|Year : 2010 | Volume
| Issue : 3 | Page : 470-475
|Comparison of FibroTest-ActiTest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis B and C
Cemile Uyar1, Fusun Zeynep Akcam1, Metin Ciris2, Onur Kaya1, Cem Kockar3, Mehmet Isler3
1 Department of Infectious Diseases, Suleyman Demirel Universty Medical Education and Research Hospital, Isparta, Turkey
2 Department of Pathology, Suleyman Demirel Universty Medical Education and Research Hospital, Isparta, Turkey
3 Department of Gastroenterology, Suleyman Demirel Universty Medical Education and Research Hospital, Isparta, Turkey
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|Date of Web Publication||22-Oct-2010|
| Abstract|| |
Aims: FibroTest and ActiTest are noninvasive tests used in determining the level of fibrosis and the degree of necroinflammatory activity in the liver. In our study, we aimed to investigate whether these tests could be alternative to liver biopsy. Materials and Methods: Fifty patients were included in the study. Serum samples were obtained and liver needle biopsy was performed on the same day. Levels of fibrosis in FibroTest and levels of activity in ActiTest, both determined via serum biochemical markers, were compared with levels of fibrosis and activity in histopathological examination. For statistical analyses, Mc Nemar chi square test and Spearman's correlation tests were used. Results: There was a significant positive correlation between fibrosis in biopsy and the level of fibrosis in FibroTest in patients with hepatitis B virus (HBV) (rho: 0.67, P < 0.0001). However, no significant correlation was determined between the activity in biopsy and the degree of activity in ActiTest (rho: 0.29, P < 0.05). No significant correlation was determined between both fibrosis and activity established in biopsy and the results of FibroTest and ActiTest in the group of patients with hepatitis C virus (HCV) (rho: 0.22, P < 0.05 and rho: 0.15, P < 0.05, respectively). Conclusion: Our results suggest that novel and safer noninvasive biochemical tests are needed as an alternative to histopathology in patients infected with HBV and HCV. Consequently, we believe that liver biopsy maintains its place as a gold standard in determining the histopathological condition of the liver.
Keywords: ActiTest, FibroTest, hepatitis B virus, hepatitis C virus, liver biopsy
|How to cite this article:|
Uyar C, Akcam FZ, Ciris M, Kaya O, Kockar C, Isler M. Comparison of FibroTest-ActiTest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis B and C. Indian J Pathol Microbiol 2010;53:470-5
|How to cite this URL:|
Uyar C, Akcam FZ, Ciris M, Kaya O, Kockar C, Isler M. Comparison of FibroTest-ActiTest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis B and C. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Jul 5];53:470-5. Available from: http://www.ijpmonline.org/text.asp?2010/53/3/470/68281
| Introduction|| |
Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the main hepatotrophic viruses leading to chronic hepatitis. Various laboratory tests are used in the follow-up and treatment of the world's leading health issue, chronic hepatitis, including serological tests, determining viral load and histopathological examination. Most important among these tests is liver biopsy, which has three main limitations, including its complications, sampling errors and variability in between pathologists. ,,,,, Most research has focused on noninvasive tests to replace liver biopsy for its complications.
Methods that allow a reliable, early, risk-free and cost-effective diagnosis are of great importance, particularly in the field of medicine. The main objective is to determine the simplest, risk-free and cost-effective diagnostic tests that give correct results. The diagnostic test should only be used if it is effective and powerful enough.  FibroTest and ActiTest are noninvasive tests suggested for use in determining the level of fibrosis and the degree of necroinflammatory activity in the liver by combining the levels of six serum biochemical markers with patient's age and sex via a patented intelligence algorithm. Six blood parameters are alpha-2-macroglobulin, haptoglobin, total bilirubin, gamma-glutamyltransferase (GGT), apolipoprotein-A1 and alanine aminotransferase (ALT). These tests were chosen because of their importance in liver fibrogenesis and fibrolytic activity. As the fibrotic process begins, there is a down-regulation of haptoglobin and apolipoprotein-A1 together with an up-regulation of the alpha-2-macroglobulin molecule.
