Non-Invasive Diagnosis Tests

Laurent Castera

Department of Hepatology, Hopital Beaujon, Assistance Publique-Hôpitaux de Paris, INSERM U773, Clichy, France

Introduction

Prognosis and management of chronic liver diseases greatly depend on the amount and progression of liver fibrosis. For many years, liver biopsy has been considered the “gold standard” for evaluation of hepatic fibrosis [1]. However, liver biopsy is an invasive procedure with rare but potentially life-threatening complications and prone to sampling errors. These limitations as well as the availability of powerful viral tools and new antiviral drugs have rapidly decreased the use of liver biopsy in viral hepatitis and led to the development of noninvasive methodologies for the assessment of fibrosis. Among the currently available noninvasive methods, there are two distinct approaches: (i) a “biologic” approach based on the dosage of serum biomarkers of fibrosis; (ii) a “physical” approach based on the measurement of liver stiffness using transient elastography (TE) [2]. Although complementary, these two approaches are based on differing rationale and conception: TE measures liver stiffness related to elasticity, which corresponds to a genuine and intrinsic physical property of liver parenchyma, whereas serum biomarkers are combinations of several not strictly liver-specific blood parameters optimized to mimic fibrosis stages as assessed by liver biopsy [3].

This chapter reviews the different methods that are currently available for the noninvasive evaluation of cirrhosis and also discusses their advantages and inconveniences for the management of patients with chronic liver disease.

Biologic Approach: Serum Biomarkers of Liver Fibrosis

Currently Available Serum Biomarkers

Many serum biomarkers have been evaluated for their ability to determine stage of liver fibrosis, mainly in patients with chronic hepatitis C virus infection (for review see [4,5]). Among the proposed markers, the so-called direct markers reflect the deposition or removal of extracellular matrix in the liver. These include glycoproteins such as serum hyaluronate, laminin, and YKL-40, collagens such as procollagen III N-peptide and type IV collagen, collagenases, and their inhibitors such as matrix metalloproteases and tissue inhibitory metalloprotease-1. So-called indirect markers include factors that can be measured in routine blood tests, such as the prothrombin index, platelet count, and ratio of aspartate aminotransferase to alanine aminotransferase (AST : ALT), which indicate alterations in hepatic function. Results from measurements of direct and indirect markers can be combined and used in diagnosis; the FibroTest® (BioPredictive, Paris, France) was the first algorithm that combined these data [6]. Several other scores [7–16] have been proposed – four are protected by patents and commercially available (Table 6.1). Nonproprietary methods use published models, based on routinely available laboratory tests.

Table 6.1 Proposed serum biomarkers for noninvasive evaluation of cirrhosis in chronic liver disease.

FibroTest® (α2-macroglobulin, GGT, apolipoprotein A1, haptoglobin, total bilirubin, age, and gender)
AST : platelet ratio (APRI) (AST and platelet count)
Enhanced Liver Fibrosis score® (ELF) (age, hyaluronate, MMP-3, and TIMP-1)
Lok index (platelet count, AST : ALT ratio, and INR)
Gotebörg University Cirrhosis Index (GUCI) (AST, INR, and platelet count)
Hepascore® (bilirubin, γGT, hyaluronate, α2-macroglobulin, age, and gender)
Fibrometers® (platelet count, prothrombin index, AST, α2-macroglobulin, hyaluronate, urea, and age)
Virahep-C model (AST, platelet count, alkaline phosphatase, and age)
FIB-4 (platelet count, ALT, and AST)
HALT-C model (hyaluronic acid, TIMP-1, and platelet count)
NAFLD Fibrosis Score (NFS) (age, BMI, hyperglycemia/diabetes, AST : ALT ratio, platelet count, and albumin)
BARD score (BMI, AST : ALT ratio, and diabetes)

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; GGT, gamma-glutamyl transpepsidase; INR, international normaized ratio; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.

Diagnostic Performances of Serum Biomarkers

Diagnostic performances for cirrhosis of these different scores are summarized in Table 6.2. The most widely used and validated are the aspartate aminotransferase: platelet ratio index (APRI; a free nonpatented index) and the FibroTest® (a patented test that is not widely available). In a meta-analysis [17], which analyzed results from 6259 HCV patients from 33 studies, the mean area under the receiver operating characteristic curve (AUROC) values from the APRI in diagnosis of cirrhosis were 0.83.

Table 6.2 Diagnostic performance of currently available serum biomarkers of fibrosis for cirrhosis (F4) in chronic liver disease.

