PRO-C3 and ADAPT Algorithm Accurately Identify Patients With Advanced Fibrosis due to Alcohol-related Liver Disease

Bjørn S. Madsen; Maja Thiele; Sönke Detlefsen; Maria Kjærgaard; Linda S. Møller; Jonel Trebicka; Mette J. Nielsen; Natasja Stæhr Gudmann; Diana J. Leeming; Morten A. Karsdal; Aleksander Krag;


Aliment Pharmacol Ther. 2021;54(5):699-708. 

In This Article

Methods and Materials

The study was performed as a prospective biopsy-controlled single-centre study. The study was approved by the ethics committee of the Region of Southern Denmark (S-20120071, S-20160021). The study adheres to the 2013 Helsinki Declaration and is registered in the Odense Patient Data Exploratory Network (OPEN) under study identification numbers OP_040 ( and OP_239 ( This report follows the Liver-Fibro STARD checklist.[17]

The liver biopsies were performed percutaneously with a 17-G Menghini suction needle (Hepafix). Biopsies were considered to be of adequate quality in the absence of cirrhosis if they were >10 mm length and contained >5 portal tracts. A single experienced pathologist evaluated the biopsies according to the Kleiner fibrosis stage and non-alcoholic fatty liver disease activity score (NAS-CRN).[18] According to the Kleiner fibrosis stage, F0 is no fibrosis, F1 is perisinusoidal or portal/periportal fibrosis, F2 is perisinusoidal fibrosis in combination with portal/periportal fibrosis, F3 is bridging fibrosis, and F4 is cirrhosis. The NAS CRN is a semi-quantitative score of steatosis (0–3), ballooning (0–2) and lobular inflammation (0–3). The ADAPT score was calculated using the previously published formula

where Diabetes is coded as 0 if absent and 1 if present.[14] FIB4 was calculated using the formula from the original publication.[19]

Biomarker Quantification

Type III collagen formation was assessed in serum samples by using the ELISA-based PRO-C3 assay from Nordic Bioscience, Herlev, Denmark as previously described.[20]

Study Population

We enrolled 426 patients with prior or current alcohol overuse for more than 1 year defined as >24 g per day for women and >36 g per day for men. Additional criteria were age 18–75 years and informed consent to undergo a liver biopsy. We consecutively recruited participants from two municipal alcohol rehabilitation centres, through advertisement in newspapers and from three liver clinics in the Region of Southern Denmark. All participants were informed in oral and writing prior to inclusion. All patients were considered to have a significant risk of alcohol-related liver disease that justified performing a liver biopsy. We revised the criteria in January 2016. Thereafter, we avoided performing a liver biopsy in patients with a liver stiffness below 6.0 kPa, as none of these patients had severe fibrosis, and instead, we categorized these individuals as not suffering from advanced fibrosis without performing a liver biopsy.[21]

Exclusion criteria were decompensated liver disease with clear clinical signs of cirrhosis, severe alcoholic hepatitis, debilitating disease with an expected survival less than 1 year, concurrent liver disease including hepatitis B and C, hepatic congestion or inability to comply with the study protocol. All investigations were performed on the same day according to standard operating procedure after an overnight of fasting. Blood samples from 154 healthy gender- and aged-matched participants were used to determine the concentration of PRO-C3 in healthy individuals.

Statistical Analysis

We used summary statistics to describe patient characteristics. Differences between continuous variables were tested by using an unpaired Students t-test or a Mann-Whitney test as appropriate. Differences between categorical variables were tested using Chi-squared test. Kruskal-Wallis and a post-hoc Dunn's test were used to identify statistical difference in PRO-C3 between fibrosis stages. By performing a multivariate logistic regression model using forced entry, we identified factors independently associated with the presence of advanced ALF. Variables with a P ≤ 0.05 in univariate analysis were included in the final multivariate analysis. The diagnostic accuracy of PRO-C3 was evaluated by AUROC. We used Delong test to compare AUROC between PRO-C3, ADAPT and FIB4 scores. Recently published cut-off values of 15.6 ng/ml for PRO-C3 and 6.3287 for the ADAPT score to detect advanced fibrosis were used to calculate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each test.[14] We developed a risk prediction score based on PRO-C3 by using a logistic regression model. We used Hosmer-Lemeshow goodness of fit with 10 quantiles and plotting of the observed and predicted values to evaluate the calibrations. P ≤ 0.05 were considered significant. We used STATA 15 (StataCorp) for the statistical analyses.