In the original ELF study patients were considered eligible
In the original ELF study , patients were considered eligible if they were due to undergo liver biopsy for the investigation of chronic liver disease, defined as abnormal biochemical liver function tests persisting for more than 6 months, ability to provide informed consent and age between 18 and 75 years. Patients were excluded from the study if their age fell outside of this range; for any disorder associated with extrahepatic fibrosis, including rheumatic, renal, or lung disease; for cardiovascular disease or cancer; for advanced cirrhosis with evidence of decompensation (Child–Pugh class C); for consumption of regular aspirin; or for hepatocellular carcinoma or drug-induced liver disease.
Paraffin embedded tissue blocks were transferred from the University of Florence, Florence, Italy, to the Department of Cellular Pathology at the Royal Free Hospital, London, UK, where the study was performed. Each tissue block was processed according to the reported CPA method and analysed for collagen content. Results of the CPA analysis were compared with the standard Ishak’s semiquantitative score  used in the original ELF study . Due to possible differences in the cut of the paraffin blocks, i.e. progressive favipiravir of the block thickness, all samples were re-staged according to the Ishak’s score. The results of the CPA analysis were then compared with the ELF panel together with relevant clinical and biochemical parameters available in the patients’ CRFs. All the above data were also re-analyzed by appropriate statistical methods and were fully reconsidered in order to find the best fit in defined mathematic cut-off.
Discussion Several studies , , , ,  were conducted using non-invasive methods for studying and monitoring patients, which have largely replaced liver biopsy in clinical practice. As mentioned previously, existing scoring systems do not represent a measurement of quantitative fibrosis, but rather a categorical description of both architecture and fibrosis and, therefore, an imperfect “gold standard” for the quantitative measurement of liver fibrosis that requires the use of continuous variables. CPA is a morphometric collagen measurement that generates continuous variables that suffer less from the variance entailed in staining procedures and differing operator experience. Non-invasive markers of liver fibrosis should hence be validated using the CPA method. The present study showed good detection of advanced fibrosis and cirrhosis in patients with chronic viral hepatitis. Recently, Fagan et al.  performed a non-invasive assessment of fibrosis severity by ELF, determining that the cut-off value of ≥9.8 identified advanced fibrosis with diagnostic accuracy, even if it is influenced by age, steatosis and histological activity. It was also the case in our study that an ELF score ≥10 was associated with advanced fibrosis (Ishak’s stage ≥ 4). In fact, the correlation between CPA and ELF is better for values up to 10 but is worse after that. ELF is a clinical method for the rapid and non-invasive assessment of fibrosis stage in chronic viral hepatitis, which is continuously subjected to extensive validation to exclude the factors influencing the fibrosis stage. Lichtinghagen et al.  measured ELF scores in 400 healthy controls and 79 CHC patients. Analysis of ELF scores in healthy subjects revealed that afternoon values were slightly higher than morning values, possibly as a result of food intake. Even if the data concerning the outcomes were poor, it is evident that clinical events occur when the ELF score is ≥9. Accordingly, Irvine et al.  showed that an ELF score indicative of advanced fibrosis (≥9.8) correlated with liver-related clinical outcomes. In our study, patients with concomitant extra-hepatic diseases that could potentially increase ELF levels were excluded. Abignano et al. , for instance, studied 210 patients with systemic sclerosis, demonstrating that ELF score, blindly, is a clinical-grade serum test that significantly correlates with fibrosis measurements in systemic sclerosis and with overall disease activity, severity, and health assessment questionnaire-disability index. Gonzalez-Lopez et al. , meanwhile, showed that procollagen type I and III aminoterminal propeptide correlated with interstitial lung disease patterns and severity in Mexican women with systemic sclerosis. A recent study by van der Voort et al.  of 531 patients applied existing cut-offs of ELF for patients and healthy controls and compared it with procollagen-3 N-terminal peptide (P3NP) test in patients with psoriasis, psoriatic arthritis and rheumatoid arthritis, and controls. ELF score (>11) and P3NP were found to be highest in patients with rheumatoid arthritis, followed by patients with psoriasis and psoriatic arthritis. Considering that ELF consists of hyaluronic acid, aminoterminal propeptide of type III collagen, and tissue inhibitor of matrix metalloproteinase 1, all pathologies that cause an increase in these markers are potentially able to determine an increase in ELF. In particular, serum hyaluronic acid level may be used as tools to differentiate between acute and chronic kidney injury . Collagen metabolism was altered in cases of acute respiratory failure . In this study, out of 5 patients with clinical outcomes, 3 patients (who had a non-liver related death) had an ELF level between 9.8 and 11.3 despite a low CPA and Ishak stage. This implies that ELF in this “gray area”, according to what observed Lichtinghagen et al.  cannot be clearly attributed to an advanced stage of fibrosis. It is plausible that such values could be a surrogate for non-liver related comorbidities that increase non-liver related morbidity and mortality, however this will need to be tested in larger cohorts. Therefore the potential utility of ELF in predicting non-liver related deaths should be further explored in larger prospective cohorts.