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Abbreviations
ALT

alanine aminotransferase

APRI

aspartate aminotransferase/platelet ratio index

AST

aspartate aminotransferase

AUROC

area under the receiver operating characteristic curve

BAAT

body mass index, alanine aminotransferase, and triglycerides

BARD

body mass index, alanine aminotransferase/aspartate aminotransferase ratio, and presence of diabetes

BMI

body mass index

CK-18

cytokeratin 18

DM

diabetes mellitus

ELF

European liver fibrosis

GGT

gamma-glutamyl transpeptidase

HA

hyaluronic acid

HAIR

hypertension, alanine aminotransferase, and insulin resistance

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

TG

triglyceride

TIMP1

tissue inhibitor of metalloproteinase 1

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver test results in the United States.[1] NAFLD is defined as the presence of steatosis in 5% or more of hepatocytes in the setting of no or little alcohol use. A thorough history is taken and an examination and laboratory testing are performed to exclude other liver diseases that also may present or coexist with hepatic steatosis. Although most patients with NAFLD have a benign course, a subset of these patients may have the progressive form of NAFLD called nonalcoholic steatohepatitis (NASH), which is characterized by the presence of ballooning degeneration and lobular inflammation with or without perisinusoidal fibrosis in addition to steatosis. Patients with NASH can progress to cirrhosis and are at increased risk of death resulting from liver disease.[2] Liver biopsy is the gold standard to confirm NASH and fibrosis. This review briefly discusses the noninvasive biomarkers that may be useful and indications for performing liver biopsy in patients with NAFLD.

Noninvasive Markers

  1. Top of page
  2. Abstract
  3. Noninvasive Markers
  4. Role of Liver Biopsy
  5. Conclusions
  6. References

NAFLD typically is characterized by a hepatocellular pattern of liver-related enzymes with mild elevations (1-2 times the upper limit of normal) in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST).[1] Despite having the disease, up to 50% of NAFLD patients can have normal ALT and AST levels.[2] Therefore, several biomarkers have been proposed to aid in the diagnosis. These markers can be divided into those that are helpful in diagnosing NAFLD, NASH, or advanced fibrosis. Table 1 summarizes the different markers, panels, and their performance characteristics. Imaging biomarkers are beyond the scope of this review because of space constraints.

Table 1. Biomarkers of NAFLD and NASH
Test Name or StudyMarkersPurpose of TestAUROC TrainingAUROC Validation
ALTALTDiagnosis of NAFLD0.68None
Steatosis testALT, α2-macroglobulin, apolipoprotein A1, haptoglobin, total bilirubin, GGT, cholesterol, TGs, glucose, age, sex, and BMIDiagnosis of NAFLD0.790.72–0.86
Liver fat scoreAST, insulin, metabolic syndrome, DM type 2, and AST/ALTDiagnosis of NAFLD0.870.86
Hepatic steatosis indexALT, AST, age, BMI, and sexDiagnosis of NAFLD0.8120.819
Fatty liver indexBMI, waist circumference, TG level, and GGTDiagnosis of NAFLD0.84None
CK-18 Distinguishing NASH from NAFLD0.930.83
NASH testAge, sex, height, weight, and serum levels of TGs, cholesterol, α2-macroglobulin, apolipoprotein A1, haptoglobin, GGT, ALT, AST, and total bilirubinDistinguishing NASH from NAFLD0.790.79
Younossi et al.[7]CK-18, cleaved CK-18, adiponectin, and resistinDistinguishing NASH from NAFLD0.910.73
Palekar et al.[8]Age, sex, AST, BMI, AST/ALT ratio, and serum HADistinguishing NASH from NAFLD0.763Not reported
HAIRHypertension, ALT, and insulin resistanceDistinguishing NASH from NAFLD in bariatric surgery patients0.9 with score of 3 associatedNot reported
NASH clinic scoring systemHypertension, DM type 2, AST > 27 IU/L, ALT > 27 IU/L, sleep apnea, and nonblack raceDistinguishing NASH from NAFLD in bariatric surgery patients0.800.75
Table 2. Biomarkers of Fibrosis in Patients with Fatty Liver Disease
Test Name or StudyMarkersPurpose of TestAUROC TrainingAUROC Validation
  1. Abbreviations: AUROC: Area under Receiver Operating Characteristic, GGT: Gamma-glutamyl transpeptidase ELF: European Liver Fibrosis Score, HA: Hyaluronic acid, BMI: Body Mass Index, DM: diabetes mellitus, AST: AST: Aspartate Aminotransferase, ALT: Alanine Aminotransferase, BARD: BMI, AST/ALT ratio and presence of diabetes, BAAT: BMI, ALT and Triglyceride, TG: Triglyceride, FIB-4: fibrosis index with 4 components: age (year) × AST (U/L)/ platelet count (109/L)× (ALT (U/L))(1/2); APRI: AST platelet ratio index.

