Emmanuel A. Tsochatzis and Massimo Pinzani
Institute for Liver and Digestive Health, University College London, Royal Free Hospital, London, UK
NAFLD affects ~20% of the general population and encompasses a wide spectrum of liver disease, from simple steatosis to necroinflammation, fibrosis, and cirrhosis [1]. Non-alcoholic steatohepatitis (NASH) is the progressive form of NAFLD and affects 15–20% of patients with NAFLD [2]. Only patients with steatohepatitis have increased liver-related mortality [1].
Data on natural history of NAFLD are still scarce: in a meta-analysis of 10 studies comprising 221 patients, 37.6% had progressive fibrosis, 41.6% had no change, and 20.8% had improvement in fibrosis over a mean follow-up of 5.3 years [3]. Age and initial necroinflammation grade were the only factors associated with progression of fibrosis [3]. From a liver point of view and in the absence of specific treatment, the hallmarks in the progression of NAFLD are the development of NASH and the diagnosis of advanced fibrosis. The former is associated with potential progressive liver disease and the latter is the hallmark for targeted interventions and screening for complications of chronic liver disease, with a particular attention to the early development of hepatocellular carcinoma (HCC).
Therefore, NAFLD is a disease of high prevalence and relatively low severity in the majority of patients. Although liver biopsy is the gold standard for diagnosing steatohepatitis and assessing disease severity, it would be inappropriate to biopsy every patient diagnosed with NAFLD, as it is a costly and invasive procedure, which is associated with patient discomfort and potential side effects. In order to address this rising need, noninvasive tests and strategies have been developed that attempt to diagnose NASH and stage the disease.
In this chapter, we review the evidence on the use of noninvasive blood tests and imaging techniques based on elastography for diagnosing NASH and staging fibrosis.
During the last few years, there has been an explosive development and use of noninvasive fibrosis tests (NILTs) [4, 5]. The NILTs can be broadly divided into three categories: simple or indirect serum markers, direct serum markers, and imaging modalities [4].
Indirect serum markers or class II biomarkers consist of the combination of routine biochemical tests, such as transaminases, platelet count and albumin, and patient demographics that are associated with fibrosis, such as age or the presence of diabetes [4]. These tests usually have dual cutoffs: a high cutoff with high specificity and a low cutoff with high sensitivity. Depending on the clinical scenario and the disease prevalence, the low or high cutoff is used at the expense of increased false positives and false negatives, respectively. If these cutoffs are combined, then the numbers of false positives and false negatives are minimized. However, a number of patients will fall in the indeterminate range of fibrosis (i.e., their score will be between the low and the high cutoff) and will need either further noninvasive testing or liver biopsy.
Direct serum noninvasive tests (class I biomarkers) are supposed to detect extracellular matrix turnover and/or fibrogenic cell changes [4]. The most common markers used in current assays involve measuring products of extracellular matrix synthesis or degradation and the enzymes that regulate their production or modification, such as hyaluronic acid, serum collagenases and their inhibitors, and profibrogenic cytokines. It should be noted that these markers are not exclusively found in liver tissue; therefore, they reflect fibrogenic processes in various other organs and their diagnostic value become questionable in aging patients with comorbidities such as atherosclerosis and chronic lung disorders. Moreover, their sensitivity is low in the initial stages of fibrosis.
Various direct and indirect tests have been combined in patented commercial algorithms that improve the diagnostic accuracy of tests when used singly. In the case of NAFLD, these are FibroTest, ELF, and FibroMeters. Among them, FibroTest (FibroSure in the United States) is the most widely validated panel: it consists of five parameters and has been studied in viral hepatitis, NAFLD, and alcohol-related liver disease (ALD) [6]. FibroMeters are a family of 6 blood tests: one for staging and one for quantifying liver fibrosis in each of the 3 main causes of liver disease (chronic viral hepatitis, ALD, and NAFLD) [7]. The ELF biomarker is a panel of direct noninvasive markers that includes hyaluronic acid, type III collagen, and tissue inhibitor of metalloproteinase-1 (TIMP-1) [8].
New imaging modalities offer better sensitivity and specificity than conventional techniques, such as ultrasound, CT, and MRI. The latter can only identify cirrhosis, based on imaging findings of coarse echo-texture, collaterals suggestive of portal hypertension and nodularity. These new modalities measure liver elasticity, or liver stiffness, based on MR or US techniques. The most widely used imaging modality is transient elastography or FibroScanTM (Echosens, Paris) [9]. Briefly, an ultrasound transducer, inducing an elastic shear wave that propagates within the liver, transmits vibrations of mild amplitude and low frequency. Pulse-echo ultrasonic acquisitions are performed to follow the shear wave and measure its speed, which is directly related to the tissue stiffness. Results are expressed in kilopascals (kPa) and correspond to the median value of ten validated measurements. The volume of liver tissue evaluated by TE is at least 100 times bigger than a liver biopsy. Moreover, TE is painless and rapid (<5 min) and thus highly acceptable for patients.
