liver Disease (NAFLD)/Non-Alcoholic Steatohepatitis (NASH)

Keywords: Non-alcoholic fatty liver disease < Hepatology, Steatohepatitis < Hepatology, Liver fibrosis < Hepatology, Liver function tests Non-alcoholic fatty liver disease, NAFLD, NASH, Steatosis, Fatty liver, Metabolic syndrome, Non-invasive assessment, Biomarkers, NAFLD fibrosis score, Transient elastography, Fibroscan

come

Introduction

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of disease ranging from simple steatosis, to inflammatory steatohepatitis (NASH) with increasing levels of fibrosis and ultimately cirrhosis. NAFLD is closely associated with obesity and insulin resistance, and is now recognised to represent the hepatic manifestation of the metabolic syndrome. Since the term NASH was first coined by Ludwig et al in 1980, [START_REF] Ludwig | Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease[END_REF] the prevalence of NAFLD has risen rapidly in parallel with the dramatic rise in population levels of obesity and diabetes, [START_REF] Vuppalanchi | Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management[END_REF] resulting in NAFLD now representing the most common cause of liver disease in the Western world. [START_REF] De Alwis | Non-alcoholic fatty liver disease: the mist gradually clears[END_REF] Despite recent advances in elucidating the complex metabolic and inflammatory pathways involved in NAFLD, the pathogenesis of steatosis and progression to steatohepatitis and fibrosis/cirrhosis is not yet fully understood. 4;5 Whilst steatosis alone appears to be associated with a relatively benign prognosis, [START_REF] Day | Natural history of NAFLD: remarkably benign in the absence of cirrhosis[END_REF] factors known to be involved in progression to more advanced and clinically relevant disease include inflammatory cytokines/adipokines, mitochondrial dysfunction and oxidative stress. [START_REF] Day | From fat to inflammation[END_REF] Insulin resistance causes impaired suppression of adipose tissue lipolysis, leading to increased efflux of free fatty acids (FFA) from adipose tissue to the liver. [START_REF] Lewis | Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes[END_REF] Hyperinsulinaemia also promotes hepatic de novo lipogenesis, which is markedly increased in NAFLD patients compared to normal individuals. [START_REF] Donnelly | Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease[END_REF] It is now recognised that FFA promote insulin resistance, inflammation and oxidative stress, 10;11 and thus rather than being harmful, hepatic triglyceride accumulation may actually be protective by preventing the harmful effects of FFA. [START_REF] Yamaguchi | Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis[END_REF] The important role of oxidative stress mechanisms, pro-inflammatory cytokines such as TNFalpha and interleukin 6, and adipokines such as leptin (proinflammatory and pro-fibrotic), and adiponectin (anti-inflammatory and insulin-sensitizing), in promoting NASH are also becoming increasingly delineated. [START_REF] Dowman | Pathogenesis of non-alcoholic fatty liver disease[END_REF] However evidence that only a minority of patients with NAFLD progress to more advanced stages of NASH suggests that disease progression is likely to depend on a complex interplay between such factors and underlying genetic predisposition. [START_REF] Petta | Non-alcoholic fatty liver disease pathogenesis: The present and the future[END_REF] The causes, epidemiology and natural history of NAFLD will be covered briefly, before discussing the established and emerging means of assessing and staging patients with NAFLD.

Causes of NAFLD

In the great majority of cases, NAFLD arises in association with one or more features of the metabolic syndrome, namely insulin resistance, glucose intolerance or diabetes, central obesity, dyslipidaemia and hypertension. [START_REF] Balkau | Comment on the provisional report from the WHO consultation. European Group for the Study of Insulin Resistance (EGIR)[END_REF][START_REF] Olufadi | Clinical and laboratory diagnosis of the metabolic syndrome[END_REF][START_REF] Reynolds | Epidemiology of the metabolic syndrome[END_REF] However, after exclusion of a history of significant alcohol intake, which is conventionally <20g/day, [START_REF] Neuschwander-Tetri | Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference[END_REF] other causes of steatosis which should be considered include nutritional causes, eg. rapid weight loss and total parenteral nutrition, rare metabolic disorders, and drug-induced steatosis. Commonly implicated agents include glucocorticoids, amiodarone, synthetic oestrogens, and highly-active anti-retroviral drugs (HAART). [START_REF] Neuschwander-Tetri | Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference[END_REF][START_REF] Preiss | Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations[END_REF][START_REF] Stefan | Causes and metabolic consequences of Fatty liver[END_REF] Steatosis is also frequently associated with hepatitis C, particularly genotype 3, and endocrine disorders such as polycystic ovary syndrome (PCOS), [START_REF] Setji | Nonalcoholic steatohepatitis and nonalcoholic Fatty liver disease in young women with polycystic ovary syndrome[END_REF];20 hypopituitarism [START_REF] Adams | Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction[END_REF] and hypothyroidism. [START_REF] Loria | Endocrine and liver interaction: the role of endocrine pathways in NASH[END_REF]

