Effect of different storage temperatures, sample collection procedures and immunoassay methods on osteocalcin measurement.

The apparent instability of measured osteocalcin has been reported as method-dependent and related to preanalytical variables such as storage temperature, and the use of anticoagulants and protease inhibitors. The aim of this study was to determine a sample collection procedure which minimised osteocalcin degradation. Blood samples from five normal individuals were collected with or without anticoagulants and protease inhibitors (heparin, EDTA, or heparin and aprotinin) and stored at 4 degrees C, -20 degrees C or -70 degrees C for up to 7 days, 28 days and 90 days respectively. Osteocalcin was measured by both a monoclonal EIA specific for intact osteocalcin and a bovine polyclonal RIA. Osteocalcin concentrations in serum and EDTA-treated samples significantly decreased by 40% (P < 0.001) with the ELISA and 72% (P < 0.001) with the RIA after 7 days storage at 4 degrees C. Similar falls were documented in these samples when stored at -20 degrees C and -70 degrees C and measured by the ELISA. Minimal changes in osteocalcin immunoreactivity were observed in either assay when heparin-treated plasma with or without aprotinin was stored at -20 degrees C or -70 degrees C for up to 90 days. The apparent instability of measured osteocalcin can be minimised using these conditions.