Our study was designed to investigate whether FibroTest and ActiTest could be alternatives to liver biopsy.
| Materials and Methods|| |
The study group consisted of 50 patients, including 25 patients with chronic hepatitis B and 25 patients with chronic hepatitis C infection followed-up at Suleyman Demirel University, School of Medicine, Infectious Diseases Clinic, between June 2005 and December 2007. Inclusion criteria were being treatment-naοve and consenting to participate.
A minimum of 4 ml of venous blood was drawn from patients into test tubes in a dark room. These blood samples were centrifuged to obtain serum and sent to a patented laboratory to study FibroTest and ActiTest over a maximum period of 24 h (Genom Laboratories, Ankara, Turkey). Equivalents of the results of FibroTest and ActiTest results in the Metavir scoring system are presented in [Table 1]. F0 and F1 indicated low and F2, F3 and F4 indicated high levels of fibrosis. Similarly, A0 and A1 indicated low and A2 and A3 indicated high levels of activity.
|Table 1 :Levels of fi brosis and acti vity according to the metavir scoring system used in serological examinati on|
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Patients underwent needle liver biopsy on the same day. Biopsy samples were examined by an investigator pathologist blinded to the results of biochemical markers. All liver biopsy specimens were fixed in 10% formol saline and then processed into parafin and stained with hematoxylin and eosin, Masson's trichrome and reticulin. Levels of fibrosis and levels of activity according to the Metavir scoring system used in histopathological examination are presented in [Table 2] and [Table 3], respectively. 
|Table 2 :Levels of fi brosis according to the metavir scoring system used in histopathological examinati on|
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|Table 3 :Levels of acti vity according to the Metavir scoring system used in histopathological examinati on (*)|
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Results of histopathological examination were considered gold standard. FibroTest and ActiTest assessments are presented in four-cell (2 x 2) tables [Table 4] and [Table 5] in such a way that the gold standard test lies in columns and tests of evaluation (FibroTest and ActiTest) lies in rows. Names of the cells are true positive (TP), false positive (FP), false negative (FN) and true negative (TN).
|Table 4 :Comparison of histopathological fi brosis results with FibroTest in pati ents with hepati ti s B and C|
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|Table 5 :Comparison of the histopathological acti vity results with Acti Test in pati ents with hepati ti s B and C|
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The following parameters were calculated to evaluate the performance of the diagnostic tests:
1. Sensitivity: Indicates the ability of the test to discriminate patients among real patients. It is calculated according to the following formula:
Sensitivity = TP/(TP + FN)
2. Specificity: Indicates the ability of the test to discriminate healthy individuals among real healthy individuals. It is calculated according to the following formula:
Specificity = TN/(TN + FP)
3. Fn Rate (FNR): Indicates false healthy results among real patients. It is calculated according to the following formula:
Fnr = (1 - SENSITIVITY)
4. Fp Rate (FPR): Indicates patients among real healthy individuals. It is calculated according to the following formula:
Fpr = (1 - SPECIFITY)
5. Positive Likelihood Ratio (L+): Indicates the ratio of correct results to false results in reporting the presence of a disease. In other words, this is the ratio of correctness in establishing a diagnosis of disease. The higher this ratio, the better is the discrimination of real patients. It is calculated according to the following formula:
L+ = SENSITIVITY/(1 - SPECIFICITY)
6. Negative Likelihood Ratio (L-): Indicates the ratio of correct diagnosis of healthiness. The smaller the ratio is, the better is the discrimination of real healthy individuals. It is calculated according to the following formula:
L = (1 - SENSITIVITY)/(SPECIFICITY)
7. Accuracy: "Accuracy" means the total ratio of correct diagnosis in terms of patients and healthy ones. This differs from the rest of the ratios in the fact that accuracy might change with disease frequency even for the same sensitivity-specificity. It increases with disease frequency (prevalence). It is calculated according to the following formula:
Accuracy = (TP + TN)/(TP + FP + FN + TN)
8. Indeed, the most important question researchers struggle to find an answer to is "What is the possibility of an individual with a positive diagnostic test being a real patient? (Or, an individual with a negative diagnostic test being really healthy?)" This is termed "Predictive Value."
