Circulating calprotectin as biomarker in neutrophil-related inflammation: Pre-analytical recommendations and reference values according to sample type

Background: Calprotectin (CLP) is a promising biomarker for the evaluation of neutrophil-related inflammation. Our aim was to establish reference values for circulating CLP in different sample types and to study the effect of pre-analytical variables. Methods: Reference values were determined in 100 healthy individuals. Pre-analytical variables were evaluated in 10 healthy controls and four rheumatoid arthritis patients with active disease and covered sample type (serum with/without gel separator, heparin, EDTA and citrate plasma), pre-centrifugation time (<2 h, 6 h, 24 h), storage condition (2-8 degrees C, 18-25 degrees C, 30 degrees C) and storage time (24 h, 72 h, 7 days). CLP measurements were performed with the EliA (TM) Calprotectin 2 assay on Phadia (TM) 200 (Thermo Fisher Scientific). Results: In healthy controls, baseline CLP concentrations in serum were more than double the concentration in EDTA and citrate plasma (0.909 mu g/mL versus 0.259 mu g/mL and 0.261 mu g/mL respectively). Heparin, EDTA and citrate stabilized CLP concentrations for up to 6 h before centrifugation, whereas significant increases in CLP levels were observed when serum was left untreated during that time period. Conclusion: Clinical studies on circulating CLP need to apply sample type-specific reference values and decision limits. To obtain reproducible CLP results in serum, more stringent pre-analytical sample handling instructions are needed.