Epidemiology

The prevalence of NAFLD is estimated to be between 20-30% in Western adults, 23;24 rising to 90% in the morbidly obese. [START_REF] Machado | Hepatic histology in obese patients undergoing bariatric surgery[END_REF] NASH, the more advanced and clinically important form of NAFLD, is less common, with an estimated prevalence of 2-3% in the general population [START_REF] Neuschwander-Tetri | Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference[END_REF] and 37% in the morbidly obese. [START_REF] Machado | Hepatic histology in obese patients undergoing bariatric surgery[END_REF] Of concern, NAFLD now affects 3% of the general paediatric population, rising to 53% in obese children, 26;27 with considerable implications for future disease burden. Steatosis was present in 70% of a large unselected cohort of patients with type 2 diabetes. [START_REF] Targher | Prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease among type 2 diabetic patients[END_REF] NAFLD affects all ethnic groups, although prevalence appears to be higher in Hispanic and European Americans compared with African-Americans. This difference remains after controlling for insulin resistance and obesity 23;29 and may be related to ethnic differences in lipid metabolism. 23;30 Patients with a diagnosis of NAFLD have been shown across several studies to have a worse outcome when compared with an age and sex-matched general population. [START_REF] Angulo | Long-term mortality in nonalcoholic fatty liver disease: is liver histology of any prognostic significance?[END_REF] Of note, the excess mortality in this group is attributable to both cardiovascular and liver-related causes. 32;33 Since the description in 1999 of the prognostic relevance of different histological types of NAFLD, [START_REF] Matteoni | Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity[END_REF] several subsequent studies have demonstrated that the presence of just simple steatosis, with no inflammation or fibrosis, is associated with a similar overall and liver-related mortality to that of an age and gender matched general population. This reinforces the need to stratify patients with NAFLD into simple steatosis or more advanced disease. More advanced disease can be defined as advancing levels of fibrosis and/or the presence/level of inflammation and hepatocyte ballooning. This distinction is pertinent as cohort studies thus far have only identified advanced fibrosis, and not inflammation, as a predictor of worse clinical outcome. [START_REF] Ekstedt | Long-term follow-up of patients with NAFLD and elevated liver enzymes[END_REF] This may be a type 2 error reflecting small sample sizes, or it may be due to additional factors such as PNPLA3 polymorphisms [START_REF] Rotman | The association of genetic variability in patatin-like phospholipase domain-containing protein 3 (PNPLA3) with histological severity of nonalcoholic fatty liver disease[END_REF] regulating the development of fibrosis.

Methods:

A systematic literature search was performed to identify studies assessing methods for the diagnosis and staging of NAFLD/NASH. Relevant articles were identified by searching the PubMed database, MEDLINE and EMBASE, limited to papers published in the English language but not date-restricted. Search terms included fatty liver, NAFLD, NASH, steatosis, AND biomarkers, noninvasive, diagnosis, assessment, staging. Additional searches were also made for each of the individual methods described, eg. NAFLD fibrosis score, transient elastography, Fibroscan, Fibrotest etc. Selected articles referenced in these publications were also examined. • they were meta-analyses, systematic reviews, or primary studies of one or more relevant diagnostic/staging tool;

Inclusion criteria:

• they included at least 30 subjects, to reduce the risk of including underpowered studies;

• liver biopsy was used as the reference standard

Data extraction

JD performed the data extraction, which was then checked by the remaining authors (PN and JT).

NAFLD: Making the diagnosis

The diagnosis of NAFLD should be strongly suspected in the presence of features such as obesity, The majority of patients with NAFLD are asymptomatic and the diagnosis suspected after finding elevated transaminases on routine testing. Hepatic steatosis is also a frequent incidental finding on ultrasound scan (US) performed for other reasons such as suspected gallstone disease. The most common symptoms are right upper quadrant discomfort and fatigue, although the latter may also be caused by OSA which is frequently observed in the typically obese population with NAFLD.

Hepatomegaly is the most common clinical finding, with signs of chronic liver disease rarely present in the absence of cirrhosis. A recent study reported the novel finding that increased dorsocervical lipohypertrophy was the anthropometric parameter most strongly associated with severity of steatohepatitis. [START_REF] Cheung | The impact of fat distribution on the severity of nonalcoholic fatty liver disease and metabolic syndrome[END_REF] Although NAFLD is often diagnosed after the finding of mildly abnormal LFTs, more than two thirds of patients have normal aminotransferase levels at any given time [START_REF] Wieckowska | Diagnosis of nonalcoholic fatty liver disease: invasive versus noninvasive[END_REF] and the entire histological spectrum of NAFLD can be observed in patients with normal alanine aminotransferase (ALT) values. 38;39 ALT is usually greater than aspartate aminotransferase (AST), and rarely more than three times the upper limit of normal. An AST:ALT ratio greater than 1.0 suggests the presence of more advanced disease. [START_REF] Angulo | Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis[END_REF] Alkaline phosphatase can be slightly elevated but is rarely the only liver function test abnormality. [START_REF] Pantsari | Nonalcoholic fatty liver disease presenting with an isolated elevated alkaline phosphatase[END_REF] Gamma-glutamyltransferase (GGT) is frequently elevated and may also be a marker of increased mortality. 42;43 Low albumin and hyperbilirubinaemia indicate advanced liver disease and are not otherwise features of NAFLD. [START_REF] Oh | Review article: diagnosis and treatment of non-alcoholic fatty liver disease[END_REF] Iron studies may show an elevated ferritin in up to 50% of patients and elevated transferrin saturation in approximately 10%. [START_REF] Angulo | Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis[END_REF] However such findings do not appear to correlate with elevated hepatic iron concentration, and the role of hepatic iron in the pathogenesis of NASH is unclear. [START_REF] Sumida | Role of hepatic iron in non-alcoholic steatohepatitis[END_REF] The Fatty Liver Index (FLI) was developed as a simple algorithm to predict fatty liver on USS in the general population. [START_REF] Bedogni | The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population[END_REF] The FLI uses four variables of BMI, waist circumference, GGT and serum triglyceride levels, and achieved an accuracy of 0.84 in detecting fatty liver. [START_REF] Bedogni | The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population[END_REF] The FLI has since been utilised by several groups in population studies of NAFLD. [START_REF] Gastaldelli | Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population[END_REF][START_REF] De Andrade | Nonalcoholic fatty liver disease in severely obese individuals: the influence of bariatric surgery[END_REF][START_REF] Balkau | Nine-year incident diabetes is predicted by fatty liver indices: the French D.E.S.I.R. study[END_REF] liver disease who underwent liver biopsy, and showed a sensitivity of 64% and specificity of 97%, rising to 91% and 93% respectively in patients with at least 30% steatosis. [START_REF] Palmentieri | The role of bright liver echo pattern on ultrasound Bmode examination in the diagnosis of liver steatosis[END_REF] However the presence of morbid obesity considerably reduces sensitivity and specificity. [START_REF] Mottin | The role of ultrasound in the diagnosis of hepatic steatosis in morbidly obese patients[END_REF] USS is unable to quantify the amount of fat present or provide any staging of disease, [START_REF] Saadeh | The utility of radiological imaging in nonalcoholic fatty liver disease[END_REF] and is operator-dependent with significant intra-and inter-observer variability. 53

