Sarah Bos

24 CHAPTER 2 were pretreated with bilirubin oxidase (2.5 U/ml, Sigma-Aldrich, Zwijndrecht, The Netherlands) for 30 minutes at 37°C to avoid interference of bilirubin with the FRETS-VWF73 assay.(24) Levels of VWF and ADAMTS13 in pooled normal plasma were set at 100%, and values obtained in test plasmas were expressed as a percentage of pooled normal plasma. Plasma markers of coagulation Levels of factor (F) VII, FVIII, prothrombin, antithrombin and protein C were assessed on an automated coagulation analyzer (ACL 300 TOP). We used factor deficient plasma for FII, FVII and FVIII, Hemosil Liquid Antithrombin for AT, and Hemosil protein C for protein C. Testing was performed according to protocols from the manufacturer (Instrumentation Laboratory, Breda, the Netherlands). Plasma markers of fibrinolysis Plasminogen activator inhibitor-1 (PAI-1) levels were determined with an ELISA kit from R&D systems (Abingdon, UK). Levels of tPA were measured using an ELISA kit Nodia (Hyphen Biomed; Amsterdam, the Netherlands). Statistical analysis Data are expressed as means (with standard deviations (SDs)), medians (with ranges), or numbers (with percentages) as appropriate. Means of two groups were compared by Student’s t-test or distributions in the two groups by Mann-Whitney U test as appropriate. Multiple groups were compared using One-way ANOVA (with the Bonferroni posttest) or Kruskal-Wallis H test (with Dunn’s posttest) as appropriate. Pearson’s correlation coefficient was used to assess correlation between variables. P values of 0.05 or less were considered statistically significant. GraphPad Prism (San Diego, USA) and IBM SPSS Statistics 23 (New York, USA) were used for analyses. Results Patient characteristics The selected group consisted of 153 patients and 44 healthy controls. We excluded 44 patients due to double diagnosis (n=10), no diagnosis (n=5), or a diagnosis which was too infrequent for further analysis (as for example Wilson’s disease, auto-immune hepatitis, secondary biliary cirrhosis) (n= 29). Our analyses were performed in samples from 109 patients and 44 healthy controls. We stratified patients according to 4 etiologies of disease categories: cholestatic disease (PBC and PSC), NASH-related cirrhosis, alcohol-related disease (ALD), and viral hepatitis.