GGTGGTDistinguishing fibrosis0.74Not reported
ELF scoreHA, TIMP1, and aminoterminal peptide of procollagen III (P3NP)Distinguishing fibrosis (severe fibrosis)Not reported0.9
Modified ELFELF score + BMI, albumin, platelet, presence of DM, and AST/ALT ratioDistinguishing fibrosis (severe)Not reported0.98
NAFLD fibrosis scoreAge, BMI, hyperglycemia, platelet count, albumin, and AST/ALT ratioDistinguishing fibrosis0.77–0.93, 0.85 in recent meta-analysisNot reported
FibrometerGlucose, AST, age, weight ferritin, ALT, and platelet countDistinguishing fibrosis0.9360.952
F0 and F1 versus F2, F3, and F40.9280.950
F0, F1, and F2 versus F3 and F40.9290.888
  F0, F1, and F3 versus F4  
Fibro testα2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, total bilirubin, and ALTDistinguishing fibrosis0860.75
F0 and F1 versus F2, F3, and F40.9320.81
F0, F1, and F2 versus F3 and F4  
BARDBMI, AST/ALT ratio, and presence of DMDistinguishing fibrosis0.810.78
BAATBMI, ALT, and TGDistinguishing fibrosis0.84Not reported
FIB-4Age, AST, ALT, and platelet countsDistinguishing fibrosis0.8020.86
APRIAST/platelet × 100Distinguishing fibrosis0.86 for significant fibrosis, 0.861 for severe fibrosis, and 0.842 for cirrhosisNot reported
AST/ALT ratio Distinguishing fibrosis0.83 with cutoff of 0.8Not reported
Table 3. Indications of Liver Biopsy in NAFLD
Indication for BiopsyStrength of Evidence
Clinical: 
Metabolic syndrome with abnormal liver enzymesStrong evidence
DM type 2 with abnormal liver enzymesStrong evidence
During bariatric surgeryStrong evidence
CholecystectomyStrong evidence
Laboratory test: 
AST > ALTStrong evidence
Low platelet countStrong evidence
Low albuminStrong evidence
Special clinical considerations: 
Older ageEmerging evidence
Family history of diabetesProposed and needs to be validated

Markers for NAFLD

The presence of coexisting risk factors such as diabetes, metabolic syndrome, and obesity with elevated liver enzymes increases the odds of the presence of NAFLD as the underlying cause of elevated ALT and/or AST levels. Although ALT is a useful test, it is by no means reliable or accurate in predicting the presence of NAFLD. Therefore, several panels of biomarkers and clinical prediction rules have been developed to improve the diagnostic accuracy further for predicting NAFLD. Some of these are listed in Table 1.[4]

