Influence of pre-analytical and analytical factors on osteoprotegerin measurements.

Osteoprotegerin (OPG), an osteoclastogenesis inhibitor implicated in bone remodelling, has emerged as a potential biomarker for cardiovascular disease. In order to implement OPG determination in the clinical laboratory, it is crucial to identify the most appropriate specimen type, preparation and measurement conditions. The present study focuses on identifying the pre-analytical variables that may influence OPG measurements. Serum and plasma (in EDTA, heparin and citrate) were collected from 45 healthy volunteers (men (n=21, 46.7%), women (n=24, 53.3%)). OPG was analysed by ELISA. The influence of the centrifugation speed, the number of freeze-thaw cycles, delay in sample processing, thermo-stability and endogenous interfering agents (haemolysis, triglycerides, bilirubin, cholesterol and RANKL) were studied. OPG concentrations were significantly lower (p<0.0001) in serum (1015±357 pg/mL) than in all plasma samples (1314±448 pg/mL in EDTA, 1209±417 pg/mL in heparin and 1260±498 pg/mL in citrate). Increasing centrifugation speed (200 g to 3000 g) did not change serum OPG concentration (p=0.88). However, OPG concentration significantly increased when centrifuged serum samples were stored at 48 h at room temperature (p<0.0001). Repeated freeze-thaw cycles did not modify OPG levels until 4 cycles (p<0.0001). Increasing time before processing the samples (2 h and 6 h) raised OPG concentrations both at room temperature (p<0.0001) or 4°C (p<0.001). Positive concentration-dependent interference of triglycerides was found in the analysed pooled samples; however, OPG concentrations were falsely diminished with haemoglobin interference. Bilirubin, cholesterol and RANKL did not interfere with OPG measurements.