Other modalities include acoustic radiation force impulse (ARFI) [10], supersonic shear imaging (SSI) [11], and MR elastography (MRE) [12]. ARFI allows the evaluation of liver stiffness in a region of interest (ROI) involving mechanical excitation of tissue, by the use of short-duration acoustic pulses, while performing a real-time B-mode conventional hepatic U/S. Results are expressed in (m/s). Although the volume of liver explored is smaller than that for TE, a critical advantage is the possibility to choose the representative area of interest, therefore avoiding large vessels and ribs. An advantage over TE is that it can be easily incorporated into a modified US machine. It should be noted that quality criteria for ARFI have not yet been established.
SSI, also named shear wave elastography, is also built on an US device and does not require an external vibrator to produce the shear wave [11]. In contrast to FibroScan and ARFI where a single shear wave is emitted at a single frequency for each measurement, in SSI a plurality of pulse wave beams at increasing depths are emitted, using a very wide frequency band from 60 to 600 Hz, therefore allowing the synchronous evaluation of several shear wave fronts over a wide frequency range. By generating a real-time color mapping of the elasticity encoded pixel by pixel in an image superimposed on the standard B-mode, SSI allows a quantitative image of the tissue elasticity. By placing an ROI in the center of the color mapping, the calculated value is the mean of values with the ROI. MRE uses a modified phase-contrast method to evaluate the propagation of the shear waves within the liver. It is a very promising technique but is not yet widely available and cost might be an important limiting factor.
A major limitation of all the aforementioned NILTs is the absence of uniformly established and validated cutoffs for specific etiologies of liver disease and fibrosis stages and the poor methodological quality of many of the published studies. In a recent meta-analysis on transient elastography, only 6 of 41 included studies had both histological evaluation and FibroScanTM measurements optimally performed, while all studies had a high risk of bias, based on quality assessment by the QUADAS tool [13].
NASH is the lynchpin between steatosis and cirrhosis in the spectrum of NAFLD and is characterized by necroinflammation, hepatocellular ballooning, and apoptosis that could lead to fibrosis and cirrhosis. Although there is a large clinical need for noninvasive assessment, the diagnosis of NASH is still largely based on liver biopsy, as there are no well-validated noninvasive tests.
The best-studied noninvasive marker for differentiating NASH is an apoptotic marker, namely, caspase generated cytokeratin 18 (CK-18) fragments in the serum. CK-18 is the major intermediate filament protein in the liver and is cleaved by effector caspases during the apoptotic process. Plasma CK-18 fragments have been evaluated in more than 10 cohort studies of patients with NAFLD, but the cutoffs for diagnosing NASH varied between studies and were not prospectively validated [14]. Feldstein evaluated CK-18 fragments in 139 patients with biopsy-proven NAFLD and 150 age-matched healthy controls and reported an AUROC of 0.83 with sensitivity and specificity of 0.75 and 0.81, respectively [15]. In the largest study to date, the initial enthusiasm on the use of CK-18 as a stand-alone test was tampered, as in a multiethnic cohort of 424 patients the sensitivity for NASH was low despite a high specificity, thus making this inadequate as a screening test [16].
All the other potential noninvasive tests for diagnosing NASH were evaluated in single studies and small cohorts of patients and therefore need further validation.
NashTestTM is a proprietary noninvasive algorithm for the diagnosis of NASH that combines 13 parameters [17]. This test categorizes patients into three groups: “not NASH,” borderline NASH, and NASH with AUROCs that range between 0.69 and 0.83.
In a Japanese study with an estimation and validation cohort and 619 patients in total, serum ferritin (≥200 ng/mL in females or ≥300 ng/mL in males), fasting insulin (≥10 μU/mL), and type IV collagen 7S (≥5.0 ng/mL) were combined in a weighted sum and formed a composite score for predicting NASH, called the NAFIC score [18]. This score had an AUROC of 0.78–0.85 for predicting NASH; however, it needs validation in Western populations as well.
In a single study, terminal peptide of procollagen III (PIIINP), which is involved in fibrogenesis, was identified as a potential biomarker of NASH using a derivation and validation cohort of 65 and 71 patients in respectively with an AUROC of 0.77–0.82 [19]. This will need validation in larger cohorts, as the sample size was low and the groups with simple steatosis/borderline NASH and NASH were disproportionate. All the aforementioned tests are summarized in Table 14.1.