Scores	Year	Etiologies	Patients (n)	F4 (%)	Cutoffs	AUROC	Sensitivity (%)	Specificity (%)	+LR	−LR
FibroTest® [6]	2001	HCV	1197	14	>0.74	0.82	63	84	4.0	0.4
APRI [7]	2003	HCV	476	17	<1.0 ≥2.0	0.89	57–89	75–93	3.6–8.1	0.1–0.5
ELF® [8]	2004	HCV	1021	NA	NA	0.89	91	69	2.9	0.4
Lok Index [9]	2005	HCV	1141	38	<0.2 ≥0.5	0.81	40–98	53–99	2.1–40.0	0.04–0.6
Hepascore® [10]	2005	HCV	211	16	>0.84	0.89	71	89	6.5	0.3
GUCI [11]	2005	HCV	179	12	>1.0	0.85	80	78	3.6	0.3
FIB-4 [13]	2007	HCV	847	17^*	<1.45 >3.25	0.85	38–74	81–98	3.9–19.0	0.3–0.6
NFS [14]	2007	NAFLD	733	27^*	<−1.455 >0.676	0.82	43–77	97–97	10.0–13.3	0.6–0.7
BARD score [15]	2008	NAFLD	669	30^*	≥2	0.81	—	—	—	—
HALT-C model [16]	2008	HCV	512	38	<0.2 >0.5	0.81	47–88	45–92	1.6–5.9	0.3–0.6
Fibrometers® [22]	2010	HCV HBV	1204	13.6	NA	0.86	44	95	8.8	0.6
^*F3–F4 patients.
AUROC, area under ROC curve; HBV, chronic hepatitis B; HCV, chronic hepatitis C; +LR, positive likelihood ratio; –LR, negative likelihood ratio; NA not availableNAFLD, non fatty alcoholic liver disease.

When compared and validated externally in patients with hepatitis C virus infection [18–22], the different patented scores have similar performances for the diagnosis of cirrhosis. In the largest study to date (n = 1307 patients with viral hepatitis) comparing prospectively the most popular patented scores (FibroTest®, FibroMeter®, HepaScore®) with the nonpatented score (APRI), the AUROCs ranged from from 0.77 to 0.86 with no significant difference between the scores [22]. Although nonpatented scores such as FIB-4 and APRI may have slightly lower performance, they are cost-free, easy to calculate, and available almost everywhere. Regarding nonalcoholic fatty liver disease (NAFLD), among the limited number of serum biomarkers available, the NAFLD fibrosis score has been the most studied thus far [14,23–26].

Advantages and Limitations of Serum Biomarkers

The practical advantages of analyzing serum biomarkers to measure fibrosis include their high applicability (>95%) and inter-laboratory reproducibility [27,28], and their potential widespread availability. However, none are liver specific – their results can be influenced by comorbid conditions and they require critical interpretation of results. For instance, FibroTest® and HepaScore® produce false positive results in patients with Gilbert’s syndrome or hemolysis, because these patients have hyperbilirubinemia [29]. Similarly, acute hepatitis can produce false positive results in the APRI, Forns index, FIB-4, or FibroMeter® tests, which all measure levels of aminotransferases.

Physical Approach: Measurement of Liver Stiffness

Transient Elastography

Principles and Reproducibility

Liver fibrosis can be staged using TE (FibroScan®, Echosens, Paris, France) [30], which measures the velocity of a low-frequency (50 Hz) elastic shear wave propagating through the liver. This velocity is directly related to tissue stiffness, the elastic modulus (expressed as E = 3ρv², where v is the shear velocity and ρ is the density of tissue, assumed to be constant). The stiffer the tissue, the faster the shear wave propagates. TE measures liver stiffness in a volume that approximates a cylinder that is 1 cm wide and 4 cm long, 25–65 mm below skin surface. The results are expressed in kilopascals (kPa), and range from 2.5 to 75 kPa; a normal value is around 5 kPa [31–33].

Diagnostic Performances of Transient Elastography

The two index studies suggesting the interest of TE in the assessment of liver fibrosis have been conducted in patients with chronic hepatitis C virus infection [34,35]. Many other groups have confirmed these results since [22,36–38], also in chronic liver diseases other than hepatitis C [39–42], including chronic hepatitis B [43–45], cholestatic liver diseases (primary biliary cirrhosis and primary sclerosing cholangitis) [46], alcoholic liver disease [47,48], and NAFLD [49–51]. As shown in Table 6.3, AUROCs range from 0.90 to 0.99 for the diagnosis of cirrhosis.

Table 6.3 Diagnostic performance of transient elastography for cirrhosis (F4) in chronic liver disease.