Staging of NAFLD

Having made a diagnosis of NAFLD the next step is to determine the severity, as that provides important information on prognosis. Historically this has required liver biopsy, although there have been many recent advances which allow non-invasive management for many patients. When staging patients with NAFLD there are two aspects to consider; (1) the level of fibrosis and (2) the level of inflammation/ballooning (table 1).

The histological spectrum of NAFLD ranges from simple steatosis through steatohepatitis to fibrosis and cirrhosis. There are no pathological changes which can definitively distinguish NAFLD from alcoholic liver disease (ALD), thus an accurate alcohol history is essential to distinguish between these two common conditions. [START_REF] Hubscher | Histological assessment of non-alcoholic fatty liver disease[END_REF] The histological changes in NAFLD are mainly parenchymal and in a perivenular location, although portal and periportal lesions may occur. [START_REF] Hubscher | Histological assessment of non-alcoholic fatty liver disease[END_REF] Simple steatosis is usually macrovesicular resulting from accumulation of triglycerides within hepatocytes. [START_REF] Oh | Review article: diagnosis and treatment of non-alcoholic fatty liver disease[END_REF] Features of steatohepatitis include hepatocellular injury, characterised by ballooned hepatocytes, with inflammation and fibrosis. [START_REF] Hubscher | Histological assessment of non-alcoholic fatty liver disease[END_REF] Mitochondrial abnormalities may occur in NASH, but rarely in simple steatosis, [START_REF] Sanyal | Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities[END_REF] supporting a role for mitochondrial defects in the pathogenesis of NAFLD-related liver injury. 54;55 The typical histological features of steatosis and inflammation often disappear in advanced disease, 56;57 thus many cases of 'cryptogenic' cirrhosis are likely caused by NASH. [START_REF] Caldwell | Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease[END_REF][START_REF] Powell | The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years[END_REF][START_REF] Poonawala | Prevalence of obesity and diabetes in patients with cryptogenic cirrhosis: a case-control study[END_REF] Hepatocellular carcinoma is a well-recognised complication of NASH-related cirrhosis, [START_REF] Marrero | NAFLD may be a common underlying liver disease in patients with hepatocellular carcinoma in the United States[END_REF];60 but can also be associated with pre-cirrhotic NAFLD. (NAS) [START_REF] Kleiner | Design and validation of a histological scoring system for nonalcoholic fatty liver disease[END_REF] is probably the most well-established. The NAS provides a composite score based on the degree of steatosis (0-3), lobular inflammation (0-3) and hepatocyte ballooning (0-2), with an additional score for fibrosis. A score of ≥5 suggests probable or definite NASH, and <3 indicates that NASH is unlikely. [START_REF] Kleiner | Design and validation of a histological scoring system for nonalcoholic fatty liver disease[END_REF] However although liver biopsy currently remains the gold standard for diagnosis of NASH, limitations of this technique include intra-observer variation 63;64 and sampling variability, 65;66 with features such as fibrosis often not uniformly distributed. [START_REF] Hubscher | Histological assessment of non-alcoholic fatty liver disease[END_REF] Non-invasive assessments of NAFLD severity Such assessments can provide information on the amount of liver fibrosis and/or the presence of NASH, features which are usually, but not always, found together. The focus on fibrosis is based on cohort studies which demonstrate that fibrosis, rather than inflammation, predicts outcome.

Several non-invasive diagnostic panels and scoring systems have been developed with varying diagnostic utility. The uneven distribution of fibrosis throughout the liver in NAFLD indicates that such scoring systems may potentially represent a more accurate reflection of global liver fibrosis severity than is permitted by the current gold standard liver biopsy, [START_REF] Angulo | The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD[END_REF] which samples only 1/50,000 th of the organ and is prone to significant sampling error. The BARD score is a simple scoring system designed to identify NAFLD patients with a low risk of advanced disease. It combines three variables of BMI, AST/ALT ratio (AAR) and the presence of Here the BARD score demonstrated an AUROC of 0.77, with sensitivity 89%, specificity 44%, NPV 95% and PPV 25%. 70 The BARD score was also validated in a Polish NAFLD cohort, where an NPV of 97% was demonstrated, [START_REF] Raszeja-Wyszomirska | Validation of the BARD scoring system in Polish patients with nonalcoholic fatty liver disease (NAFLD)[END_REF] but appeared less useful in a Japanese cohort, where the AUROC was 0.73 with NPV 77%. [START_REF] Fujii | Applicability of BARD score to Japanese patients with NAFLD[END_REF] The BARD score is easily calculated and thus represents a simple tool for excluding the presence of advanced fibrosis in NAFLD patients.

The AST-to-platelet ratio index (APRI), [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF] AST/ALT ratio, [START_REF] Williams | Ratio of serum aspartate to alanine aminotransferase in chronic hepatitis. Relationship to cirrhosis[END_REF] and FIB-4 score [START_REF] Vallet-Pichard | FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotest[END_REF] have previously demonstrated utility in the non-invasive assessment of fibrosis in a number of chronic liver diseases. Several recent studies have also examined the role of these markers in NAFLD, as will be described.