TABLE 14.1 Noninvasive tests for diagnosing NASH in patients with non-alcoholic fatty liver disease
| Test | Number of studies | Components | AUROC |
| CK-18 fragments | >5 | CK-18 | 0.83 |
| NashTestTM | 1 | Age, sex, height, weight, triglycerides, cholesterol, a2-macroglobulin, ALT, GGT, ALT, AST, bilirubin, haptoglobin, apolipoprotein a1 | 0.79 |
| NAFIC | 1 | Ferritin, fasting insulin, type IV collagen | 0.78–0.85 |
| PIIINP | 1 | PIIINP | 0.77–0.84 |
Therefore, despite the huge clinical need, the tools for noninvasive diagnosis of NASH still remain inadequate. The fact that the diagnosis of NASH does not necessarily incorporate fibrosis and the considerable interobserver variability in the histological diagnosis of NASH further complicate this issue. A combination of biomarkers that evaluate apoptosis, necroinflammation, and fibrosis might be the most appropriate tool for diagnosing NASH and currently remains an unmet need.
Various combinations and algorithms of potential serum biomarkers have been used in NAFLD as shown in Table 14.2. The combination of simple laboratory parameters with clinical variables has resulted in simple scores that can be used at the point of care and have an excellent negative predictive value of >95% for advanced fibrosis (≥F3) in unselected cohorts of NAFLD patients. The most commonly used scores are the NAFLD fibrosis score [20] and FIB4 [21]. NAFLD fibrosis score incorporates age, BMI, presence of diabetes, AST, ALT, platelet count, and albumin, whereas FIB4 consists of age, AST, ALT, and platelet count. They both have dual cutoffs, that is, a low cutoff with a high sensitivity and a high cutoff with a high specificity. Their main clinical utility derives from the low cutoff, which can be used to rule out patients from further investigations and/or liver biopsy. Most importantly, they have been validated against clinical outcomes; in a retrospective cohort study of 320 patients with almost 10 years of follow-up, patients with intermediate and high risk of advanced fibrosis according to the NAFLD fibrosis score had a hazard ratio of 4.2 and 9.8 for death compared to the low-risk group [22].
TABLE 14.2 Noninvasive fibrosis tests in patients with non-alcoholic fatty liver disease
| Test | Components | Fibrosis stage assessed | Cutoff | AUROC/other measures |
| Nonproprietary serum tests | ||||
| AST/ALT ratio | AST, ALT | F4 | 1 | NA |
| APRI | AST, platelet count | F2, F4 | F2: <0.5, >1.5 | F4: 0.85 |
| F4: <1, >2 | ||||
| FIB-4 | Age, AST, ALT, platelet count | F3 | <1.45, >3.25 | 0.85 |
| NAFLD fibrosis score | Age, BMI, diabetes. AST, ALT, platelet count, albumin | F3 | < −1.455, >0.676 | 0.88 |
| BARD | BMI, AST/ALT ratio, diabetes | F3 | ≥2 | NA |
| Hepascore | Age, sex, a2-macroglobulin, hyaluronic acid, bilirubin, GGT | F3 | 0.37 | 0.84 |
| Proprietary serum tests | ||||
| FibroTestTM | Bilirubin, haptoglobin, GGT, a2-macroglobulin, apolipoprotein A | <0.3, >0.7 | NPV:0.98 | |
| PPV: 0.60 | ||||
| ELFTM | Hyaluronic acid, PIIINP, TIMP-1 | F2, F3, F4 | F3: 9.5 | 0.90 |
| FibroMeterTM | Age, weight, glucose, AST, ALT, platelet count, ferritin | F3 | <0.611, >0.715 | 0.94 |
| Elastography modalities | ||||
| FibroScanTM | US based | All stages | F4: >10.5 | 0.92 |
| ARFITM | US based | All stages | To be defined | >0.90 |
| Shear wave elastography | US based | All stages | No specific data on NAFLD | >0.90 |
| MR elastography | MR based | All stages | No specific data on NAFLD | >0.90 |
APRI and the ratio of aspartate aminotransferase to alanine aminotransferase (AST/ALT) are other simple scores that diagnose cirrhosis rather than advanced fibrosis and have not been extensively validated in NAFLD [23]. The BARD score was created from the retrospective analysis of 827 patients with NAFLD and consists of body mass index (BMI), presence of diabetes, and AST/ALT ratio [24]. A cutoff point of 2 is used to predict advanced fibrosis; however, the diagnostic accuracy is lower than NAFLD fibrosis score and FIB4.