Positive Predictive Value (PPV) is the ratio of being sick indeed when the diagnostic test also indicates disease.
PPV = TP/(TP + FP)
9. Negative Predictive Value (NPV) is the ratio of being healthy indeed when the diagnostic test also indicates disease.
NPV = TN/(TN + FN)
10. Another frequently used method of comparison is the "Youden Index (J)." It suggests that the J-value:
a. varies between -1 and +1,
b. indicates that the test has no diagnostic power if it is lower than zero,
c. is higher if the diagnostic test is more effective.
It is calculated according to the following formula:
Youden's index (J) = (SENSITIVITY) + (SPECIFICITY) - 1
The presence of any difference between diagnostic tests (FibroTest and ActiTest) and real results (histopathology) was analyzed using the Mc Nemar chi-square test in order to detect the judgement abilities, i.e. performance of diagnostic tests. Consistency of the two tests (gold standard and the test of assessment) was evaluated by calculating the kappa coefficient. All statistical analyses were carried out using the statistical package for social sciences (SPSS Inc., Chicago, IL, USA), with a two-tailed P-value of 0.05 used as a threshold for significance. Kappa statistical evaluation categories were as follows:
Values equal to or lower than 0.10 Weak - consistence
Values in between 0.11 and 0.30 - Poor consistence
Values in between 0.31 and 0.50 - Moderate consistence
Values in between 0.51 and 0.70 - Good consistence
Values equal to or greater than 0.71 - Perfect consistence
This study was conducted according to the ethical standards set by the Declaration of Helsinki that promote respect for all human beings and protect their health and rights.  After informing the participants about the purpose of the trial (investigation, research, study) and about where and how the obtained data would be used, written consents were obtained. All participants in the research survey gave their consent for the release of information for public use.
| Results|| |
Patients consisted of 20 males and five females in the hepatitis B group and 11 males and 14 females in the hepatitis C group. The mean length of biopsy sample was 12 ± 0.51 mm, and included a mean number of 7.88 ± 2.42 portal spaces in patients with chronic HBV. The mean length of the biopsy sample was 14 ± 0.56 mm, and included a mean number of 8.92 ± 3.29 portal spaces in patients with chronic HCV.
The parameters regarding the diagnostic performance of FibroTest in levels of fibrosis equal to or greater than 2 (advanced) and ActiTest in levels of activity equal to or greater than 2 (advanced) are shown in [Table 4] and [Table 5].
The relationship between fibrosis and activity levels of FibroTest and ActiTest and those determined in biopsy were investigated using Spearman's correlation test. Correlation results are presented in [Table 6].
|Table 6 :Correlati on of histopathological fi brosis and necroinfl ammatory acti vity scores with FibroTest and Acti Test in pati ents with hepati ti s B and C|
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It was determined that the histopathological fibrosis scores and levels of fibrosis in FibroTest did not differ in the group of patients with HBV (Mc Nemar chi-square test, P = 0.05, kappa-value = 0.34) [Table 4]. A significant positive correlation was established between these two tests [Spearman's correlation coefficient (rho) = 0.67, P < 0.001] [Table 6]. No significant differences were determined between the scores of histopathological activity and ActiTest activity levels (Mc Nemar chi-square test, P = 0.08); kappa-value = 0.36 [Table 5]. No significant correlation was established between these two tests [Spearman's correlation coefficient (rho) = 0.29, P > 0.05].