The APRI was originally developed for use in chronic hepatitis C, [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF] Increased serum GGT level has also been shown to be associated with advanced fibrosis in NAFLD, with a study of 50 NAFLD patients demonstrating an AUROC of 0.74 for the prediction of advanced fibrosis of 0.74. Using a cut-off serum GGT value of 96.5 U/L, GGT predicted advanced fibrosis with 83% sensitivity and 69% specificity. [START_REF] Tahan | Serum gamma-glutamyltranspeptidase distinguishes non-alcoholic fatty liver disease at high risk[END_REF] FibroMeter is a panel of serum markers which was originally developed for staging fibrosis in chronic HCV. [START_REF] Cales | A novel panel of blood markers to assess the degree of liver fibrosis[END_REF] The NAFLD fibrosis score (NFS) is a panel comprising six variables of age, hyperglycaemia, BMI, platelet count, albumin and AST/ALT ratio, which was constructed using a large panel of 733 biopsy-proven NAFLD patients across several centres worldwide. Two cut-off scores were generated to predict the likelihood of the presence or absence of advanced fibrosis respectively. [START_REF] Angulo | The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD[END_REF] In the original study, by applying the low cut-off score (-1.455), the NFS had an NPV of 93% and 88% in the estimation and validation groups respectively for excluding the presence of advanced fibrosis. By applying the high cut-off score (0.676), PPVs of 90% and 82% in the estimation and validation groups respectively were achieved for predicting the presence of advanced fibrosis. The AUROC was 0.84, and application of this model to the study population would have avoided liver biopsy in 75% of patients, with a correct prediction in 90%. [START_REF] Angulo | The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD[END_REF] In the recent study by McPherson et al, the NFS demonstrated an AUROC of 0.81 with NPV 92% and PPV 72%, which was the highest PPV of the four tests examined. 70 Cales et al demonstrated an AUROC of 0.884 for significant fibrosis, 0.932 for severe fibrosis, and 0.902 for cirrhosis. [START_REF] Cales | Comparison of blood tests for liver fibrosis specific or not to NAFLD[END_REF] Studies in East Asian populations have also demonstrated good accuracy for excluding advanced fibrosis, with NPVs of 89% and 91% demonstrated in Japanese [START_REF] Fujii | Applicability of BARD score to Japanese patients with NAFLD[END_REF] and Chinese 83 NAFLD cohorts respectively. The NFS also demonstrated excellent accuracy at excluding fibrosis in morbidly obese subjects with NAFLD undergoing bariatric surgery, where NPVs of 98%, 87% and 88% for excluding advanced, significant and any fibrosis respectively were demonstrated. [START_REF] Qureshi | The utility of the "NAFLD fibrosis score" in morbidly obese subjects with NAFLD[END_REF] In a recent meta-analyses, NFS achieved pooled AUROC, sensitivity and specificity of 0.85 (0.80-0.93), 0.90 (0.82-0.99) and 0.97 (0.94-0.99) for the identification of NASH with advanced fibrosis. [START_REF] Musso | Meta-analysis: Natural history of nonalcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity[END_REF] The NAFLD fibrosis score thus facilitates the identification of NAFLD patients with more advanced disease who require ongoing follow-up, and considerably reduces the requirement for liver biopsy in the minority of patients with an indeterminate score. 70