Serum ferritin has also been proposed as a marker of NASH; in the NASH CRN cohort of 628 patients, a serum ferritin >1.5 the upper limit of normal was an independent predictor of advanced fibrosis. In a cohort of 111 patients, serum ferritin was independently associated with both the presence of NASH and more severe fibrosis [25]. Clearly, these data need further validation and ideally ferritin should be incorporated in one of the algorithms of fibrosis assessment.
Finally, the Hepascore has been evaluated in a single study and showed an acceptable diagnostic accuracy ≥F3, although not significantly better than NAFLD fibrosis score and FIB4 [26].
The FibroTestTM, the FibroMeter NAFLDTM, and the ELFTM score are the proprietary panels that have been used for fibrosis assessment in NAFLD (Table 14.2). The FibroTestTM, which has been best validated in CHC, uses a dual cutoff to “rule in” or “rule out” patients with advanced fibrosis, but up to a third of patients will fall in the indeterminate range of values and thus will require further assessment and/or biopsy [27]. The FibroMeter NAFLDTM had an AUROC of 0.94 for predicting significant fibrosis in 235 patients with NAFLD and performs better than the NAFLD fibrosis score and APRI [28]. ELFTM was evaluated in 192 adults and had a sensitivity of 0.8 and a specificity of 0.93 for advanced fibrosis [29]. The diagnostic accuracy of ELFTM was even higher in pediatric patients with NASH, with an AUROC of 0.95. All the aforementioned proprietary panels need independent validation by groups not involved in their creation and also in non-Caucasian populations.
Transient elastography using FibroScan is the most widely evaluated imaging tool for fibrosis assessment in NAFLD. Although not as widely validated as in patients with CHC, FibroScan in patients with NAFLD has excellent diagnostic accuracy for the diagnosis of cirrhosis and acceptable but not ideal accuracy for the diagnosis of fibrosis stage, F2 [30]. Although not definitely proven, it appears that steatosis influences the liver stiffness measurements, thus resulting in slightly lower values than CHC. It should be noted that there are no validated cutoff values for specific fibrosis stages and this remains a weakness of FibroScanTM [13].
Using the conventional M probe, liver stiffness measurements are uninterpretable in 19% of the cases and this is mainly due to increased waist circumference and BMI, as is frequently the case in patients with NAFLD [31]. This drawback of FibroScanTM in patients with NAFLD was recently addressed by the development of a dedicated XL probe for patients with high BMI. The use of this probe in patients with high BMI reduces the rate of failures from 10 to 2% and also the rate of unreliable results from 33 to 25%, as shown in a cohort of 193 patients with NAFLD [32]. Pairwise examination showed that liver stiffness measurement by the XL was lower than by the M probe; thus, a separate set of diagnostic cutoff values need to be validated for the XL probe [32]. The controlled attenuation parameter (CAP) incorporated in the new FibroScanTM machines can also quantify steatosis, although this requires further validation [33]. As with other noninvasive fibrosis tests, FibroScanTM is reliable for diagnosing the extremes of the fibrosis spectrum (no fibrosis/cirrhosis) but of less value in intermediate fibrosis stages.
ARFITM was compared with elastography in two small studies with <100 patients in NAFLD and was of similar diagnostic accuracy [23]. It resulted in less exam failures compared to FibroScanTM using the M probe, whereas there the success rates were similar when compared to the XL probe. Clearly, larger studies in NAFLD and the validation of specific cutoffs are needed.
There are no dedicated studies of SSI and MRE in patients with NAFLD to date. In a recent study comparing SSI with FibroScan and ARFI in 349 patients with chronic liver disease, SSI had a significantly higher accuracy than FibroScan for ≥F3 and a significantly higher accuracy than ARFI for ≥F2 [11]. MRE has shown AUROCs of >0.90 in cohorts of patients with various etiologies of liver disease; however, the inclusion of normal control might have resulted in the overestimation of diagnostic accuracy [12]. Both SSI and MRE need further validation in patients with NAFLD.
NAFLD is the most common cause of abnormal transaminases, and its prevalence will further increase given the current epidemic of obesity. Noninvasive strategies are urgently needed to diagnose NASH and stratify patients who will need secondary referral and specialist follow-up. Although the noninvasive diagnosis of NASH is still an unmet need, risk stratification is possible with simple noninvasive tests that consist of simple laboratory indices and clinical information. FIB4 and NAFLD fibrosis score can rule out advanced fibrosis with a negative predictive value of >95% and thus prevent unnecessary referrals in a significant number of patients. More refined noninvasive tests, such as elastography, FibroTestTM, or ELFTM, could be used as second tier tests to further characterize patients, although this sequential approach will need prospective validation. The development of such noninvasive algorithms, with the incorporation of efficacy and cost-effectiveness data, will likely limit the need for liver biopsy in a selected few in the future.