Preanalytical effects on the ELF score and its component biomarkers; aminoterminal peptide of type III procollagen (PIIINP), hyaluronic acid (HA) and tissue inhibitor of metalloproteinases-1 (TIMP-1)

BACKGROUND: The Enhanced Liver Fibrosis (ELF) score, which combines aminoterminal peptide of type III procollagen (PIIINP), hyaluronic acid (HA) and tissue inhibitor of metalloproteinases-1 (TIMP-1), is a clinically relevant tool for assessing liver fibrosis. This study examines the effect of preanalytical factors on the measurement of PIIINP, HA, TIMP-1 and the ELF score. METHODS: Serum pools spanning the measurement range of ELF biomarkers were spiked with increasing concentrations of hemolysate. Additionally, blood samples from 30 donors were centrifuged at 0-, 4-, 8-, and 24-hours post-collection. The influence of these factors was assessed by calculating the percentage difference in measured concentrations compared to baseline, and the underlying mechanisms were investigated. RESULTS: PIIINP showed significant susceptibility to hemolysis, with a 10% reduction in analyte concentration already at 0.5 g/L free hemoglobin. HA and TIMP-1 remained stable even at 10 g/L, and the ELF score up to 4 g/L. The effect on PIIINP cannot be explained by proteolytic activity, or assay components, but may be linked to direct effects of hemoglobin on the PIIINP molecule. Pre-centrifugation delays up to 8 h had minimal impact on PIIINP and HA measurements, whereas TIMP-1 concentrations fluctuated dramatically already after 4 h, but this had a negligible impact on the ELF score. CONCLUSION: Hemolysis significantly interferes with PIIINP measurement, and centrifugation delays particularly affect TIMP-1 measurement in serum, whereas the ELF-score remains robust. These findings highlight the need for thorough investigation of preanalytical factors of an assay alongside the manufacturer's instruction.