ii) Fibrosis Biomarkers

The Original ELF (European Liver Fibrosis) test is a panel of automated immunoassays to detect three markers of matrix turnover in serum: hyaluronic acid (HA), tissue inhibitor of metalloproteinase 1 (TIMP1), and aminoterminal peptide of pro-collagen III (P3NP), used in combination with age. [START_REF] Rosenberg | Serum markers detect the presence of liver fibrosis: a cohort study[END_REF] The simplified ELF panel excludes age but has a similar diagnostic performance. The addition of five simple markers -BMI, presence of diabetes/impaired fasting glucose, AST/ALT ratio, platelets and albumin -to the ELF test improved diagnostic accuracy further, with area under the receiver operating curve AUROCs of 0.98, 0.93 and 0.84 for the diagnosis of severe, moderate and no fibrosis respectively. [START_REF] Guha | Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: Validating the European Liver Fibrosis Panel and exploring simple markers[END_REF] The ELF panel may also represent a useful prognostic tool, with a one unit change in ELF score shown to be associated with a doubling of the odds of significant liver-related mortality or morbidity at 6 year follow-up. [START_REF] Parkes | Enhanced liver fibrosis test can predict clinical outcomes in patients with chronic liver disease[END_REF] FibroTest is another validated marker for the quantitative assessment of fibrosis in NAFLD, ALD and chronic viral hepatitis. [START_REF] Poynard | Meta-analyses of FibroTest diagnostic value in chronic liver disease[END_REF] Combining five biochemical markers of haptoglobin, α2-macroglobulin, apolipoprotein A1, total bilirubin and GGT, corrected for age and gender, a mean standardized AUROC of 0.84 for advanced fibrosis in NAFLD patients was demonstrated using FibroTest in one meta-analyses. Importantly the diagnostic value was found to be similar for the diagnosis of both intermediate and extreme fibrosis stages, with no significant difference between the AUROC of the intermediate adjacent stages F2 vs. F1 to that of the extreme stages F3 vs. F4 or F1 vs. F0. [START_REF] Poynard | Meta-analyses of FibroTest diagnostic value in chronic liver disease[END_REF] FibroTest can also be combined with two other panels -SteatoTest and NASHTest -to form the FibroMax panel (BioPredictive), which provides a simultaneous and complete estimation of the liver injury in NAFLD. [START_REF] Poynard | Meta-analyses of FibroTest diagnostic value in chronic liver disease[END_REF] FibroTest/FibroMax have now been widely adopted as a non-invasive alternative to liver biopsy. [START_REF] Munteanu | Noninvasive biomarkers for the screening of fibrosis, steatosis and steatohepatitis in patients with metabolic risk factors: FibroTest-FibroMax experience[END_REF] Other potential fibrosis biomarkers include type VI collagen 7s domain and hyaluronic acid (HA), with the latter also representing a constituent of the ELF test. [START_REF] Sakugawa | Clinical usefulness of biochemical markers of liver fibrosis in patients with nonalcoholic fatty liver disease[END_REF] In a separate cohort of 72 patients, the AUROC curves for type IV collagen 7s domain and HA were 0.767 and 0.754 respectively for the detection of advanced fibrosis in NASH. However after multiple regression analysis only type IV collagen 7s domain was independently associated with advanced fibrosis in this study. [START_REF] Yoneda | Type IV collagen 7s domain is an independent clinical marker of the severity of fibrosis in patients with nonalcoholic steatohepatitis before the cirrhotic stage[END_REF] Other studies of HA in NAFLD using varying cut-off levels have demonstrated AUROCs of between 0.89 and 0.97 for detecting advanced fibrosis. [START_REF] Hashimoto | The characteristics and natural history of Japanese patients with nonalcoholic fatty liver disease[END_REF][START_REF] Kaneda | Hyaluronic acid levels can predict severe fibrosis and platelet counts can predict cirrhosis in patients with nonalcoholic fatty liver disease[END_REF][START_REF] Suzuki | Hyaluronic acid, an accurate serum marker for severe hepatic fibrosis in patients with non-alcoholic fatty liver disease[END_REF] Kaneda et al demonstrated HA to have an AUROC, NPV, sensitivity and specificity of 0.97, 100%, 100% and 89% respectively for detecting severe fibrosis, with a lower AUROC of 0.87 demonstrated for type IV collagen. In this study the platelet count alone was an independent predictor of cirrhosis, with an AUROC of 0.98, and sensitivity, specificity, PPV and NPV of 100%, 95%, 76% and 100% respectively using a cut-off value of 16 x 10 4 /microL. [START_REF] Kaneda | Hyaluronic acid levels can predict severe fibrosis and platelet counts can predict cirrhosis in patients with nonalcoholic fatty liver disease[END_REF] Lesmana et al also demonstrated the utility of levels of HA and type IV collagen to differentiate between mild (F1-2) and advanced fibrosis (F3-4), [START_REF] Lesmana | Diagnostic value of a group of biochemical markers of liver fibrosis in patients with non-alcoholic steatohepatitis[END_REF] and in a separate study of 80 NAFLD patients the combination of HA with AST, AAR, age, gender and BMI demonstrated an AUROC of 0.763 for distinguishing simple steatosis and NASH. [START_REF] Palekar | Clinical model for distinguishing nonalcoholic steatohepatitis from simple steatosis in patients with nonalcoholic fatty liver disease[END_REF] In a small study of serum extracellular matrix components in 30 NAFLD patients, serum laminin >282ng/ml was shown to have an accuracy of 87%, sensitivity 82%, specificity 89%, PPV 82% and NPV 89% for identifying the presence of NASH with fibrosis. When combined with type IV collagen, both specificity and PPV increased to 100%, but with lower sensitivity and NPV of 64% and 83% respectively. [START_REF] Santos | Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease[END_REF]

(iii) Radiological Assessment

Although many imaging modalities have been evaluated in NAFLD, their major focus has been Together with FibroTest, these three panels comprise the FibroMax panel described previously. [START_REF] Poynard | Meta-analyses of FibroTest diagnostic value in chronic liver disease[END_REF] Many other potential serum biomarkers have been identified which are typically markers of the key mechanisms believed to be involved in NASH pathogenesis, such as inflammation, oxidative stress, apoptosis and insulin resistance. [START_REF] Oh | Review article: diagnosis and treatment of non-alcoholic fatty liver disease[END_REF] Inflammation is associated with an increase in tumour necrosis factor-alpha (TNF-α) and decreased adiponectin expression (measured by ELISA) and this cytokine imbalance does appear to correlate with NASH 110-112 although the accuracy or clinical usefulness of these markers is yet to be determined. 113 respectively. [START_REF] Musso | Meta-analysis: Natural history of nonalcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity[END_REF] An AUROC of 0.88 for diagnosis of NASH was also demonstrated in a morbidly obese population, where, in addition, CK-18 levels were observed to fall significantly following bariatric surgery. 138 Plasma homocysteine (Hcy) levels were shown to distinguish NASH from simple steatosis with good accuracy in a Turkish study of 71 NAFLD patients. Using a threshold of 11.935 ng/mL, sensitivity and specificity were 91.7% and 95.7% with an AUROC of 0.948 for predicting NASH. 141 Serum prolidase enzyme activity (SPEA) catalyzes the final step of collagen breakdown by liberating free proline for collagen recycling, and is reported to be of hepatic origin. 142 In a Turkish study of 54 NAFLD patients, SPEA was significantly greater in patients with NASH than those with simple steatosis or controls, and positively correlated with the grade of liver fatty infiltration, lobular inflammation, stage of fibrosis and NAFLD activity score. 142 In this study the SPEA was shown to be superior to AST, ALT, or the AAR for distinguishing NASH from simple steatosis, with an AUROC of 0.85, and also the most useful of these tests for predicting lobular inflammation, NAFLD activity score, and fibrosis. 142 Other novel biomarkers which may be of potential utility in the diagnosis of NASH include plasma pentraxin 3 levels 143 and tissue polypeptide specific antigen. 144 Further study of these markers is warranted.