Markers for NASH

The diagnosis of NASH without a liver biopsy remains the most significant clinical challenge in the evaluation of a patient with hepatic steatosis. Several biomarkers have been examined to distinguish between simple steatosis and NASH. Some of the inflammatory markers include serum C-reactive protein, interleukin-6, ferritin, hyaluronic acid (HA), tumor necrosis factor α, leptin, adiponectin, and resistin.[4] Apoptosis plays a key role in the pathogenesis of NASH. Among the markers of apoptosis, plasma cytokeratin 18 (CK-18) is emerging as one of the promising biomarkers for the noninvasive detection of NASH.[4] Since oxidative stress also plays an important role in the pathogenesis of NASH, several biomarkers of oxidative stress have been investigated. Among these, oxidized low-density lipoprotein, thiobarbituric acid–reacting substances, superoxide dismutase, and glutathione peroxidase dismutase have been examined.[4] The combination of biomarkers of apoptosis and oxidative stress has been studied and has led to modest improvement in the discrimination of NASH from steatosis alone.

The NASH test combined 13 variables [age, sex, height, weight, and serum levels of triglycerides (TGs), cholesterol, α2-macroglobulin, apolipoprotein A1, haptoglobin, gamma-glutamyl transpeptidase (GGT), ALT, AST, and total bilirubin] to achieve an area under the receiver operating characteristic curve (AUROC) of 0.79 with a specificity, sensitivity, positive predictive value, and negative predictive value of 94%, 33%, 66%, and 81%, respectively.[4] Younossi et al. used a panel including 2 forms of CK-18, adiponectin, and resistin.[7] This panel achieved an AUROC of 0.91 in the training set; however, the AUROC dropped to 0.73 in the validation cohort.[4, 7] Palekar et al. developed a panel that included 6 variables: age, sex, AST, body mass index (BMI), AST/ALT ratio, and serum HA.[8] This model achieved an AUROC of 0.76.[4, 8] Other panels also have been proposed to identify the presence of NASH in patients who are undergoing bariatric surgery; panels that may be useful in this select group of patients included the hypertension, alanine aminotransferase, and insulin resistance (HAIR) system and the NASH clinical scoring system (see Table 1).

Markers for Advanced Fibrosis and Cirrhosis

The use of biomarkers or panels for predicting advanced fibrosis in patients with NAFLD is much closer to routine clinical practice than the use of biomarkers for diagnosing NASH without a liver biopsy assessment.[9] Single biomarkers that have been shown to predict fibrosis are HA and type IV collagen S, which have AUROCs of 0.82 and 0.80 and negative predictive values of 84% and 78%, respectively.[4]

Two of the most promising tests for diagnosing advanced fibrosis in NAFLD are the European liver fibrosis (ELF) score and the NAFLD fibrosis score.[4, 6, 10] The ELF score includes HA, tissue inhibitor of metalloproteinase 1 (TIMP1), aminoterminal peptide of procollagen 3, and age. The simplified ELF score excluded the age and performed similarly in predicting fibrosis in NAFLD with an AUROC of 0.90. The addition of 5 parameters—BMI, albumin, platelets, presence of diabetes mellitus (DM), and AST/ALT ratio—improved the AUROC to 0.98. The NAFLD fibrosis score is helpful in the clinical setting because it uses routinely available variables in the clinical setting, including age, BMI, hyperglycemia, platelet count, serum albumin, and AST/ALT ratio. The NAFLD fibrosis score achieved AUROCs between 0.81 and 0.937. Its usefulness is not in diagnosing the stage of fibrosis but in predicting the presence of bridging fibrosis and cirrhosis in patients with NAFLD. Other notable biomarkers in predicting fibrosis in NAFLD are listed in Table 2, but they are not discussed in detail because of space constraints. Finally, simple tests such as the AST/platelet ratio and the AST/ALT ratio also have shown fair to good diagnostic test performance characteristics that may be applied clinically[4] (Table 1).

In summary, the ELF score panel and the NAFLD fibrosis score seem to have good test performance characteristics for predicting advanced fibrosis, including bridging fibrosis, cirrhosis, or both.