It was determined that the histopathological fibrosis scores and levels of fibrosis in FibroTest did not differ in the group of patients with HCV (Mc Nemar chi square test, P = 0.35); kappa-value = 0.15 [Table 4]. No significant correlation was established between these two tests [Spearman's correlation coefficient (rho) = 0.22, P > 0.05] [Table 6]. No significant differences were determined between the activity scores determined in biopsy and ActiTest activity levels (Mc Nemar chi square test, P = 0.39); kappa-value = 0.14 [Table 5]. No significant correlation was established between these two tests [Spearman's correlation coefficient (rho) = 0.15, P > 0.05] [Table 6].
| Discussion|| |
FibroTest and ActiTest have entered clinical practice as an alternative to biopsy in several countries since September 2002 because they are noninvasive and easily performed compared to liver biopsy. 
Several studies have suggested the use of FibroTest as a noninvasive alternative to liver biopsy for assessing fibrosis in patients with HCV and ActiTest for assessing necroinflammatory activity. ,,, However, there are a limited number of studies in patients with chronic hepatitis B, and one study including 209 patients has concluded that FibroTest and ActiTest are noninvasive markers providing correct results in HBV-related activity and fibrosis.  A systematic review by Gebo et al. in 2002 has suggested that FibroTest is paramount in demonstrating minimal and advanced fibrosis and cirrhosis but fairly successful in predicting moderate fibrosis. In our study, we investigated whether FibroTest and ActiTest could be alternatives to liver biopsy.
Sensitivity of FibroTest in demonstrating F2 or greater fibrosis was found to be 100% in patients with hepatitis B, whereas this rate was 67% in patients with hepatitis C. Although the specificity of FibroTest is similar in patients with hepatitis B and C, the L+ values indicate that patients with hepatitis B have higher values compared to patients with hepatitis C. The higher this ratio the better is the discrimination of real patients. According to these parameters, FibroTest is better in differentiating advanced fibrosis and patients in hepatitis B than in hepatitis C.
FibroTest has a better diagnostic power in both patients with hepatitis B and patients with hepatitis C, according to the Youden's index. This results from the fact that this index has been calculated as greater than zero, which indicates the presence of diagnostic power. It may be suggested that FibroTest provides more effective results in patients with hepatitis B than in patients with hepatitis C because the Youden's index is greater in patients with hepatitis B compared to that in patients with hepatitis C.
Patients with both hepatitis B and C obtained similar histopathological and statistical results in FibroTest according to the results of the Mc Nemar analysis. Both P-values indicate no significant difference (P > 0.05). Kappa-values show that FibroTest is 0.34, consistent with the histopathological diagnosis in patients with hepatitis B. On the other hand, the consistency of FibroTest with histopathology is only 0.15 in patients with hepatitis C.
The sensitivity of ActiTest was found to be 67% in patients with hepatitis B, which is quite higher than the sensitivity determined in patients with hepatitis C. The specificity of ActiTest is similar in patients with hepatitis B and C. In ActiTest, patients with hepatitis B have slightly higher L+ values than patients with hepatitis C. The higher this ratio the better is the discrimination of real patients. These parameters suggest that ActiTest is comparatively better in determining the higher acitivity in hepatitis B than in hepatitis C.
The diagnostic power of ActiTest has supported its use in patients with both hepatitis B and C according to the Youden's index. This results from the fact that this index has been calculated as greater than zero, which indicates the presence of diagnostic power. It may be suggested that ActiTest provides more effective results because the Youden's index is greater in patients with hepatitis B compared to that in patients with hepatitis C.
Patients with both hepatitis B and C obtained similar histopathological results in ActiTest according to the results of Mc Nemar analysis. Both P-values indicated no difference (P > 0.05). In other words, ActiTest has demonstrated histological activity similar to histopathology in patients with hepatitis B and C. Kappa-values showed that ActiTest was 0.36, consistent with histopathology in patients with hepatitis B, whereas this figure was 0.14 in patients with hepatitis C.
According to kappa-values, both FibroTest and ActiTest were found to be moderately consistent with the histopathological diagnosis in patients with hepatitis B, and the rate of consistency was found to be quite low in patients with hepatitis C.