It should be highlighted that although some of the non-invasive serum markers described above are already widely used in the assessment and staging of NASH, further validation of their use in this setting will increase confidence in their utility (table 2).

ii. Radiological Assessment

Contrast-enhanced ultrasound using Levovist is the first imaging technique to demonstrate efficacy in distinguishing between simple steatosis and NASH. In a study of 64 patients with either normal liver, NAFLD or NASH, this modality was able to diagnose NASH with an AUROC of 100%. 145 The accumulation of Levovist microbubbles in the liver parenchyma was shown to be decreased in NASH but not in NAFLD or chronic viral hepatitis, with the decrease seen in NASH correlating with fibrosis rather than steatosis. Severe decrease was seen in both NASH and ASH livers, especially The diagnostic yield using CT is similar to that of US in NAFLD. Unenhanced CT shows low attenuation of the steatotic liver in contrast to the spleen, and the severity of steatosis has been shown to correlate with the liver:spleen (L/S) attenuation ratio. 146 A CT L/S cut-off of value of 0.8 yielded 100% specificity and 82% sensitivity for diagnosing macrovesicular steatosis of 30% or greater, 147 although a CT L/S cut off of 1.0 for defining steatosis has been used in some studies. 148 Accuracy of unenhanced CT is greatly reduced with lesser degrees of steatosis. 147 Other pathologies, such as hepatic siderosis, may also alter attenuation values leading to misdiagnosis, 149;150 and the radiation exposure associated with CT limits its use in younger patients and in longitudinal studies. 149 Fibroscan has also recently demonstrated utility in detecting and quantifying steatosis, using a novel attenuation parameter termed "Controlled Attenuation Parameter" (CAP), which was devised to specifically target the liver using a process based on

DISCUSSION

Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).

Table 2 Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). For more information, visit: www.prisma-statement.org.

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  diabetes and obstructive sleep apnoea (OSA), however other causes should always be considered before attributing abnormal liver function tests (LFTs) to NAFLD alone (fig1). Alternative diagnoses which should be excluded by history and serological testing include the viral hepatitides, excess alcohol consumption, haemochromatosis, autoimmune liver disease, alpha-1 antitrypsin deficiency, Wilson's disease and drug-induced liver dysfunction.

65 ; 66 a)

 6566 Assessment of fibrosis i) Demographic factors and simple blood testsSeveral diagnostic panels have been developed to facilitate the non-invasive assessment of NAFLD and differentiation between different stages of disease. These are generally based on a number of laboratory measurements, often in combination with clinical parameters such as age, sex and BMI. Such scoring systems have generally demonstrated greater utility in the detection of advanced fibrosis than intermediate and early stages of fibrosis, a group potentially more likely to benefit from therapeutic interventions.[START_REF] Wieckowska | Diagnosis of nonalcoholic fatty liver disease: invasive versus noninvasive[END_REF]

  weighted sum (BMI ≥ 28 = 1 point, AAR of ≥ 0.8 = 2 points, DM = 1 point), to generate a score from 0-4. In the original study, a score of 2-4 was shown to be associated with an odds ratio for advanced fibrosis of 17 and a negative predictive value of 96%.[START_REF] Harrison | Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease[END_REF] A further study of the BARD score in 138 patients with biopsy-proven NAFLD revealed an area under the receiver operating curve (AUROC) of 0.67 (95% CI, 0.56-0.77), with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 51.4%, 77.2%, 45.2% and 81.3% respectively.[START_REF] Ruffillo | Comparison of NAFLD fibrosis score and BARD score in predicting fibrosis in nonalcoholic fatty liver disease[END_REF] In a recent study including 145 patients with biopsy-proven NAFLD, McPherson et al compared the diagnostic performance of five simple non-invasive tests (BARD score, NAFLD fibrosis score, FIB-4 score, AST to platelet ratio index (APRI) and ALT/AST ratio), for the identification of NASH-related advanced fibrosis.

  Of these various algorithms FIB-4 and the NAFLD fibrosis score (NFS) have been validated most widely with demonstrably superior test characteristics.70;85

  Younossi et al evaluated the diagnostic utility of several ELISA-based assays in patients with biopsy-proven NASH. This study found that the levels of cleaved CK-18 (M30 antigen), and intact CK-18 (M65) predicted histological NASH with 70% sensitivity, 83.7% specificity, AUROC 0.711, and 63.6% sensitivity, 89.4% specificity, and AUROC 0.814 respectively. Histological NASH was found to be predicted by a combination of four ELISA-based tests -cleaved CK-18, a product of the subtraction of cleaved CK-18 level from intact CK-18 level, serum adiponectin, and serum resistin -with a sensitivity 95.5%, specificity of 70.2%, and AUROC of 0.91. 139 CK18 fragment has also been combined with ALT levels and the presence of the metabolic syndrome in the 'Nice model', a composite model where AUROCs of 0.83-0-88 were diagnosis of NASH, as defined by a NAS score ≥5, in a morbidly obese population. 140

  of bridging fibrosis (F3), however the same stage of fibrosis in chronic viral hepatitis showed only a mild decrease or normal uptake of Levovist. These differences may be explained both by changes in Kupffer cell function and differences between pericellular and periportal fibrosis in provoking disturbance of Levovist microbubble accumulation. Although this remains an experimental technique at present, evolving imaging modalities may thus soon permit the non-invasive differentiation between steatosis and NASH.

Conclusions 26 21 FUNDINGFunding 27

 262127 Provide a general interpretation of the results in the context of other evidence, and implications for future research. Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review.