Authors	Year	Etiologies	Patients (n)	F4 (%)	Cutoffs (kPa)	AUROC	Sensitivity (%)	Specificity (%)	+LR	−LR
Castera et al. [35]	2005	HCV	183	25	12.5	0.95	87	91	9.7	0.1
Ziol et al. [34]	2005	HCV	251	19	14.6	0.87	86	96	23.1	0.1
Corpechot et al. [46]	2006	PBC-PSC	95	16	17.3	0.96	93	95	18.6	0.07
Gomez-Dominguez et al. [40]	2006	Mixed	94	17	16.0	0.94	89	96	22.25	0.11
Ganne-Carrie et al. [41]	2006	Mixed	775	15	14.6	0.95	79	95	15.8	0.11
Foucher et al. [42]	2006	Mixed	354	13	17.6	0.96	77	97	25.66	0.23
Fraquelli et al. [39]	2007	Mixed	200	12	11.9	0.90	91	89	8.27	0.1
Coco et al. [43]	2007	HCV HBV	228	20	14.0	0.96	78	98	39.0	0.22
Lupsor et al. [37]	2008	HCV	324	21	11.9	0.94	87	91	9.7	0.1
Arena et al. [36]	2008	HCV	150	19	14.8	0.98	94	92	11.3	0.1
Yoneda et al. [49]	2008	NAFLD	97	9	17.0	0.99	100	97	33.33	0
Nguyen-Khac et al. [48]	2008	ALD	103	32	19.5	0.92	86	84	5.37	0.16
Nahon et al. [47]	2008	ALD	147	54	22.7	0.87	84	83	5.24	0.19
Marcellin et al. [45]	2009	HBV	173	8	11.0	0.93	93	87	7.0	0.08
Chan et al. [44]	2009	HBV	161	25	9.0	0.93	98	75	3.92	0.02
Wong et al. [51]	2010	NAFLD	246	10	10.3	0.95	92	88	7.5	0.1
Degos et al. [22]	2010	HCV, HBV, HCV-HIV	1307	14	12.9	0.90	72	89	6.8	0.3
Zarski et al. [71]	2012	HCV	382	14	12.9	0.93	77	90	7.7	0.25
ALD, alcoholic liver disease; AUROC, area under ROC curve; HBV, chronic hepatitis B; HCV, chronic hepatitis C; +LR positive likelihood ratio; −LR negative likelihood ratio; NAFLD, non-fatty alcoholic liver disease; PBC-PSC, primary biliary cirrhosis primary sclerosing cholangitis.

Several meta-analyses [52–55] have confirmed the high diagnostic performance of TE for cirrhosis, with mean AUROC values of 0.94 [54]. In a meta-analysis of 40 studies (32 papers and 8 abstracts), sensitivity and specificity values were 0.83 and 0.89 for patients with cirrhosis, respecively. However, only 9 studies (comprising 1364 patients) had acceptable standards for liver biopsy and TE, which limit the conclusions. It will therefore be important to perform meta-analyses of data from individual patients.

TE thus appears to be a reliable method for the diagnosis of cirrhosis, better at excluding than at predicting cirrhosis. For instance, in a population of 1007 patients with different chronic liver diseases, a cutoff value of 14.6 kPa yielded positive and negative predictive values of 74% and 96%, respectively [41]. Interestingly, proposed cutoff values for cirrhosis ranged from 11 kPa in patients with hepatitis B virus infection to 22.7 kPa in patients with alcoholic liver disease. Some researchers have proposed that cutoff values be adapted based on causes of liver disease [41]. However, differences among cutoff values could result from differences in prevalence of cirrhosis among study populations (ranging from 8% to 25%). A cutoff value for one population might not be applicable to another, which has a different prevalence of disease. Most studies used single cutoff values for patients with cirrhosis or advanced fibrosis, but more information can be obtained when values are interpreted as a continuum. For example, when liver stiffness values range from 2.5 to 7 kPa, fibrosis is likely mild or absent, whereas when values are above 13 kPa, cirrhosis is likely [56].

Advantages and Limitations of Transient Elastography

Advantages to TE include a short procedure time (<5 minutes), immediate results, and the ability to perform the test at the bedside or in an outpatient clinic – it is not a difficult procedure to learn. However, the interpretation of TE results should be always in the hands of an expert clinician and should be made with information regarding patient demographics, disease etiology, and essential laboratory parameters as well as manufacturer’s recommendations (number of valid shots ≥10; success rate (the ratio of valid shots to the total number of shots) ≥60%; and interquartile range (IQR, reflecting the variability of measurements) less than 30% of the median liver stiffness measure (LSM) value (IQR/LSM ≤30%) [56].

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