It has been reported that abnormal values (e.g., too high or too low) of the components of (serum markers: haptoglobin, apolipoprotein-A1, alpha-2-macroglobulin, GGT, total bilirubin, ALT) FibroTest and ActiTest may have an impact on the test results and result in false interpretations.  Components of FibroTest and ActiTest in our patients were reexamined in the light of this information. No abnormal laboratory data were determined that would affect the test results. Therefore, it was concluded that the tests did work correctly but did not reflect the results of liver biopsy adequately.
Certain case reports in the literature have also demonstrated false negativities of biopsy. ,, These reports have suggested that histopathological values with lower scores compared to noninvasive tests were biased due to patient features (esophageal varicosities, low thrombocyte count, signs of liver cirrhosis in ultrasonography). A prospective study has shown that 18% of the inconsistencies of FibroTest and ActiTest with liver histology result from errors in performing the biopsy and 2% from errors in FibroTest-ActiTest. 
In the study by Poynard et al., biopsy samples shorter than 15 mm were compared and FibroTest-ActiTest was found to have higher accuracy rates, and the inconsistency between these tests and histology was observed to result from sampling errors. Therefore, authors have suggested that fibrogenic-necrotic damages of liver are quantitatively better established with the use of biochemical markers than with liver biopsy. 
A single biopsy sample does not always show all features of the disease in viral hepatitis-like diseases that do not involve all parts of the liver equally. Although a consensus has not been established on what should the size of the biopsy sample be for optimal evaluation, it has been suggested that the sample should be 20 mm in length and include five to 11 portal spaces. ,,
In our study, the mean length of biopsy material was 12 ± 0.51 mm and the mean number of portal spaces 7.88 ± 2.42 in patients with chronic HBV, and 14 ± 0.56 mm and 8.92 ± 3.29, respectively, in patients with chronic HCV. In a recent study demonstrating that adequate number of portal spaces were associated with biopsy samples as much as they are associated with sample length, samples including 7 ± 1.8 portal spaces were appropriate for pathological examination.  In the light of the related literature, we believe that the biopsy samples and pathological evaluation are adequate in this study. Various fibrosis and necroinflammatory activities in our patients are shown in [Figure 1],[Figure 2],[Figure 3],[Figure 4].
|Figure 1 :Mild interface hepati ti s and fi brous expansion of the portal area, hepati ti s B. Level of fi brosis: F1; level of acti vity: A1 (H and E, ×100)|
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|Figure 2 :Severe interface hepati ti s and portal to portal bridging of some portal areas, hepati ti s C. Level of fi brosis: F2; level of acti vity: A3 (H and E, ×100)|
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|Figure 3 :Mild interface hepati ti s and marked bridging, hepati ti s B. Level of fi brosis: F3; level of acti vity: A1 (H and E, ×100)|
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|Figure 4 :Cirrhosis, hepati ti s B. Level of fi brosis: F4 (reti culin stain, ×200)|
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According to the results of our study, FibroTest and ActiTest gave results consistent with histopathological examination in the presence of advanced fibrosis and activity in patients with hepatitis B. The consistency is lower in patients infected with HCV. According to the results of correlation presented in [Table 6], FibroTest results correlate with histopathology only in hepatitis B. No significant correlation was established between histopathology and the results of ActiTest in hepatitis B and results of both FibroTest and ActiTest in hepatitis C.
Consequently, currently liver biopsy maintains its importance as the gold standard in fibrosis and histopathological activity. Novel and safer diagnostic tests are needed in determining fibrosis and activity, which could be alternative to histopathology, particularly in patients with hepatitis C in whom biopsy is contraindicated.
| Acknowledgment|| |
The authors are grateful to Ersin Uskun (Associated Prof., Department of Public Health) for her help with the statistical analysis.
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Fusun Zeynep Akcam
Department of Infectious Diseases, Suleyman Demirel University Medical Education and Research Hospital, Isparta-32260
Source of Support: Suleyman Demirel University, Scientific Research Commission, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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