24 From:

 24 Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(6): e1000097. doi:10.1371/journal.pmed1000097

  The AST/ALT ratio (AAR) is calculated using two widely available laboratory liver function tests. In addition to its utility as an individual marker, the AAR is also a component of several other fibrosis scoring systems including the NAFLD Fibrosis score and BARD score. Despite its simplicity, using a cut-off of 0.8 McPherson et al demonstrated an AUROC of 0.83, with sensitivity 74%, specificity 78%, and NPV of 93% for the diagnosis of advanced fibrosis in NAFLD using the AAR.70 The United States Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) recently investigated the utility of readily available clinical and laboratory variables to predict histological severity of NASH in >600 patients with biopsy-proven NAFLD. In this study, a combination of serum AST, ALT and the AAR performed only modestly (AUROC 0.59) for predicting steatosis, but was able to predict cirrhosis with an AUROC of 0.81. However the addition of demographic data,

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but its utility in NAFLD has since been studied by a number of groups. Using this score, Cales et al demonstrated an AUROC of 0.866 for significant fibrosis, 0.861 for severe fibrosis and 0.842 for cirrhosis in a study of 235 NAFLD subjects.

[START_REF] Cales | Comparison of blood tests for liver fibrosis specific or not to NAFLD[END_REF]

However significantly lower values were obtained in other studies, where AUROCs of 0.564 for significant fibrosis, 0.568 for advanced fibrosis,

[START_REF] Loaeza-Del-Castillo | AST to platelet ratio index (APRI) for the noninvasive evaluation of liver fibrosis[END_REF]

and 0.786 for predicting cirrhosis

[START_REF] Fujii | Noninvasive laboratory tests proposed for predicting cirrhosis in patients with chronic hepatitis C are also useful in patients with non-alcoholic steatohepatitis[END_REF]

were demonstrated. In their study of 145 NAFLD patients, McPherson et al reported an AUROC of 0.67 for the diagnosis of advanced fibrosis.

70

comorbidities, and several other routinely measured laboratory tests increased the AUROCs to 0.79 for NASH and 0.96 for cirrhosis.

[START_REF] Neuschwander-Tetri | Clinical, laboratory and histological associations in adults with nonalcoholic fatty liver disease[END_REF]

The FIB-4 test combines age with three standard biochemical values (platelets, ALT and AST) to assess fibrosis. In NAFLD FIB-4 has demonstrated similar results to the AST/ALT ratio where, using a cut-off of 1.3, an AUROC of 0.86, sensitivity 85%, specificity 65% and NPV of 95% were demonstrated for the diagnosis of advanced fibrosis.

70

In a US-based comparison of several noninvasive markers of fibrosis in 541 NAFLD patients, FIB-4 had the highest AUROC of 0.802, with PPV and NPV of 80% and 90% respectively for diagnosis of advanced fibrosis. In this study, AUROCs for the NAFLD fibrosis score, AAR, APRI, AST:platelet ratio and BARD score were 0.768, 0.742, 0.730; 0.72, and 0.70 respectively.

[START_REF] Shah | Comparison of noninvasive markers of fibrosis in patients with nonalcoholic fatty liver disease[END_REF]

  weight, fasting glucose, AST, ALT, ferritin, and platelet count), and in a study of 235 NAFLD patients demonstrated AUROCs of 0.943 for significant fibrosis, 0.937 for severe fibrosis and 0.904 for cirrhosis respectively. The sensitivity, specificity, PPV and NPV of FibroMeter for diagnosing significant fibrosis were 78.5%, 95.9%, 87.9 and 92.1%.[START_REF] Cales | Comparison of blood tests for liver fibrosis specific or not to NAFLD[END_REF]

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However, FibroMeter NAFLD has since been developed which has shown good diagnostic accuracy in staging NASH-related fibrosis. This panel combines 7 variables (age,

  In a cohort of 112 NAFLD subjects, 14 these two biomarkers were able to exclude advanced fibrosis with AUROCs of 0.82 and 0.80, and NPVs of 83.6 and 77.9% respectively. These biomarkers also demonstrated PPVs of 86% and 92% and AUROCs of 0.83 and 0.80 for discriminating NASH from simple fatty liver.

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  sensitivity 79% for distinguishing early-stage NASH. In this study, the combination of serum adiponectin level with HOMA-IR and type IV collagen 7S demonstrated a sensitivity of 94% and specificity of 74% for diagnosing NASH.114 Hui et al also reported significantly lower adiponectin levels and higher HOMA-IR in patients with NASH compared to subjects with simple steatosis, and demonstrated an AUROC of 0.79 for the combination of these markers for distinguishing between steatohepatitis and steatosis.115 However the relationship between adiponectin levels and severity of hepatic fibrosis remains to be established.116;117 The inflammatory marker C-reactive protein (CRP), measured by immunometric assay, has demonstrated mixed results in NASH. While some studies have shown a significant increase in high-sensitivity CRP levels in NASH patients compared to controls, 118;119 another study demonstrated no significant difference. 120 CRP also lacks specificity for hepatic inflammation.Thioredoxin (TRX) is stress-inducible thiol-containing protein which may represent a clinically useful indicator of oxidative stress.127 In a small study of 57 patients, Sumida et al demonstrated significantly elevated serum TRX levels in patients with NASH compared to those with simple steatosis and healthy controls, with an AUROC of 0.785 for distinguishing NASH from simple steatosis.127 Similar findings have been reported elsewhere, with a correlation between serum TRX and ferritin levels also observed.128 Apoptosis plays an important role in the liver injury observed in NAFLD, 129 and cytokeratin-18 (CK-18) represents a useful marker of this process. In a US multi-centre validation study including 139 NAFLD patients and 150 controls, Feldstein et al demonstrated that plasma CK-18 levels measured by ELISA were significantly higher in patients with biopsy-proven NASH than in those with a borderline diagnosis and normal controls, with an AUROC of 0.83 for NASH diagnosis. CK-18 was an independent predictor of both NASH and severity of disease.