Role of Liver Biopsy

  1. Top of page
  2. Abstract
  3. Noninvasive Markers
  4. Role of Liver Biopsy
  5. Conclusions
  6. References

Liver biopsy is still the gold standard for diagnosing NASH and assessing the stage of fibrosis in patients with NAFLD. However, it has many limitations, including cost, sampling error, morbidity, and, rarely, death. The position statement on NAFLD and NASH based on the 2009 European Association for the Study of Liver Diseases special conference recommended liver biopsy when noninvasive markers suggest advanced fibrosis and in indeterminate cases when the suspicion of fibrosis remains.[11] Liver biopsy was also recommended when bariatric surgery or cholecystectomy is performed. The recently published practice guidelines on the management of nonalcoholic fatty liver disease by the American Association for the Study of Liver Diseases, the American College of Gastroenterology, and the American Gastroenterological Association recommended liver biopsy in patients who are at increased risk of steatohepatitis and fibrosis, such as those with metabolic syndrome.[12] On the basis of several studies highlighted earlier, we recommend that liver biopsy be considered when the likelihood of the presence of NASH and advanced fibrosis is increased significantly among patients with NAFLD. We also propose that the presence of metabolic syndrome and diabetes increases the likelihood of the presence of NASH on liver biopsy.[13] Therefore, these patients with NAFLD who have coexisting diabetes and/or metabolic syndrome and elevated ALT or AST levels may be considered for a liver biopsy examination (Table 3).[9] This will help in the early identification of patients who are at the highest risk of progressive liver disease.[14] Prompt recognition of NASH and the institution of emerging treatment strategies (such as vitamin E, exercise, and weight loss) in these patients will halt the progression of liver disease. Furthermore, biopsy-proven NASH patients also are candidates for more aggressive lifestyle interventions, which will reduce the risk of future cardiovascular and liver disease progression. Other subpopulations of NAFLD patients who may be considered for liver biopsy include those with AST levels greater than ALT levels, older patients,[16] those with low platelet counts or serum albumin levels when there is diagnostic uncertainty, and those with a competing diagnosis that requires exclusion, such as drug-induced liver injury. Figure 1 displays the diagnostic algorithm that we recommend for assessing the need for performing liver biopsy in patients with NAFLD. We use routinely available models or markers that increase the pretest likelihood of finding more advanced liver disease on liver biopsy. These tests can aid in clinical decision making for patients with NAFLD. Some of these markers are a high AST/ALT ratio, a high AST/platelet ratio, low albumin levels, and low platelet levels. In addition, we take into account the preferences of our patients by helping them make an informed decision by reviewing their risk of progression and the clinical usefulness of the information that will be obtained with liver biopsy. This patient-centered approach is especially important in the subgroup of patients for whom there are emerging data suggesting an increased risk of NASH or fibrosis, such as the elderly and patients who have a family history of diabetes or cirrhosis[15] (Fig. 1).

image

Figure 1. Evidence-based recommendations regarding when to perform liver biopsy in patients with NAFLD.

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Genetic testing for risk stratification of patients with NAFLD is not yet ready for routine clinical practice.

Conclusions

  1. Top of page
  2. Abstract
  3. Noninvasive Markers
  4. Role of Liver Biopsy
  5. Conclusions
  6. References

Biomarkers and clinical prediction rules for refining the management paradigm of NAFLD are emerging. Metabolic syndrome and diabetes are the key determinants of advanced histological examinations in NAFLD. Therefore, liver biopsy may be considered in this subset of patients with NAFLD. Until further refinement of biomarkers and clinical prediction rules, liver biopsy should be considered in patients with NAFLD who have an increased risk of advanced fibrosis and when the diagnosis is uncertain. Incremental advances in the noninvasive diagnosis of NASH will continue to shape the management paradigm of NAFLD and will change clinical practice in the coming years.

References

  1. Top of page
  2. Abstract
  3. Noninvasive Markers
  4. Role of Liver Biopsy
  5. Conclusions
  6. References