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	R e R e sensitivity and specificity for NASH were 0.82 (0.78-0.88), 0.78 (0.64-0.92), and 0.87 (0.77-0.98)
	v i e w v i e adiponectin/leptin ratio demonstrated an AUROC of 0.82 for distinguishing between NASH and simple steatosis. 123 w
	Indicators of oxidative stress, including lipid peroxidation products (measured using a
	spectrofluorometric method), vitamin E levels (measured by reverse phase high performance liquid
	chromatography), and copper-to-zinc superoxide dismutase and glutathione peroxidase (GSH-Px)
	activity (measured by commercial antioxidant assay kits), have been investigated as surrogate
	markers of NASH. However most studies to date have been small with mixed results, 113;124;125 and
	it is not yet clear whether or not oxidative stress in the liver is accurately reflected in the serum. 126

Shimada et al observed the serum adiponectin level to be significantly lower in patients with early-stage NASH (3.6 mug/mL) than in those with simple steatosis group (6.0 mug/mL), with an AUROC of 0.765, sensitivity 68% and Interleukin-6 (IL-6) is another marker of inflammation which has been shown to be elevated in NASH. 121 In a study comparing 43 NASH patients, 40 subjects with steatosis and 48 controls, normal levels of IL-6 were highly specific in confirming the absence of NASH, with an AUROC of 0.817 for distinguishing NASH from simple steatosis. The same study also demonstrated significantly elevated levels of vascular endothelial growth factor (VEGF) concentrations in NASH, although the AUROC of 0.678 was lower than for IL-6. 122 IL-6 levels were also independently associated with fibrosis in a study by Lemoine et al, where the combination of HOMA-IR with the 130 Other studies have corroborated these findings, 131-137 suggesting that CK-18 represents a potentially useful biomarker for the diagnosis and differentiation of NASH from simple steatosis. CK-18 recently received independent validation for diagnosing NASH in a 2010 meta-analyses, where pooled AUROC,

Table 1

 1 work-up of patients with NAFLD is detailed in figure2. This combination of inexpensive non-invasive algorithms prior to more detailed examinations should hopefully permit a cost-effective and efficient way of investigating what is likely to be a large number of potential patients.ConclusionsNAFLD now represents the most common cause of liver disease in the Western world, and rising levels of obesity, diabetes and the metabolic syndrome render it an increasingly important cause of morbidity and mortality. Whilst simple steatosis carries a relatively benign prognosis, a significant proportion of patients will progress to NASH and later cirrhosis with risk of hepatocellular carcinoma. Although liver biopsy remains the gold standard for disease assessment, the development of risk scores, biomarker panels and ultrasound modalities has resulted in much improved identification of at risk patients without recourse to use of liver biopsy on a routine basis. Methods for assessing fibrosis and NASH Techniques to non-invasively distinguish NASH from simple steatosis remain largely experimental although show much promise for the future. Several methods are available for the non-invasive diagnosis/exclusion of fibrosis in patients with NAFLD. The BARD score is the simplest scoring system to calculate in the clinic, but the NAFLD fibrosis score can also be easily calculated by entering the relevant details into a freely available online calculator 70 (http://nafldscore.com).Currently FibroTest/FibroMax, FibroMeter and the ELF test are each only available from a single laboratory at significant cost, which has limited their use to mainly specialist liver centres. JK Dowman is a Wellcome Trust Clinical Research Fellow and is therefore receiving funding from the Wellcome Trust. However the content of this article has not been influenced by this funding source. JW Tomlinson is an MRC senior clinical fellow and has no conflict of interest. PN Newsome is Principal Investigator in a Phase II randomised placebo-controlled trial funded by the Wellcome Trust investigating the efficacy of Liraglutide in NASH.

	Vibration Control Transient Elastography

Table 2

 2 Non-invasive techniques for diagnosis of a) NASH, and b) fibrosis in patients with NAFLD in descending order of diagnostic accuracy a. AUROCs of 0.866 for significant fibrosis, 0.861 for severe fibrosis and 0.842 for cirrhosis.[START_REF] Cales | Comparison of blood tests for liver fibrosis specific or not to NAFLD[END_REF] AUROCs of 0.73 for advanced fibrosis in study of 541 NAFLD pts, 80 and 0.67 in study of 145 patients. 70 Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 6 Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations. 9-20 Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). n/a Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency. n/a Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). n/a Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]).

	Diagnostic	Size of cohort used in studies /	Popularity
	technique	Comments
	Contrast-enhanced	Study including 64 patients with normal	Use currently limited to
	ultrasound	liver, NAFLD or NASH, demonstrated	clinical trials
		AUROC of 100% for diagnosis of NASH. 145
	NASHTest	Study of 257 pts: 160 in training group	Use mainly confined to
		and 97 in validation group. Specificity,	specialist liver centres.
		sensitivity, PPV and NPV of 94%, 33%,
		66% and 81% for diagnosis of NASH.
		Can be combined with SteatoTest and
		FibroTest in Fibromax. 109
	CK-18	Multi-centre validation study including	Use currently limited to
		139 NAFLD pts demonstrated AUROC of 0.83 for NASH diagnosis. 130 Recently	clinical trials but holds promise for more
		received independent validation for	widespread application.
		diagnosing NASH in meta-analyses
		demonstrating pooled AUROC,
		sensitivity and specificity for NASH of 0.82, 0.78, and 0.87. 85

Alimentary Pharmacology & Therapeutic

Confirm diagnosis

Repeat LFTs to check if still abnormal ALT men >30 women >19 Liver screen and USS Hep B and C serology, ferritin/transferrin saturation, liver autoantibodies (AMA, ASMA, ANA), alpha-1-anti-trypsin levels, ceruloplasmin if <40.

Liver screen positive Manage as per condition

n/a

Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale.

5-6

Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

5-6

Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

5

Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

5-6

Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

6

Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.

5-6

Risk of bias in individual studies

12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.